Influence of polyunsaturated fatty acids on fluconazole susceptibility and drug efflux in Candida krusei by Abdullahi Temitope Jamiu Submitted in fulfilment of the requirements for the degree Magister Scientiae In the Department of Microbial, Biochemical and Food Biotechnology Faculty of Natural and Agricultural Sciences University of the Free State Bloemfontein South Africa February 2021 Supervisor: Prof. C.H. Pohl-Albertyn Co-Supervisors: Prof. J. Albertyn Mr. E. Bisschoff Page | 0 DISSERTATION SUMMARY Globally, fungal infections affect more than one billion people annually, with an estimated mortality of approximately two million people. The genus Candida is a highly heterogeneous group containing several opportunistic pathogens responsible for the increasing number of life-threatening mycoses, especially in immunocompromised subjects, including cancer patients, human immunodeficiency virus (HIV) positive patients, and organ transplant recipients. Compared to antibacterial counterparts, the number of available antifungals is limited, and the increase in antifungal resistance further complicates this. Resistance or tolerance towards all the available classes of antifungals has been reported. Strikingly, Candida krusei exhibits innate resistance to fluconazole (FLC) and rapid adaptive resistance to other antifungal drugs. Moreover, the role of efflux pumps (e.g. ATP-binding cassette 1, Abc1p) in this inherent FLC resistance remains unclear. This yeast also forms biofilms, which are potentially more resistant to antifungal drugs than planktonic counterparts. Additionally, there is paucity of information on the ability of exogenous polyunsaturated fatty acids (PUFAs) to overcome intrinsic antifungal resistance, such as in the case of C. krusei. In order to address this lack of knowledge, we firstly determined the susceptibility profiles of biofilms of C. krusei strains (and a C. albicans reference strain) towards FLC and five PUFAs [i.e. oleic acid (OA), linoleic (LA), gamma-linolenic acid (GLA), arachidonic acid (AA), and eicosapentaenoic (EPA)]. Our results showed that the antifungal activity of FLC against these strains is concentration-dependent, with C. krusei UFS Y-0277 displaying the least susceptibility. Moreover, the antifungal effect of the PUFAs is dependent on the strain, as well as on the chain length and dose of the fatty acid, with LA and GLA showing the most favourable activity. Upon combination therapy assay, we found that either of the two superior PUFAs (LA or GLA) potentiates the action of FLC towards the biofilms of the most-resistant strain of C. krusei. An initial attempt to examine the mechanism responsible for this revealed that the combination treatments induce the production of extracellular vesicles, cell membrane damage, and cell rupture. A subsequent membrane integrity assay confirmed this deleterious impact on the cell membrane. Our results also showed that antioxidants are capable of protecting C. krusei biofilms from the deleterious effects of the combination treatments. Additionally, these treatments had an inhibitory influence on the activity of efflux pumps, which was directly proportional to the concentration of PUFA used. Furthermore, our in vitro findings were corroborated by in vivo assays in a Caenorhabditis elegans infection model, which demonstrated that the combination treatments promote the overall survival and significantly reduce the intestinal fungal burden of infected nematodes. These observations may reiterate the combination of fatty acids with conventional antifungal drugs as a favourable therapeutic strategy deserving of increased traction and research for resistance reversal and infection Page | 1 control. We also aimed to establish a Clustered Regularly Interspaced Short Palindromic Repeats- Cas associated protein 9 (CRISPR-Cas9)-mediated gene-editing system for C. krusei since the absence of such system has impeded genome engineering, resistance, and virulence studies in this yeast. This was performed through the adaptation of a previously designed C. albicans-specific, CRISPR-Cas9 system (HIS-FLP type). This system's efficacy for gene- editing was validated by the successful homozygous deletion of two auxotrophic marker genes, URA3 and ADE2, in this yeast. Using the adapted system, we attempted to construct a Green Fluorescent Fusion (GFP) fusion of Abc1p to assess the influence of AA and FLC on the localisation, expression, and activity of Abc1p – in order to gain better insights into the role of this efflux pump in FLC resistance. However, this was unsuccessful, possibly due to the failure of the yeast to incorporate the supplied ABC1-GFP fusion donor DNA (dDNA). Hence, we resorted to using western blot analysis and efflux pump assay. Results obtained demonstrate that FLC increases the expression and functionality of Abc1p, suggesting that this transporter plays a role in FLC resistance. However, AA reduces the expression of Abc1p, and abrogates its activity in a dose-dependent manner, even in the presence of FLC. These findings highlight AA as a potential inhibitor of Abc1p and lent credence to the role of this transporter in FLC resistance. Taken together, this study demonstrates the FLC-potentiating activity of PUFAs against an intrinsically-resistant C. krusei in vitro and in vivo in a C. elegans infection model – which may pave the way for future studies into novel therapeutic strategies. It also establishes a successful development of a CRISPR-Cas9 system for C. krusei. Although preliminary findings demonstrate the involvement of Abc1p in FLC resistance and show the potential of AA as an inhibitor of this transporter, further studies are necessary for a definitive assertion. Keywords: Candida krusei, biofilm, antifungal resistance, polyunsaturated fatty acids, fluconazole susceptibility, combination therapy, Caenorhabditis elegans, CRISPR-Cas9 system, Abc1p Page | 2 LAY SUMMARY Every year, fungal infections, although less studied compared to bacterial infections, kill up to two million people. More worrisome is that many fungi are becoming increasingly resistant due to the extended and indiscriminate use of the limited available antifungals. For example, Candida krusei, a yeast (fungus) displays a natural resistance to FLC (fluconazole) – the most commonly used antifungal due to its affordability, low toxicity, and excellent efficacy. This yeast also develops an acquired (secondary) resistance to other antifungal drugs. Such resistance increases the risks of treatment failures, resulting in long-term hospitalisation, increased economic burden, and reduced quality of lives. Hence, there is an urgent need to develop effective therapeutic options and the complementary usage of antifungals (e.g. FLC) with natural compounds, such as polyunsaturated fatty acids (PUFAs), might offer more effective therapy. Hence, we evaluated the effect of the combination of FLC and PUFAs on FLC resistance of C. krusei. We found that when combined with FLC, either of two PUFAs (linoleic acid or gamma-linolenic acid) potentiates the susceptibility of C. krusei biofilms to FLC (i.e. combination of a PUFA with FLC enhanced the killing of C. krusei compared to when FLC is used alone). Furthermore, we designed a gene-editing tool (CRISPR-Cas9 system) for C. krusei, a system which was previously absent in this yeast. Our study also demonstrated that Abc1p transporter is vital for FLC-resistance in C. krusei and that arachidonic acid (a type of PUFA) is a potential inhibitor of this transporter. Together, this study provides answers to some key research questions and sets the pace for future investigations into overcoming antifungal resistance. Page | 3 DECLARATIONS I, Abdullahi Temitope Jamiu, declare that the Master’s degree dissertation or interrelated, publishable manuscripts/published articles, or coursework Master’s degree mini-dissertation that I herewith submit for the Master’s degree qualification in Microbiology at the University of the Free State is my independent work and that I have not previously submitted to any faculty or institution of higher education for the attainment of any qualification. I, Abdullahi Temitope Jamiu, hereby declare that all royalties regarding intellectual property that was developed during the course of, and/or in connection with the study at the University of the Free State, will accrue to the University. ---------------------------------------- Abdullahi T. Jamiu abdullahijamiu45@gmail.com Page | 4 DEDICATION In the Name of Allah, the most Beneficent, the entirely Merciful. All praises and adorations are due the Lord of all worlds. This work is dedicated to Allah (SWT) for bestowing me with the wellbeing, knowledge, and tenacity to complete this dissertation; His beloved Prophet Muhammad (peace and blessing be upon him, his household and companions), the quintessential role model and a mercy to mankind; and all sincere seekers of beneficial knowledge in all realms of life. “This is a favour of Allah. He grants it to whomever He wills. And Allah is the Lord of infinite bounty.” (Qur’an 62 vs 4) Page | 5 ACKNOWLEDGEMENTS I would like to express my heartfelt gratitude to the following persons and institutions:  Prof. Carolina Pohl-Albertyn, for her unmatched guidance, support, motivation, and confidence throughout this study. For wonderfully unleashing my potential. I am truly grateful.  Prof. Jacobus Albertyn, for his stellar mentorship, valuable input and everyday support  Mr. Eduvan Bisshoff, for his brilliant input and guidance with the molecular aspect of this study  Prof. Olihile Sebolai, for his assistance and everyday support  Dr. Oluwasegun Kuloyo, for being a fantastic mentor and wingman  Dr. Ruan Fourie, for his valuable discussions and pace-setting  Dr. Sabiu Saheed, for his kindness, motivation, and moral support  Ms Nthabiseng Mokoena, for her help with the nematodes  Mrs Aurelia Jansen, at the UFS Yeast Culture Collection  Everyone in the Pathogenic Yeast Research Group, for the great moments shared together  Ms Hanlie Grobler, for her assistance with electron microscopy  Dr. Obinna Ezeokoli, for his assistance with protein analyses  Dr. Wunmi Ogundeji, for her help with fluorescence microscopy  Ms Toluwase Adedoja and Ms Gloria Kankam, for their kindness and support  Dr. Samuel Folorunso, for his support  The Department of Microbial, Biochemical and Food Biotechnology, thank you for the enabling environment Financial assistance: The financial assistance of the National Research Foundation (NRF) towards this research is hereby acknowledged (grant number 117435). Opinions expressed and conclusions arrived at, are those of the author and are not necessarily to be attributed to the NRF. The financial assistance of the South African Fryer Oil Initiative (SAFOI) is also acknowledged. Page | 6 Personal Acknowledgements: A very special thank you to the following people: o My parents, Alhaji Ishaq Jamiu and Alhaja Khadijah Jamiu, for their unwavering love, kindliness, prayer, and support. Most importantly, thank you for always believing in me! o My uncles, Mr. Kunle Adedigba and Mr. Rahman Olarinde, for their altruism, support, and guidance. o My siblings, thank you for your affection and never-ending support. o The special one, thank you for being a wonderful backroom supporter! o My entire family, I thank you for your support. o Musa Akanbi, for leading the way! o All dear friends, brothers, and sisters in faith, you guys are amazing! o To others far and wide, thank you for being part of this story “I am, because we are’’. Page | 7 ETHICAL CLEARANCE This research was approved by the Biosafety & Environmental Research Ethics Committee of the University of the Free State with ethical clearance number: UFS-ESD2019/0029 Page | 8 RESEARCH OUTPUT  Symposia & Conferences: AT Jamiu, J Albertyn, OM Sebolai, CH Pohl. Polyunsaturated fatty acids potentiate the susceptibility of Candida krusei to fluconazole via the distortion of membrane integrity and efflux pump activity. World Antimicrobial Awareness Week (WAAW) 2020 Virtual Symposium. 19 – 20 November 2020 [Oral presentation]. AT Jamiu, J Albertyn, OM Sebolai, O Kuloyo, N Mokoena, CH Pohl. Arachidonic acid increases the susceptibility of Candida krusei to fluconazole. American Society for Microbiology (ASM) Microbe 2020 Conference. 18 – 22 June 2020 [ePoster]. AT Jamiu, O Kuloyo, N Mokoena, J Albertyn, CH Pohl. Anti-biofilm activity of unsaturated fatty acids with fluconazole. Canadian Fungal Research Network 2020. 29 – 30 July 2020 [Oral presentation]. AT Jamiu, O Kuloyo, N Mokoena, J Albertyn, CH Pohl. Influence of polyunsaturated fatty acids on fluconazole susceptibility of Candida krusei. University of the Free State Postgraduate Academic Conference. 20 September 2019 [Oral presentation]. AT Jamiu, O Kuloyo, N Mokoena, J Albertyn, CH Pohl. Influence of polyunsaturated fatty acids on fluconazole susceptibility of Candida krusei. Young Scientist Symposium on Infectious Diseases. 27 – 28 May 2019 [Poster].  Publication: AT Jamiu, J Albertyn, OM Sebolai, CH Pohl (2020) Update on Candida krusei, a potential multidrug-resistant pathogen, Medical Mycology, 59:14-30, https://doi.org/10.1093/mmy/myaa031 Page | 9 LIST OF FIGURES Chapter 1 Fig. 1 Schematic representation of targets of representative of various antifungal classes (Adapted from Lupetti et al. 2002; Robbins et al. 2017). Fig. 2 Description of the mechanism of action of flucytosine (Obtained from Kabir and Ahmad 2013). Fig. 3 Simple illustration of the outcome of mono- and combination therapeutic approaches Chapter 2 Fig. 1 An illustration of the components and fundamental antifungal resistance mechanisms of fungal biofilm. A typical biofilm has reduced resistance to drugs due to inherent factors, such as increased cell density, presence of persister cells, modulated physiology, extracellular polymer matrix, overexpressed and modified drug targets, and enhanced efflux pump activity (Adapted from Costa-Orlandi et al. 2017). Fig. 2 Quantification of biofilms of Candida krusei and Candida albicans. (A) Metabolic activity of biofilms measured by XTT reduction assay. (B) Biofilm biomass quantified by CV assay. (C) BSA index of biofilms determined using XTT and CV values. Fig. 3 The effect of various concentrations of fluconazole on the metabolic activity of biofilms of C. krusei strains and C. albicans SC5314 after incubation at 37oC for 48 h, using XTT reduction assay. Fig. 4 Effect of varying concentrations of various fatty acids (A- oleic acid, B- linoleic acid, C- gamma- linolenic acid, D- arachidonic acid, E- eicosapentaenoic) on the metabolic activity of C. krusei strains and C. albicans SC5314 biofilms after incubation at 37oC for 48 h, using XTT reduction assay. Fig. 5 The biofilm inhibitory activity and structures of unsaturated fatty acids. Fig. 6 The effect of fluconazole in the presence or absence of LA (A) or GLA (B) on the metabolic activity of C. krusei UFS Y-0277 biofilm after incubation at 37oC for 48 h, using XTT reduction assay. Fig. 7.1 Scanning electron micrographs of C. krusei UFS Y-0277 biofilms under various treatment conditions after incubation at 37oC for 48 h. Fig. 7.2 Scanning electron micrographs of C. krusei UFS Y-0277 biofilms under various treatment conditions after incubation at 37oC for 48 h (x8000). Fig. 8 Fluorescence of C. krusei UFS Y-0277 cells stained with propidium iodide dye after exposure to various treatment conditions. Fluorescence of cells exposed to fluconazole in the presence or absence of LA (A) or GLA (B). Fig. 9 Fluorescence micrographs of C. krusei UFS Y-0277 cells stained with propidium iodide dye after exposure to various treatment conditions. Fig. 10 Biomass of C. krusei UFS Y-0277 biofilms after exposure to the combination of FLC and LA (A) and FLC+GLA (B) in the presence or absence of antioxidants (BHT or TPGS). Page | 10 Fig. 11 Rhodamine 6G efflux in C. krusei UFS Y-0277 biofilms after treatment with fluconazole in the presence or absence of 0.1 mM LA (A), 1 mM LA (B), 0.1 mM GLA (C) or 1 mM GLA (D). Fig. 12 Survival of infected Caenorhabditis elegans after treatment with linoleic acid (A) or gamma- linolenic acid (B) in the presence or absence of fluconazole. OP50 represents uninfected nematodes fed with Escherichia coli OP50 (uninfected group). Fig. 13 Fungal burden of infected Caenorhabditis elegans after treatment with linoleic acid (A) or gamma-linolenic acid (B) in the presence or absence of fluconazole. Chapter 3 Fig. 1 Mechanisms used to repair double-stand breaks (Obtained from Saha et al. 2019). Fig. 2 An illustration of the procedure followed for primer designs for the assembly of fragments with NEBuilder® HiFi DNA Assembly kit (https://international.neb.com/). Fig. 3 A schematic representation of the NEBuilder® HiFi DNA Assembly reaction (https://international.neb.com/). Fig. 4 A schematic summary of the steps involved in the construction of CK pADH99 plasmid. Fig. 5 A workflow for the preparation of CK pADH147 plasmid. Fig. 6 A flow chart of the steps followed to design Cas9 and gRNA expression cassettes. (A) CK pADH99 plasmid is digested with restriction enzyme MssI to generate an intact Cas9 cassette (B) The 5' (Fragment A) and 3' (Fragment B) regions of the gRNA cassette are prepared from pADH110 and CK pADH147, respectively, by PCR with appropriate primers and oligonucleotide. Fig. 7 A schematic representation of the steps followed to design a donor DNA. Fig. 8 A plasmid map of pADH99 showing components such as C. albicans 5’-HIS1 region, flippase recognition target (FRT) region, CAS9 gene under the control of C. albicans ENO1 promoter, and an overlapping portion of the nourseothricin N-acetyltransferase (NAT) marker gene. Fig. 9 Profile of pADH99 plasmid digested with NcoI and SmaI restriction enzymes. Fig. 10 Amplification of 5’-HIS1 region of Candida krusei. Fig. 11 Amplification of ENO1 promoter region of Candida krusei. Fig. 12 Synthesis of flippase recognition target (FRT) fragment. Fig. 13 Screening of transformants for CK pADH99 plasmid with XbaI restriction enzyme. Fig. 14 Screening of transformants for CK pADH99 plasmid with BamHI restriction enzyme. Fig. 15 A map of pADH110 plasmid depicting an overlapping portion of the nourseothricin N- acetyltransferase (NAT) marker gene and SNR52 promoter (of gRNA). Page | 11 Fig. 16 A map of pADH147 plasmid showing gRNA scaffold, flippase recognition target (FRT) region, and 3’-HIS1 region of C. albicans. Fig. 17 Linearisation of pADH147. Fig. 18 Amplification of 3’-HIS1 region of Candida krusei. Fig. 19 Screening of transformants for CK pADH147 plasmid with Bgll and HindIII restriction enzymes. Fig. 20 A representation of de novo pyrimidine ribonucleotide biosynthetic pathway. Fig. 21 Construction of Cas9 cassette. Fig. 22 Construction of the first component (Fragment A) of gRNA cassette. Fig. 23 Construction of the second component (Fragment B) of gRNA cassette specific for URA3. Fig. 24 Construction of complete URA3-specific gRNA cassette. Fig. 25 Synthesis of an intact URA3 donor DNA (dDNA). Fig. 26 Uracil-deficient minimal medium plate with the transformed and wildtype colonies. Fig. 27 Gel profile of representatives of ura3Δ/Δ mutants. Fig. 28 Comparison of the colonial morphology of ura3Δ/Δ mutant and wildtype strain. Fig. 29 Microscopic comparison of the phenotype of ura3Δ/Δ mutant (B) and wildtype strain (A). Fig. 30 A representation of de novo purine biosynthetic pathway. Fig. 31 Construction of the second component (Fragment B) of gRNA cassette specific for ADE2 gene. Fig. 32 Construction of complete ADE2-specific gRNA cassette. Fig. 33 Synthesis of intact ADE2 donor DNA (dDNA). Fig. 34 Adenine-deficient minimal medium plate showing growth of ade2Δ/Δ mutant. Fig. 35 Gel profile of ade2Δ/Δ mutant (Lane 2), wildtype (Lane 1), and white transformant (Lane 3). Fig. 36 Comparison of the colonial morphology of ade2Δ/Δ mutant and wildtype strain. Fig. 37 Microscopic comparison of the phenotype of ade2Δ/Δ mutant (B) and wildtype strain (A). ade2Δ/Δ mutant appear predominantly in yeast form. Fig. 38 Successful excision of CRISPR-Cas9 cassette from the genome of the mutants. Fig. 39 Schematic representation of the complete CRISPR-Cas9 system used for gene editing in Candida krusei. Page | 12 Chapter 4 Fig. 1 Nucleotide sequence alignment of selected regions of C. krusei ABC1 and ABC11 genes. Fig. 2 A section of a vector map depicting the components of ABC1-GFP donor DNA. Fig. 3 Construction of the components of CRISPR-Cas9 cassette. Fig. 4 Synthesis of components of ABC1-GFP donor DNA. Fig. 5 Gel profile showing successful synthesis and amplification of intact ABC1-GFP donor DNA (~2277 bp). Fig. 6 Gel profile depicting unsuccessful incorporation of ABC1-GFP donor DNA into the genome of the transformants. Fig. 7 SDS-PAGE profile of proteins from Candida krusei biofilms following exposure to various treatments. Fig. 8 Confirmation of Abc1p protein and assessment of its expression level with western blot analysis following exposure to various treatments. Fig. 9 Rhodamine 6G efflux in C. krusei UFS Y-0277 biofilms after treatment with 0.1 mM arachidonic acid (A) and 1 mM arachidonic acid (B) in the presence or absence of fluconazole (32 μg/ml). Page | 13 LIST OF TABLES Chapter 1 Table 1 Selected examples of combination therapy with fatty acids against pathogenic fungi Chapter 2 Table 1 The MIC50 and SMIC50 of fluconazole against biofilms Table 2 MIC50 and SMIC50 of unsaturated fatty acids against C. krusei and C. albicans biofilms Chapter 3 Table 1 Description of HIS-FLP plasmids constructed by Nguyen and co-workers (2017) Table 2 Primers used in this study Table 3 Reaction mixture for KAPA Taq PCR kit (KAPA Biosystems) Table 4 Reaction mixture for KAPA HiFi PCR kit (KAPA Biosystems) Table 5 Reaction mixture for KOD Hot Start DNA polymerase kit (Novagen®) Table 6 PCR condition for KAPA Taq PCR kit (KAPA Biosystems) Table 7 PCR condition for KAPA HiFi PCR kit (KAPA Biosystems) Table 8 PCR condition for KOD Hot Start DNA polymerase kit (Novagen®) Table 9 Reaction mixture for digestion reaction Chapter 4 Table 1 Plasmids used in this study Table 2 Reaction mixture for KOD Hot Start DNA polymerase kit (Novagen®) Table 3 PCR condition for KOD Hot Start DNA polymerase kit (Novagen®) Table 4 Primers used in this study Page | 14 TABLE OF CONTENTS DISSERTATION SUMMARY ................................................................................................ 1 LAY SUMMARY .................................................................................................................... 3 DECLARATIONS .................................................................................................................. 4 DEDICATION ........................................................................................................................ 5 ACKNOWLEDGEMENTS ..................................................................................................... 6 ETHICAL CLEARANCE ........................................................................................................ 8 RESEARCH OUTPUT .......................................................................................................... 9 LIST OF FIGURES ............................................................................................................. 10 LIST OF TABLES ................................................................................................................ 14 TABLE OF CONTENTS ...................................................................................................... 15 CHAPTER 1 ....................................................................................................................... 19 SECTION A ........................................................................................................................ 20 Motivation…… ................................................................................................................ 20 SECTION B ........................................................................................................................ 22 SECTION C ........................................................................................................................ 40 1.1 Introduction .............................................................................................................. 40 1.2 Antifungal drugs and their mechanistic profiles ......................................................... 40 1.2.2 Polyenes ........................................................................................................ 42 1.2.3 Echinocandins ............................................................................................... 43 1.2.4 Nucleoside/pyrimidine analogs ...................................................................... 43 1.2.5 Allylamines and Morpholines ......................................................................... 44 1.3 Combination therapy with fatty acids ........................................................................ 45 1.4 General conclusions for Chapter 1 ........................................................................... 47 1.5 Research Aim and Objectives .................................................................................. 49 1.6 References ............................................................................................................... 50 CHAPTER 2 ....................................................................................................................... 61 2.1 Abstract .................................................................................................................... 63 2.2 Introduction .............................................................................................................. 64 2.3 Materials and Methods ............................................................................................. 66 2.3.1 Strains used ................................................................................................... 66 2.3.2 Drug and fatty acids ....................................................................................... 66 2.3.3 Biofilm formation ............................................................................................ 67 2.3.3.1 XTT reduction assay .................................................................................. 67 2.3.3.2 Crystal violet assay .................................................................................... 68 2.3.4 Determination of the minimum biofilm inhibitory concentration of fluconazole 68 2.3.5 Determination of the minimum biofilm inhibitory concentration of fatty acids .. 69 Page | 15 2.3.6 Determination of the potentiating effect of fatty acids on fluconazole susceptibility .................................................................................................................... 69 2.3.7 Morphological examination of treated biofilms................................................ 70 2.3.8 Influence of fatty acids on membrane integrity of C. krusei ............................ 70 2.3.9 Influence of antioxidants on the potentiating effect of the combination treatments. ...................................................................................................................... 71 2.3.10 Influence of fatty acids on efflux pump activity of C. krusei............................. 71 2.3.11 In vivo evaluation of the potentiating effect of fatty acids on fluconazole activity…….. .................................................................................................................... 72 2.3.11.1 Nematode propagation and bacterial culture ........................................... 72 2.3.11.2 Infection of C. elegans ............................................................................ 72 2.3.11.3 C. elegans treatment assay .................................................................... 72 2.3.11.4 Evaluation of fungal burden within C. elegans ........................................ 73 2.3.12 Statistical analysis ......................................................................................... 73 2.4 Results and Discussions .......................................................................................... 73 2.4.1 Biofilm formation and quantification ............................................................... 73 2.4.2 Determination of minimum biofilm inhibitory concentration of fluconazole ...... 76 2.4.3 Determination of the minimum biofilm inhibitory concentration of fatty acids .. 77 2.4.4 Polyunsaturated fatty acids potentiate the susceptibility of C. krusei biofilm to fluconazole ...................................................................................................................... 81 2.4.5 Morphological examination of treated biofilms................................................ 82 2.4.6 Influence of fatty acids on membrane integrity of C. krusei ............................ 86 2.4.7 Antioxidants rescue biofilm from the toxicity of combination treatments ......... 88 2.4.8 Influence of fatty acids on efflux pump activity of C. krusei............................. 89 2.4.9 Combination treatments prolong the lifespan of infected nematodes ............. 91 2.4.10 Combination treatments reduce the fungal burden of infected nematodes ..... 94 2.5 Conclusions .............................................................................................................. 95 2.6 References ............................................................................................................... 96 CHAPTER 3 ..................................................................................................................... 112 3.1 Abstract .................................................................................................................. 113 3.2 Introduction ............................................................................................................ 114 3.3 Materials and Methods ........................................................................................... 117 3.3.1 Strains used ................................................................................................. 117 3.3.2 In silico analyses .......................................................................................... 117 3.3.3 Plasmids and primers used .......................................................................... 117 3.3.4 Polymerase chain reaction (PCR) amplification ........................................... 119 3.3.5 Genomic DNA extraction ............................................................................. 121 3.3.5.1 DNA extraction with Zymo Research kit ................................................... 121 3.3.5.2 DNA extraction with a manual method ...................................................... 121 3.3.6 Agarose gel electrophoresis ......................................................................... 122 Page | 16 3.3.7 Gel extraction............................................................................................... 122 3.3.8 Restriction digest ......................................................................................... 122 3.3.9 DNA assembly using NEBuilder® ................................................................ 123 3.3.10 Bacterial transformation ............................................................................... 124 3.3.11 Plasmid extraction and purification ............................................................... 125 3.3.11.1 Miniprep – lysis by boiling method ........................................................ 125 3.3.11.2 Plasmid purification ............................................................................... 125 3.3.12 Minimum fungicidal concentration (MFC) for nourseothricin ......................... 126 3.3.13 Construction of a HIS-FLP type CRISPR-Cas9 system for C. krusei ............ 126 3.3.13.1 Adaptation of pADH99 plasmid ............................................................. 126 3.3.13.2 Propagation of pADH110 ...................................................................... 128 3.3.13.3 Adaptation of pADH147 ........................................................................ 128 3.3.14 Validation of the system ............................................................................... 129 3.3.14.1 Deletion of URA3 gene ......................................................................... 129 3.3.14.2 Deletion of ADE2 gene ......................................................................... 133 3.3.15 Removal of CRISPR-Cas9 cassette ............................................................. 134 3.4 Results and Discussions ........................................................................................ 135 3.4.1 Constructing a HIS-FLP type CRISPR-Cas9 system for C. krusei ................ 135 3.4.1.1 Adapting pADH99 plasmid ....................................................................... 135 3.4.1.2 Propagating pADH110 ............................................................................. 141 3.4.1.3 Adapting pADH147 ................................................................................... 141 3.4.2 Validating the adapted system ..................................................................... 144 3.4.2.1 Deleting URA3 gene ................................................................................. 145 3.4.2.2 Deleting ADE2 gene ................................................................................. 152 3.4.3 Removing CRISPR-Cas9 cassette ............................................................... 158 3.4.4 The complete system ................................................................................... 159 3.5 Conclusions ............................................................................................................ 161 3.6 References ............................................................................................................. 161 CHAPTER 4 ..................................................................................................................... 169 4.1 Abstract .................................................................................................................. 171 4.2 Introduction ............................................................................................................ 172 4.3 Materials and Methods ........................................................................................... 173 4.3.1 Strains used ................................................................................................. 173 4.3.2 Drug and fatty acids ..................................................................................... 173 4.3.3 Construction of ABC1-GFP mutant with CRISPR-Cas9 system ................... 173 4.3.3.1 CRISPR-Cas9 cassettes for ABC1-GFP fusion ........................................ 177 4.3.3.2 Design of ABC1-GFP fusion donor DNA .................................................. 178 4.3.3.3 Transformation ......................................................................................... 180 4.3.4 Influence of arachidonic acid and fluconazole on Abc1p expression ............ 180 Page | 17 4.3.4.1 Biofilm formation ...................................................................................... 180 4.3.4.2 Protein extraction and visualisation of Abc1p on SDS-PAGE ................... 180 4.3.4.3 Western blot analysis and Immunodetection of Abc1p ............................. 181 4.3.5 Influence of arachidonic acid and fluconazole on the activity of Abc1p ........ 181 4.3.6 Statistical analysis ....................................................................................... 182 4.4 Results and Discussions ........................................................................................ 182 4.4.1 Constructing an ABC1-GFP mutant with CRISPR-Cas9 system .................. 182 4.4.1.1 CRISPR-Cas9 cassettes for ABC1-GFP fusion ........................................ 182 4.4.1.2 Designing ABC1-GFP fusion donor DNA .................................................. 183 4.4.1.3 Transformation ......................................................................................... 185 4.4.2 Influence of arachidonic acid and fluconazole on Abc1p expression ............ 185 4.4.3 Abc1p activity is increased by fluconazole but extenuated by arachidonic acid in a dose-dependent manner......................................................................................... 187 4.5 Conclusions ............................................................................................................ 189 4.6 References ............................................................................................................. 189 CHAPTER 5 ..................................................................................................................... 195 5.1 Influence of polyunsaturated fatty acids on in vitro fluconazole susceptibility of C. krusei….. ........................................................................................................................... 196 5.2 Influence of polyunsaturated fatty acids on the survival and fungal burden of infected C. elegans ......................................................................................................................... 198 5.3 Establishment of a CRISPR-Cas9 genome editing tool for C. krusei ...................... 199 5.4 Influence of arachidonic acid and fluconazole on the expression and function of Abc1p…. ........................................................................................................................... 200 5.5 References ............................................................................................................. 201 APPENDIX A: ETHICAL CLEARANCE FORM.................................................................. 209 APPENDIX B: DEPOSITED MUTANTS’ FORMS ............................................................. 210 Page | 18 CHAPTER 1 LITERATURE REVIEW Candida krusei as a drug-resistant pathogen and influence of fatty acids on drug resistance in Candida species Page | 19 SECTION A Motivation Some members of the Candida genus are part of the commensal microbiota of humans; they colonise the gastrointestinal and urogenital tracts, oral cavity, mucosal as well as cutaneous surfaces in healthy individuals (Filler and Sheppard 2006; Bizerra et al. 2008). These yeasts are typically innocuous, with their growth and spread well controlled by coexisting microbiota, intact epithelial barriers and defences of the innate immune system (Kabir and Ahmad 2013). However, under certain circumstances, such as mucosal barrier disruption, immune system impairment, usage of broad-spectrum antibiotics, or a combination thereof, Candida spp. may proliferate and multiply to cause various opportunistic infections, ranging from self-limiting topical diseases to life-threatening systemic infections (Samaranayake and MacFarlane 1990; Dixon et al. 1996). The incidence of life-threatening candidal infections has markedly increased over the years due to the increasing population of immunosuppressed patients, such as HIV/AIDS and cancer patients, premature neonates, and organ transplant recipients (Pfaller and Diekema 2007). Candida albicans remains the primary cause of invasive candidiasis; however, the epidemiology has changed in recent years, with 35 to 65% of all cases of infections attributed to non-albicans Candida (NAC) species (i.e. C. krusei, C. tropicalis, C. parapsilosis, and C. glabrata) (Trick et al. 2002; Poikonen et al. 2010; Chi et al. 2011; da Silva et al. 2013; Sadeghi et al. 2018). To date, fungal infections are mainly treated with either azole, echinocandin or polyene antifungals. Among these, azoles such as posaconazole, fluconazole (FLC), voriconazole, and itraconazole are the most commonly used in the treatment and prevention of mycoses, due to their broad-spectrum activity (Falci and Pasqualotto 2013). These compounds inhibit lanosterol 14α-demethylase (Erg11p), an enzyme important for ergosterol biosynthesis (Kathiravan et al. 2012). Its inhibition results in the depletion of ergosterol and accumulation of toxic methylated sterols, which ultimately result in the arrest of cell growth (Sheehan et al. 1999; Weete et al. 2010). Fluconazole remains the most widely used azole for the treatment of candidiasis; however, its fungistatic nature, as well as widespread and extended use, has led to the development of resistance among fungi (Shukla et al. 2016). More worrisome, Candida krusei, a member of the highly heterogeneous Candida genus, exhibits innate resistance to this drug, with more than 97% of isolates displaying resistance (Whaley et al. 2017). Many studies have attributed the mechanism of inherent FLC resistance in this yeast to the low affinity of Erg11p for FLC (Venkateswarlu et al. 1997; Orozco et al. 1998; Fukuoka et al. 2003). The role of efflux pump transporters, including Abc1p, in this inherent resistance remains controversial and warrants further studies. Page | 20 Additionally, like other common Candida spp., C. krusei forms recalcitrant biofilms with higher antifungal resistance compared to planktonic cells (Hacioglu et al. 2018). This enhanced resistance could be explained by the inherent complexity of biofilm owing to its sophisticated structures and functions (Finkel and Mitchell 2011; Ramage et al. 2012). Moreover, their presence on medical implants increases the risks of systemic infections, seeds recurrent infections, and results in treatment failures, increased economic burden, and reduced quality of lives (Ramage et al. 2006). This highlights an urgent need to develop novel antifungal treatment approaches and combination therapy may be one such option. The study of putative antifungal-resistance related genes, for example, ABC1 and ERG11, using molecular tools would provide valuable insights into the roles of these genes in antifungal resistance. These insights may consequently guide the preservation of current antifungal drugs and inspire the development of novel therapeutic strategies. However, such molecular study is impeded in C. krusei by the absence of a facile, precise, and efficient genome engineering tool like CRISPR technology. Furthermore, although polyunsaturated fatty acids (PUFAs) have been reported to increase the sensitivity of intrinsically-susceptible C. albicans and C. dubliniensis biofilms to antifungal drugs such as clotrimazole, amphotericin B, and FLC (Ells et al. 2009; Thibane et al. 2012b; Kuloyo et al. 2020), whether exogenous PUFAs could reverse intrinsic antifungal resistance in C. krusei remains to be investigated. This study was, therefore, conducted to address the aforementioned knowledge gaps. Page | 21 SECTION B This section was published in Medical Mycology; the writing and reference style of the journal was followed. The candidate, Abdullahi Temitope Jamiu, conducted the literature study and wrote the manuscript. The supervisor and co-authors reviewed and provided constructive feedbacks on the manuscript. Citation: AT Jamiu, J Albertyn, OM Sebolai, CH Pohl (2020) Update on Candida krusei, a potential multidrug-resistant pathogen, Medical Mycology, 59:14-30, https://doi.org/10.1093/mmy/myaa031 License and copyright:  This article is licensed under Creative Commons Attribution Non-Commercial No Derivatives license (CC BY-NC-ND).  Copyright of this article is ceded to Oxford University Press. However, authors retain the following rights: o The right to use all or part of the article and abstract, for personal use, including their own classroom teaching purposes. o The right to use all or part of the article and abstract, in the preparation of derivative works, extension of the article into book-length or in other works, provided that a full acknowledgement is made to the original publication in the journal. o The right to include the article in full or in part in a thesis or dissertation, provided that this is not published commercially. Page | 22 Page | 23 Page | 24 Page | 25 Page | 26 Page | 27 Page | 28 Page | 29 Page | 30 Page | 31 Page | 32 Page | 33 Page | 34 Page | 35 Page | 36 Page | 37 Page | 38 Page | 39 SECTION C The published article described in SECTION B and this section form the complete literature review for this dissertation. This section is followed by general conclusions of chapter 1, and the aim and objectives of this dissertation 1.1 Introduction Undoubtedly, antimicrobial resistance is an enormous public health crisis responsible for escalated therapeutic failures, increased hospitalisation, high morbidity and mortality, and amplified economic burden (Prestinaci et al. 2015; Shrestha et al. 2018; Dadgostar 2019). By extension, antifungal resistance amongst several pathogenic fungal species, especially Candida species, poses a considerable threat to human and veterinary medicine (Moran et al. 2010; Arastehfar et al. 2020; Bhattacharya et al. 2020). Additionally, the number of antifungal drugs available is limited compared to antibacterial counterparts. This is partially due to the eukaryotic nature of both fungi and humans, which in turn makes the development of safe, less toxic and broad-spectrum antifungal agents a more challenging endeavour (Campoy and Adrio 2017). As a result, other therapeutic approaches are being explored. One such strategy is combination therapy which has been harnessed against pathogens in various forms, including combination of conventional antifungal drugs with appropriate non-antimicrobial compounds (e.g. fatty acids, calcineurin inhibitors, phytochemicals) (Ells et al. 2009; Shrestha et al. 2015; Sharifzadeh et al. 2018; Jia et al. 2019). The use of fatty acids (FAs) as antifungal compounds, especially as adjuvants that potentiate the activity of known antifungals, is of interest in the current study. Certain FAs have been reported to exhibit antiviral, antibacterial, and antifungal activity (Chanda et al. 2018). Such antimicrobial properties are usually dependent on various factors, including the length of carbon chain and degree of unsaturation. Interestingly, FAs can also be used as adjuncts to enhance the efficacy of antimicrobial agents. Polyunsaturated fatty acids (PUFAs), such as AA and stearidonic acid (SDA), have been found to increase the susceptibility of biofilms of Candida spp. to antifungal drugs, including amphotericin B, clotrimazole and FLC (Ells et al. 2009; Thibane et al. 2012b; Mishra et al. 2014; Kuloyo et al. 2020). Although the precise mechanisms of action of these adjunct FAs remain unclear, they have been implicated to induce membrane disorganisation, increase oxidative stress and interfere with ATP synthesis (Ells et al. 2009; Thibane et al. 2012b; Kuloyo et al. 2020). 1.2 Antifungal drugs and their mechanistic profiles Among the available classes of antifungals, only three classes are effective for the treatment of obstinate invasive candidal infections, and these include the azoles, polyenes, and Page | 40 echinocandins. Nucleoside analogues, allylamines, and morpholines are usually used for topical treatment or adjuvants with other antifungal drugs (Zhanel et al. 1997; Finch and Warshaw 2007). The descriptions and mechanisms of action of these drugs are discussed herein. 1.2.1 Azoles Azoles are heterocyclic compounds with at least one nitrogen atom in their (five-membered) rings. The azoles are excellent inhibitors of the cytochrome P450 enzyme, Erg11p encoded by ERG11 in Candida and Cryptococcus spp., and CYP51 in Aspergillus spp., a key enzyme involved in the conversion of lanosterol to ergosterol during the biosynthesis of ergosterol (Kathiravan et al. 2012). More specifically, the iron atom within the heme group of the active site of the enzyme is bound by the free nitrogen atom of the azole ring, thus preventing the activation of oxygen and as a result inhibits the synthesis of ergosterol from lanosterol (Hitchcock, 1991). Like cholesterol in animals, ergosterol is an essential component of fungal cell membranes, playing an important role in maintaining membrane fluidity and stability, inhibition of its biosynthesis by azoles results in the depletion of ergosterol and accumulation of toxic methylated sterol, 14α-methyl-3,6-diol, and this results in the disruption of cell membrane fluidity and stability, increased membrane permeability and arrest of cell growth (Fig. 1) (Sheehan et al. 1999; Weete et al. 1999). Furthermore, azoles have also been reported to exert antifungal effects via the inhibition of hyphal development, inactivation of vacuolar ATPases, as well as through the induction of oxidative and nitrosative stress (Odds et al. 1986; Zhang et al. 2010; Arana et al. 2010; Kabir and Ahmad 2013; Peng et al. 2018; Dbouk et al. 2019). Azoles are classified as imidazole or triazole based on the number and arrangement of their nitrogen atoms. Whilst imidazole has two non-adjacent nitrogen atoms; triazole has three adjacent nitrogen atoms in its five-membered rings (Arnold et al. 2010; Campestre et al. 2017). The imidazoles (bifonazole, clotrimazole, econazole, ketoconazole) are limited to topical treatment of fungal infections, due to their poor water solubility and severe side effects when used orally and/or systemically. Ketoconazole can be used systemically; however, it is less preferred to the triazoles due to severe associated toxicity (Maertens 2004). The limitations of the imidazoles led to the development of the first generation (FLC, itraconazole) and second- generation (voriconazole, posaconazole, isavuconazole) triazoles that generally exhibit a broader spectrum of activity due to the presence of triazole structure instead of the imidazole ring. Additionally, in comparison to the imidazoles, they have improved safety profiles due to their increased affinities for the target enzyme (Girmenia 2009; Mast et al. 2013). Among azoles, FLC is the most widely used azole for the treatment of candidiasis because of its affordability, broad-spectrum activity, high water-solubility, high bioavailability, and good tolerance with few side effects (Grant and Clissold 1990; Andriole 2000; Falci and Pasqualotto Page | 41 2013). However, its fungistatic nature, widespread, misuse, and extended use, have led to increased resistance among yeasts (Shukla et al. 2016). At present, the azole with the broadest activity is posaconazole, which has high effectiveness against invasive candidiasis (Campoy and Adrio 2017). Fig. 1 Schematic representation of targets of representative various antifungal classes (Adapted from Lupetti et al. 2002; Robbins et al. 2017). 1.2.2 Polyenes The polyenes are amphiphilic macrolides consisting of a 20 to 40 carbons macrolactone ring, conjugated with a d-mycosimine group (Mayers 2009). They are fungicidal and are produced by Streptomyces species (Moen et al. 2009; Kabir and Ahmad 2013). Like azoles, polyenes, such as amphotericin B, nystatin, and natamycin, affect the fungal cell membrane; they exert antifungal effect by binding and forming complexes with ergosterol. This results in the formation of transmembrane channels, plasma membrane disruption, leakage of monovalent ions, as well as other intracellular cell contents, and ultimately, fungal cell death (Hossain and Ghannoum 2001; Andes 2003; Yadav et al. 2012). More recently, a detailed structural and biophysical study has highlighted polyenes’ mode of action to be beyond complex formation with ergosterol. The study emphasized that polyenes bind and directly extract ergosterol from the fungal cell membrane (Fig. 1). This consequently hinders the essential cellular functions of ergosterol, resulting in increased membrane permeability, membrane leakage and consequently, cell death (Anderson et al. 2014). Furthermore, polyenes also exert anti-mycotic action via the production of reactive oxygen species, which results in oxidative damage and impairment of fungal membranes (Mesa-Arango et al. 2012; Mesa-Arango et al. 2014; Page | 42 Scorzoni et al. 2017). Polyenes were the first antifungal drugs for clinical use and they possess the broadest spectrum of activity against fungal pathogens. However, their clinical use is hindered due to their poor distribution in the body and associated high (renal) toxicity. Such toxicity is due to their slight affinities for the ergosterol homologues, cholesterol in mammalian cells (Paterson et al. 2003; Lemke et al. 2005). Despite this, polyene resistance is very uncommon, and they (especially amphotericin B) remain good therapeutic options when an infection resists treatment with azoles and echinocandins (Mora-Duarte et al. 2002). Furthermore, over the years, concerted efforts have been made to alleviate polyene- associated toxicities. An example is amphotericin B's lipid formulations in liposomes or disc- like or ribbon-like lipid complexes to reduce its toxicity (Dupont 2002; Chandrasekar 2011). Moreover, new semisynthetic polyenes with lower toxicity and better water solubility than amphotericin B, and better activity against amphotericin B-resistant C. albicans have also been developed (Kakeya et al. 2008; Santo 2010). 1.2.3 Echinocandins The echinocandins are semisynthetic amphiphilic lipopeptides derived from fungi, such as Glarea lozoyensis (caspofungin), Aspergillus nidulans var. echinulatus (micafungin), and Coleophoma empetri (anidulafungin) (Vazquez and Sobel 2006; Eschenauer et al. 2007; Campoy and Adrio 2017; Ksiezopolska and Gabaldon 2018). The echinocandins exert antifungal effects by inhibiting the biosynthesis of β-1,3-D-glucan, a vital component of the fungal cell wall via the non-competitive inhibition of β-1,3-D-glucan synthase (encoded by FKS genes). The inhibition of this enzyme leads to the formation of a defective fungal cell wall, disruption of cell wall integrity, cell lysis, and consequent cell death (Sanguinetti et al. 2015). Despite their expensive costs and absence of oral forms, the three echinocandins remain the best therapeutic options for treating candidaemia and invasive candidiasis because they: (i) have fungicidal activities against all Candida spp. (including azole and polyene-resistant strains); (ii) show no interaction with other drugs; and (iii) do not cause severe side effects due to the absence of their target, β-1,3-D-glucan synthase in mammalian cells (Pfaller et al. 2003; Theuretzbacher 2004). Interestingly, however, azole therapy is preferred for certain medical conditions, such as urinary tract candidiasis, meningitis, and ophthalmitis because the echinocandins are not excreted into the urine, do not effectively cross the blood-brain barrier, and do not effectively penetrate the ocular system, respectively (Pappas et al. 2018). 1.2.4 Nucleoside/pyrimidine analogues Flucytosine or 5-fluorocytosine (5-FC), a derivative of cytosine, is the only antifungal drug that inhibits the syntheses of nucleic acid and protein (Onishi et al. 2000). It is a prodrug, and it only exerts antifungal effects after its conversion to 5-fluorouracil (5-FU). This prodrug (5-FC) is transported into fungal cells via cytosine permease (Fcy2p) and converted to 5-fluorouracil Page | 43 (5-FU) in fungal cells by cytosine deaminase (Fcy1p), an enzyme not found in mammalian cells. The fluorouracil is converted into 5-fluorouridine monophosphate (FUMP) by uracil phosphoribosyltransferase (Fur1p). The FUMP produced can be incorporated directly into RNA (ribonucleic acid), in place of the normal uridine triphosphate, and this results in the inhibition of fungal protein synthesis (Vermes et al. 2000; Kabir and Ahmad 2013). Alternatively, 5-FU can be converted into 5-fluorodeoxyuridine monophosphate (5-FdUMP), which inhibits DNA (deoxyribonucleic acid) synthesis via the inhibition of thymidylate synthase, an important enzyme for DNA synthesis. The inhibition of DNA synthesis results in the blockage of cell division and ultimately, fungal cell death (Fig. 2) (Waldorf and Polak 1983; Morio et al. 2017). Flucytosine is effective against Candida and Cryptococcus spp.; however, it is less ideal for primary therapy due to rapid resistance development amongst yeasts. Notably, it is more appropriate as an adjunct than a primary therapy, and when combined with amphotericin B, it is effective for the treatment of cryptococcosis (Zhanel et al. 1997). Fig. 2 Description of the mechanism of action of flucytosine (Obtained from Kabir and Ahmad 2013). 1.2.5 Allylamines and Morpholines In addition to the azoles and polyenes, other classes of antifungal that affect the fungal cell membrane are the allylamines and morpholines. The allylamines (e.g. naftifine, terbinafine) exert fungicidal effects by non-competitively inhibiting the squalene epoxidase enzyme encoded by ERG1 gene (Fig. 1). This enzyme catalyses the conversion of squalene to 2,3- squalene epoxide, which is converted to lanosterol, and then to ergosterol after a series of enzymatic steps. Thus, the inhibition of this enzyme leads to the blockage of ergosterol biosynthesis, depletion of ergosterol, and accumulation of squalene (Andriole 2000; Denning Page | 44 and Hope 2010). The accumulation of squalene rather than the depletion of ergosterol results in increased membrane permeability, altered cell membrane, and ultimate cell death (Ryder 1988; Campoy and Adrio 2017; Abdel-Kader and Muharram 2017). Morpholines (e.g. amorolfine) also exert antifungal and fungistatic effects by blocking the ergosterol biosynthetic pathway. This is done via the inhibition of two enzymes, Δ7-8-isomerase (Erg2p) and Δ14-reductase (Erg24p), this results in the depletion of cell membrane ergosterol and accumulation of toxic sterols (Fig. 1) (Polak 1992). Amorolfine is usually used for topical treatment of mycoses, and it is effective against yeasts, some moulds and even some bacteria (e.g. Actinomyces spp.) (Gupta et al. 2003; Finch and Warshaw 2007). 1.3 Combination therapy with fatty acids As a result of the increase in antifungal resistance, complicated by the paucity of available antifungals and host toxicity, the exploitation of alternative treatment approaches is imperative. One such approach is through combination therapy and this has been harnessed in various forms, including the combination of two antifungal drugs (Graybill et al. 1995; Olver et al. 2006; Schilling et al. 2008; DiDone et al. 2011; Chen et al. 2013); the combination of an antifungal drug and a non-antimicrobial compound (Ells et al. 2009; Gamarra et al. 2010; da Silva et al. 2013; Shrestha et al. 2015; Hacioglu et al. 2018; Sharifzadeh et al. 2018; Jia et al. 2019); and combination of appropriate non-antimicrobial compounds (Bae and Rhee 2019). Whilst antagonism is sometimes possible, combination therapy, if the compounds exhibit synergy, is usually more effective and provides greater benefits compared to monotherapy. Its benefits include increased efficacy, due to the complementary effects of both agents; reduced evolution of resistance; decreased drug(s) dosage, which translates to decreased host toxicity; and microbicidal activity, which may result from the combination of two fungistatic agents (Chang et al. 2017; Prasad et al. 2017). As previously discussed in Section B, a considerable number of studies have reported the synergistic effects of various combinations of antifungal drugs and that of antifungal drugs with non-antimicrobial agents. Here, we briefly review available reports on the synergistic activity of fatty acids with antifungal drugs against Candida spp. Fatty acids (FAs) are organic molecules characterised by a hydrophilic carboxyl group (-COOH) at one end and a hydrophobic methyl group (-CH3) at the other end, thus they are amphipathic in nature. These molecules are important building blocks of cellular lipids and membranes, and they regulate various signalling pathways, are involved in the storage of energy (adipose tissues), are essential for the synthesis and functions of hormones (Calder 2015; Pohl et al. 2011). Fatty acids generally have chain lengths between 4 and 28 carbon atoms; those with <8 carbons, 8 to 12 carbons, and >12 carbons are classified as short-chain, medium-chain, and long-chain FAs, respectively. Additionally, FAs are also classified as either saturated or unsaturated, Page | 45 depending on the presence or absence of a double bond. Unsaturated FAs are further classified as monounsaturated (MUFAs) or polyunsaturated FAs (PUFAs), the former possess just a single double bond, while the latter contains more than one double bond (Pohl et al. 2011; Yoon et al. 2018). Interestingly, certain FAs exhibit antimicrobial activity against viruses, bacteria, and fungi (Chanda et al. 2018). This antimicrobial property is usually dependent on various factors such as carbon chain length, degree of unsaturation (including number, location, and spatial property of double bonds), and structure of the fatty acid (Desbois and Smith 2010). More specifically, the chief mechanistic action of antibacterial FAs is through the alteration of cellular lipids and membranes, and disruption of several cellular processes such as oxidative phosphorylation where FAs bind to electron carriers, disrupt electron transport, and consequently decrease membrane potential and proton gradient (Galbraith and Miller 1973; Yoon et al. 2018). The major target of antifungal FAs is the fungal membrane, where they are incorporated into the lipid bilayer, resulting in increased membrane fluidity and permeability, perturbation of membrane functions and structure, and ultimately, cell death (Avis and Belanger 2001; Pohl et al. 2011; Mishra et al. 2014). Additionally, increased oxidative stress, resulting from lipid peroxidation, has been attributed to the insertion of PUFAs into fungal membranes (Thibane et al. 2012a). Fatty acids may also directly inhibit membrane proteins, such as glucosyltransferase (Won et al. 2007; Zhou et al. 2018). Furthermore, antifungal FAs can also disrupt fatty acid metabolism, protein synthesis and topoisomerase activity (Pohl et al. 2011). Interestingly, some antifungal FAs have also been reported to influence virulence factors, such as biofilm formation, hyphal growth, secreted aspartyl proteinases, and lipases, without affecting fungal growth (Muthamil et al. 2020). Such anti-virulence effect, without the inhibition of microbial growth, will considerably reduce selective pressure and result in a reduced rate of antimicrobial resistance development. For this reason, FAs may also be used as adjuncts to complement conventional antimicrobial agents that are highly prone to pathogen resistance due to their influence on microbial growth (Pierce and Lopez-Ribot 2013; Vila et al. 2017; Wall and Lopez-Ribot 2020). Unsurprisingly, the synergism of FAs and conventional antifungals has been demonstrated. Polyunsaturated fatty acids such as AA and SDA, have been reported to increase the susceptibility of C. albicans and C. dubliniensis biofilms to antifungal drugs, such as amphotericin B. These fatty acids possibly exert additive effects via the disruption of membrane organisation and increased oxidative stress, leading to apoptosis (Ells et al. 2009; Thibane et al. 2012a). Similarly, Mishra and co-workers (2014) have demonstrated the synergism of AA with FLC and terbinafine. Additionally, Bae and Rhee (2019) reported the synergistic activity of caprylic acid (a medium-chain fatty acid found in palm oil and coconut oil) with carvacrol or thymol against C. albicans. The observed synergistic effect was attributed Page | 46 to membrane disruption and inhibition of efflux pumps by these compounds (Bae and Rhee 2019). Further, a more recent study by Kuloyo and co-workers (2020) demonstrated that AA potentiates the susceptibility of FLC to C. albicans via interference with methionine and ATP synthesis pathways. Table 1 depicts cases of combination therapy with fatty acids against some pathogenic fungi. Table 1 Selected examples of combination therapy with fatty acids against pathogenic fungi Type Combination Fungus Suggested Reference mechanism Fatty acids and Arachidonic acid C. albicans Influences Ells et al. 2009 antifungal and amphotericin C. dubliniensis ergosterol and B or clotrimazole unsaturation content; Increases oxidative stress Stearidonic acid C. albicans Not known Thibane et al. and amphotericin C. dubliniensis 2012b B Arachidonic acid C. glabrata Influences Mishra et al. 2014 and fluconazole C. parapsilosis prostaglandin or terbinafine C. tropicalis production Arachidonic acid C. albicans Interferes with Kuloyo et al. and fluconazole methionine ATP 2020 production and methionine synthesis Fatty acids and Caprylic acid and C. albicans Influences Bae and Rhee non- carvacrol or membrane 2019 antimicrobials thymol integrity and efflux pump activity 1.4 General conclusions for Chapter 1 The epidemiology of candidiasis has changed with a shift to non-albicans Candida (NAC) species, including C. krusei. This epidemiological shift is partly explained by the increasing resistance of NAC species to antifungal drugs. Candida krusei can cause life-threatening infections in immune-compromised patients, such as those with hematologic malignancies. Those using prolonged azole prophylaxis are also at higher risk. The teleomorph of C. krusei, Page | 47 Pichia kudriavzevii has been given the Generally Regarded as Safe status by the United States Food and Drug Administration (FDA). It is used for the production of various food products, including chocolate. However, this needs to be revisited, given the pathogenic potential of C. krusei. The widespread use and misuse of the limited antifungal arsenal against an ever-increasing number of fungal infections (due to the rise in the number of immunocompromised and terminally ill individuals) have continued to create selective pressure for resistance development amongst fungal pathogens. Understanding the mechanisms of antifungal resistance of these pathogens is crucial for effective management of their infections, proper use of the limited antifungals, and insights into future drug development. For instance, drugs or adjuvants that are efflux pump inhibitors can be developed to tackle drug resistance due to overexpression of efflux pumps. The paucity of antifungal agents coupled with the problem of antifungal resistance, host toxicity, as well as difficulty in antifungal drug development partially due to the eukaryotic nature of both fungi and humans, have heightened fungal infections treatment failures which in turn prompt researchers to exploit alternative therapeutic options. One of these numerous alternatives is combination therapy, and if synergism is obtained, it exhibits better activity than monotherapy (Pierce and Lopez-Ribot 2013) (Fig. 3). This combination therapy has been explored and exploited in various forms, including the co-administration of an antifungal drug(s) with fatty acids. More so, anti-virulent fatty acids represent excellent adjuvant candidates, because, unlike conventional drugs, they do not influence cell viability, and thus have a lower ability to induce resistance due to low selective pressure (Wall and Lopez-Ribot 2020). This research area's full exploitation is envisaged as being worthwhile and could be used as an efficient tool to combat the menace of antifungal resistance. Fig. 3 Simple illustration of the outcome of mono- and combination therapeutic approaches Page | 48 1.5 Research Aim and Objectives Based on this background and due to the apparent knowledge gaps, the aim of this dissertation is to investigate the influence of polyunsaturated fatty acids on fluconazole susceptibility and drug efflux in Candida krusei. The specific objectives to achieve this aim are listed below:  Objective 1: Establish the susceptibility profiles of clinical and environmental isolates of C. krusei to fluconazole and unsaturated fatty acids with varying degrees of unsaturation [oleic acid (18:1), linoleic acid (18:2), gamma-linolenic acid (18:3), arachidonic acid (20:4), and eicosapentaenoic acid (20:5)] (Chapter 2).  Objective 2: Determine the potentiating effect of fatty acids on fluconazole susceptibility in C. krusei and examine the underlying mechanisms of the observed effect (Chapter 2).  Objective 3: Examine the potentiating effect of the combination of fatty acid and fluconazole against C. krusei in a C. elegans infection model (Chapter 2).  Objective 4: Develop a CRISPR-Cas9 genome editing system for C. krusei (Chapter 3).  Objective 5: Determine the influence of exogenous arachidonic acid and fluconazole on the expression, localisation, and activity of Abc1p efflux pump in C. krusei (Chapter 4). 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BMC Research Notes 11:135. https://doi.org/10.1186/s13104-018-3242-8 Page | 60 CHAPTER 2 Polyunsaturated fatty acids potentiate the activity of fluconazole against Candida krusei in vitro and in vivo in a Caenorhabditis elegans model Page | 61 RESEARCH OUTPUT A manuscript from this chapter was submitted to Antimicrobial Agents and Chemotherapy for consideration for publication. Page | 62 2.1 Abstract Although Candida albicans remains the major cause of invasive candidiasis, the incidence of infections caused by non-albicans Candida species, including C. krusei, is increasing and demands urgent public health attention. Candida krusei exhibits innate resistance to fluconazole (FLC) while also rapidly displaying adaptive resistance to other antifungal drugs. Moreover, this yeast has the propensity to form a recalcitrant biofilm with increased resistance. Hence, there is a need to develop novel therapeutic strategies to combat infections caused by this pathogen. One such approach is through combination therapy with natural compounds such as polyunsaturated fatty acids (PUFAs). This study was conceptualised to investigate the effect of PUFAs on FLC susceptibility of C. krusei biofilms and the conserved nature of this effect in the Caenorhabditis elegans infection model. This was carried out by exposing C. krusei biofilms to FLC in the presence and absence of linoleic acid (LA) or gamma-linolenic acid (GLA). The effect of these treatments on biofilm formation, cell ultrastructure, membrane integrity, oxidative stress, and efflux pump activity was evaluated. In addition, the ability of the PUFAs to prolong the survival and reduce the fungal burden of infected C. elegans was assessed. Our results showed that both PUFAs potentiate the susceptibility of C. krusei biofilms to FLC in vitro via cell membrane damage, induction of oxidative stress, and disruption of efflux pump activity. This potentiating effect was also observed in vivo in C. elegans. Taken together, PUFAs show potential as antifungal potentiating agents against intrinsically FLC- resistant C. krusei, both in vitro and in vivo. This may pave the way for future studies into novel therapeutic options for overcoming increasing antifungal resistance. Keywords: Candida krusei, biofilm, antifungal resistance, polyunsaturated fatty acids, fluconazole, susceptibility, combination therapy, Caenorhabditis elegans Page | 63 2.2 Introduction Globally, Candida species remain the fourth causative agents of hospital-acquired systemic infections and the leading cause of nosocomial fungaemia, with more than 250,000 and 50,000 annual attributable cases of infections and deaths, respectively (Pfaller and Diekema 2007; Leroy et al. 2009; Kullberg and Arendrup 2015). Although C. albicans is the primary cause of invasive candidiasis, the epidemiology is evolving with an increasing number of infections being attributed to non-albicans Candida (NAC) species, including C. krusei. This epidemiological shift may be partly explained by the increasing resistance of NAC species to antifungal agents (Chi et al. 2011; da Silva et al. 2013; Sadeghi et al. 2018). Fluconazole, an ergosterol biosynthesis disruptor – via the inhibition of lanosterol-14α-demethylase, encoded by ERG11, is the most commonly used drug for the prevention and treatment of superficial and systemic candidiasis due to its affordability, broad-spectrum activity, high water-solubility, high bioavailability, and good tolerance with few side effects (Grant and Clissold 1990; Andriole 2000; Falci and Pasqualotto 2013). However, due to its fungistatic nature and extended use, its efficacy is challenged by rapid resistance development among many fungal species (Shukla et al. 2016). More alarmingly, C. krusei isolates display intrinsic resistance to FLC, with more than 97% isolates displaying resistance (Whaley et al. 2017), while also rapidly developing acquired resistance to other antifungal drugs, including the echinocandins (Forastiero et al. 2015). Hence, this yeast can be regarded as a potential multidrug-resistant pathogen (Arendrup and Perlin 2014). The resistance mechanisms of this yeast and other clinically significant Candida spp. include, but not limited to, the overexpression of antifungal targets (e.g. Erg11p), alteration of drug targets, and reduction in intracellular drug concentration (e.g. due to biofilm formation) (Pappas et al. 2016; Robbins et al. 2017). A biofilm consists of surface-attached populations of sessile cells that are enclosed in a self- produced extracellular polymer matrix (Wang et al. 2009). Like bacterial biofilm, biofilm formation in Candida spp. contributes to antifungal resistance development by reducing effective drug penetration and concentration, and consequently increasing the non- susceptibility of fungal species to antifungal drugs, especially azoles, polyenes and nucleoside analogues (Ramage et al. 2012; Desai et al. 2014). Moreover, it has also been documented that Candida biofilms could be 1000-fold more resistant to antifungal drugs compared to planktonic counterparts (Hacioglu et al. 2018). This is unsurprising because unlike free-living planktonic cells, biofilms have reinforced functional and structural arsenal, such as enhanced efflux pump activity (e.g. Cdr1p, Cdr2p), which ultimately reduces the intracellular concentration of drugs; genetic changes of drug targets; extracellular polymer matrix (comprising of polysaccharides, glycoproteins, and signalling molecules), which decreases the penetration of drugs; dense population of cells within biofilms; modification of sterol Page | 64 contents of fungal membranes; and presence of phenotypic variants, known as persister cells, that are tolerant to drugs (Fig. 1) (Finkel and Mitchell 2011; Ramage et al. 2012). Furthermore, biofilms also play important roles in the virulence, infection, and protection of microorganisms from host defences (Baillie and Douglas 2000; Samaranayake et al. 2002). Indeed, their presence on medical implants, such as catheters and dentures, increases the risk of invasive infections and represent a possible source of recurrent infections, mainly because of their resilience and refractory nature (Ramage et al. 2006). Moreover, detached cells from biofilms have been reported to be responsible for more significant mortality than planktonic yeast cells (Uppuluri et al. 2010; Ramage et al. 2012). Consequently, there is an urgent need for the development of novel treatment approaches against fungal biofilms, and the combination of conventional antifungals with PUFAs might be a probable option since PUFAs have known antibacterial and antifungal properties (Huang and Ebersole 2010; Thibane et al. 2010; Chanda et al. 2018; Beavers et al. 2019; Kim et al. 2020; Muthamil et al. 2020). Additionally, in contrast to monotherapy, combination therapy may offer enhanced drug efficacy and bioavailability; alleviates host toxicity; decrease antimicrobial resistance development; and induce microbicidal activity, in the case of two fungistatic agents (Carradori et al. 2016; Chang et al. 2017; Prasad et al. 2017). Furthermore, previous studies in our research group have found that PUFAs (e.g. arachidonic acid) increase the susceptibility of C. albicans and C. dubliniensis biofilms to FLC, clotrimazole, and amphotericin B (Ells et al. 2009; Thibane et al. 2012b). Other studies have also reported the antifungal activity of fatty acids-monotherapy and their additive effects with other antifungal and non-antifungal compounds against Candida spp. biofilms (Mishra et al. 2014; Bae and Rhee 2019). Although several mechanisms, such as increased oxidative stress and disruption of membrane organisation, have been implicated in this activity, there is a dearth of information on the ability of exogenous PUFAs to overcome intrinsic antifungal resistance, such as in the case of C. krusei. With this as background, this study was conceptualised to investigate the potentiating effects of fatty acids with FLC against C. krusei biofilms while also determining the underlying mechanisms of the observed effects. In addition, the ability of PUFAs to potentiate the activity of FLC in vivo, in a Caenorhabditis elegans infection model, is also investigated. Page | 65 Fig. 1 An illustration of the components and fundamental antifungal resistance mechanisms of fungal biofilm. A typical biofilm has reduced resistance to drugs due to inherent factors, such as increased cell density, presence of persister cells, modulated physiology, extracellular polymer matrix, overexpressed and modified drug targets, and enhanced efflux pump activity (Adapted from Costa-Orlandi et al. 2017). 2.3 Materials and Methods 2.3.1 Strains used Four yeast strains, previously phenotypically-identified as either Candida krusei (UFS Y-0801, UFS Y-0217, UFS Y-0277) or Pichia kudriavzevii (UFS Y-0637) (test strains), as well as Candida albicans SC5314 (reference strain), were obtained from the Yeast Culture Collection of the University of the Free State, Bloemfontein, South Africa. The yeasts were revived on Yeast Malt extract (YM) agar plates (10 g/l glucose, 3 g/l yeast extract, 3 g/l malt extract, 5 g/l peptone, 16 g/l agar) at 30oC for 24 h. The identities of these isolates were re-confirmed genotypically by D1/D2 sequencing. A glycerol stock (15%) was prepared for each strain and stocks were stored at -80oC for future use. 2.3.2 Drug and fatty acids Fluconazole (FLC) was obtained from Sigma-Aldrich (St. Louis, MO, USA), a stock of 5 mg/ml was prepared in dimethyl sulfoxide (DMSO) and stored at -20oC. Unsaturated fatty acids, Page | 66 including Oleic Acid (OA) (18:1), Linoleic Acid (LA) (18:2), Gamma-Linolenic Acid (GLA) (18:3), Arachidonic Acid (AA) (20:4), and Eicosapentaenoic Acid (EPA) (20:5) were also obtained from Sigma-Aldrich (St. Louis, MO, USA), a stock (10 mM) of each fatty acid was prepared in ethanol and stored at -20oC. 2.3.3 Biofilm formation Biofilms of test and reference strains were formed with slight modifications of previously described methods (Ramage et al. 2001; Mishra et al. 2014). Briefly, a loopful of cells of each yeast from YM agar plates was inoculated separately into 5 ml sterile Yeast Nitrogen Base (YNB) broth (10 g/l glucose, 6.7 g/l YNB), and was incubated at 30oC for 24 h. After incubation, cells were harvested and washed twice with sterile Phosphate Buffered Saline [PBS; 10 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM sodium chloride (pH 7.4) (Sigma- Aldrich, St. Louis, MO, USA)] by centrifugation (3000 x g, 5 min) (Eppendorf, Germany). Thereafter, cells were re-suspended in 5 ml sterile PBS, counted with a haemocytometer and standardised to a final concentration of 1.0 x 107 cells/ml in 5 ml filter sterilised (0.20 μm cellulose acetate filter, GVS Life sciences ME, USA) YNB broth. A volume of 100 μl of standardised cell suspension was dispensed into a flat-bottom 96-well tissue culture-treated (polystyrene) microtiter plate (Corning Incorporated, Costar®, U.S.) and incubated for 90 min at 37oC, to allow the cells to adhere to the well surface. Cell-free wells containing only sterile YNB broth without cells were included as blank. Following the adhesion phase, the media (containing non-adherent cells) was aspirated with a pipette, the wells re-filled with 200 μl of sterile YNB broth and the plates were incubated at 37oC for 48 h to allow biofilm formation. After biofilm formation, biofilm metabolic activity and biomass were quantified by XTT reduction and crystal violet (CV) assays, respectively. 2.3.3.1 XTT reduction assay The XTT reduction assay used for the assessment of biofilm metabolic activity was a slight modification of previously described methods (Kuhn et al. 2003; Al-Fattani et al. 2006). Briefly, XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino) carbonyl]-2H tetrazolium hydroxide) (Sigma-Aldrich, St. Louis, MO, USA) was prepared (1 mg/ml in PBS), filter sterilised, and stored at -20oC. One (1) mM of menadione (Sigma-Aldrich, St. Louis, MO, USA) solution in acetone was also prepared, filter sterilised, and kept at -20oC. Prior to each assay, both XTT and menadione solution were thawed. After biofilm formation, spent medium was aspirated with a pipette from the biofilm-coated wells, and each well was washed once with 200 μl of sterile PBS. Subsequently, a mixture of XTT (46.30 μl) and 1 mM menadione (3.7 μl) was dispensed into each well and incubated at 37oC in the dark for 3 h. Following incubation, the water-soluble formazan product of XTT was measured at 492 nm using an EZ Read 800 microplate reader (Biochrom, England). The average of the blank absorbance Page | 67 values was deducted from experimental values to eliminate background interferences and normalise data. 2.3.3.2 Crystal violet assay The crystal violet (CV) assay used for the quantification of biofilm biomass was a modification of previously described methods (Jin et al. 2003; O’Toole 2011; Hacioglu et al. 2018). Briefly, spent medium was aspirated from the biofilm-coated wells, the wells were washed once with 200 μl of sterile PBS and air-dried for 45 minutes at room temperature. The washed wells were stained with 110 μl of aqueous CV (0.1% v/v) (Merck Chemicals Pty. Ltd, South Africa) solution for 45 minutes at room temperature. Afterwards, the CV reagent was aspirated from each well, and the wells were washed twice by carefully submerging the microtiter plate in water, without disturbing the formed biofilm. Following this washing step, the biofilm was immediately de- stained with 200 μl of 30% acetic acid for 45 minutes at room temperature. After the de- staining procedure, 100 μl of the solubilised CV was transferred to a new microtiter plate and measured at 595 nm using an EZ Read 800 microplate reader (Biochrom, England). The average of blank absorbance values was deducted from experimental absorbance values to eliminate background interference and normalise values. Additionally, biofilm specific activity (BSA) was implemented to compare the data obtained from XTT and CV assays. This index was calculated with the formula indicated below. (𝑋𝑇𝑇+𝐶𝑉) BSA = (𝑋𝐶𝑅) × { } 2 Where XCR indicates XTT:CV ratio 2.3.4 Determination of minimum biofilm inhibitory concentration of fluconazole The minimum biofilm inhibitory concentration (MBIC) of FLC against test and reference strains was determined with slight modifications of previously described microtiter-based methods (Ramage et al. 2001; Mishra et al. 2014). Briefly, biofilms were prepared as described above. Following the adhesion phase, media (containing non-adherent cells) was aspirated. The wells were re-filled with 200 μl of serially two-fold diluted concentrations (128 to 16 μg/ml) of FLC in sterile YNB broth (prepared from 5 mg/ml stock) and incubated at 37oC for 48 h to allow biofilm formation. Drug-free wells (containing appropriate DMSO concentration in sterile YNB broth) and biofilm-free wells (containing sterile YNB broth only without cells) were included as negative control and blank, respectively. Next, the wells were washed once with 200 μl sterile PBS, then XTT reduction assay was performed (Kuhn et al. 2003), as described above. The average of blank absorbance values was subtracted from experimental (E) and negative control (N) values to eliminate background interferences and normalise values. The % inhibition was calculated as indicated below. Page | 68 𝑁−𝐸 % inhibition = ( ) × 100 𝑁 Where N and E represent the negative control and experimental values, respectively. The MBIC50 was defined as the lowest concentration that resulted in at least 50% reduction in biofilm metabolic activity (compared to drug-free control), while the sub-inhibitory concentration (SMBIC50) was regarded as any concentration below the MBIC50. 2.3.5 Determination of minimum biofilm inhibitory concentration of fatty acids The MBIC of fatty acids against test and reference strains was determined with slight modifications of previously described microtiter-based methods (Ramage et al. 2001; Mishra et al. 2014). Cells were prepared as described above. Following incubation at 37oC for 90 min to allow cell adhesion, the media was aspirated. Afterwards, 200 μl of serially ten-fold diluted concentrations (1 to 0.01 mM) of unsaturated fatty acids [Oleic Acid (OA, 18:1), Linoleic Acid (LA, 18:2), Gamma-Linolenic Acid (GLA, 18:3), Arachidonic Acid (AA, 20:4), and Eicosapentaenoic Acid (EPA, 20:5)] (Sigma-Aldrich, St. Louis, MO, USA) in sterile YNB broth was dispensed into designated wells and incubated at 37oC for 48 h to allow biofilm formation. Fatty acid-free wells (containing appropriate ethanol concentration in sterile YNB) and biofilm- free wells (containing only sterile YNB broth without cells) were included as negative control and blank, respectively. Subsequently, the wells were washed once with 200 μl sterile PBS and biofilm metabolic activity was examined with XTT reduction assay (Kuhn et al. 2003). The average of the blank absorbance values was deducted from the experimental and negative control values to eliminate background interferences and normalise data. The % inhibition, MBIC50, and SMBIC50 were determined as described above. 2.3.6 Determination of the potentiating effect of fatty acids on fluconazole susceptibility The effects of FLC and fatty acids on biofilms of the least-susceptible C. krusei strain (UFS Y- 0277) was determined. Cells of this strain were prepared as described above. Following incubation at 37oC for 90 min to allow cell adhesion, media was aspirated, then 200 μl of YNB broth containing both FLC at 32 μg/ml and fatty acid (LA or GLA) at 0.1 mM was dispensed into designated wells and incubated at 37oC for 48 h to allow biofilm formation. Negative control (DMSO and EtOH, DE) and blank (PBS) were included. Afterwards, the wells were washed once with 200 μl sterile PBS, and the biofilm metabolic activity was examined with XTT reduction assay (Kuhn et al. 2003). The average of the blank absorbance values was subtracted from the experimental and negative control values to eliminate background interferences and normalise values. The % inhibition was calculated as described above, and the potentiating effect of the combination was determined by comparing the % inhibition values of the combination treatment with that of individual treatments. Page | 69 2.3.7 Morphological examination of treated biofilms Biofilm of the least-susceptible strain, C. krusei UFS Y-0277, was formed in the presence of FLC (32 μg/ml), fatty acid (LA or GLA) at 0.1 mM, a combination of both [FLC (32 μg/ml) + fatty acid (0.1 mM)], or YNB broth (control) as described above, in a flat bottom 6 well culture plates (Corning Incorporated, USA). After biofilm formation, the biofilm was washed once with sterile PBS, and 5 mm2 rectangular sections of the bottom of the wells were excised, placed in sterile PBS, and prepared for SEM analysis according to the protocol of Swart and co- workers (2010). Briefly, each sample was fixed with the primary fixative, 3% (v/v) glutardialdehyde (Merck, Darmstadt, Germany) buffered with 0.1 M sodium phosphate buffer (pH 7.0) for 3 h. Secondary fixation was done using a buffered solution of 1% osmium tetroxide (Merck, Darmstadt, Germany) for another 1 h. The samples were rinsed with sodium phosphate buffer after each of the fixation steps. Next, the samples were dehydrated with varying concentrations of ethanol (50%, 70% and 95%) for 20 min each and twice with 100% ethanol for 1 h. After dehydration, the samples were dried with a critical point dryer (Tousimis, Maryland, USA), that uses pressurised liquid CO2, at 35 – 40oC, for 1.5 hours. Following drying, samples were mounted on metal stubs and gold coated with a sputter coater (Bio-Rad, United Kingdom) to become electrically conductive. The specimens were then viewed and imaged with a JEOL JSM-7800F SEM (Tokyo, Japan). 2.3.8 Influence of fatty acids on membrane integrity of C. krusei The effect of various treatments on the membrane integrity of cells within C. krusei UFS Y- 0277 (least-susceptible strain) biofilm was analysed using propidium iodide assay with a few modifications of previous protocols (Ogundeji et al. 2016; Bae and Rhee 2019). Briefly, biofilms of the least-susceptible were formed in the presence of either FLC (32 μg/ml), fatty acid (LA or GLA) at 0.1 mM, a combination of both [FLC (32 μg/ml) + fatty acid (0.1 mM)], or DE (control), as described above, in a flat bottom 6 well culture plates. After biofilm formation, each biofilm was washed once, re-suspended in sterile PBS, and vortexed thoroughly to disrupt the biofilm (Gulati et al. 2018). The resulting suspension was standardised to a final concentration of 1.0 x 107 cells/ml in filter sterilised PBS. Subsequently, 99 μl of the standardised suspension was transferred to a black 96-well microtiter plate (Thermo Scientific, Denmark) containing 1 μl propidium iodide stain (200 μg/ml). The plate was incubated at 37oC for 30 min, then the extent of cell membrane damage was quantified by measuring the red fluorescence of stained cells at an excitation wavelength of 485 nm and an emission wavelength of 635 nm using a Fluoroskan Ascent Fluorimeter (Thermo Scientific, China). Additionally, the fluorescence of the stained cells for all the treatment conditions was visualised using an inverted (fluorescence) CKX53 Microscope (Olympus, Japan) combined Page | 70 with a 130 W high-pressure mercury lamp (Olympus, Japan). Red fluorescence was visualised using a blue filter. 2.3.9 Influence of antioxidants on the potentiating effect of the combination treatments Cells were prepared as described above. Following incubation at 37oC for 90 min to allow cell adhesion, media was aspirated, then 200 μl of YNB broth containing the combination of FLC at 32 μg/ml and 0.1 mM fatty acid (LA or GLA) with or without 2 mM α-tocopherol polyethylene glycol succinate (TPGS, vitamin E) or 2 mM butylated hydroxytoluene (BHT) (Sigma-Aldrich, St. Louis, MO, USA) was dispensed into designated wells (Ak and Gülçin 2008). The wells were incubated at 37oC for 48 h to allow biofilm formation. Negative control and blank were included. Accordingly, the wells were washed once with 200 μl sterile PBS, the biofilm biomass was examined with CV assay (Jin et al. 2003; O’Toole 2011; Hacioglu et al. 2018). The percentage biofilm biomass relative to the untreated (negative) control was determined. 2.3.10 Influence of fatty acids on efflux pump activity of C. krusei The influence of fatty acids on the efflux pump(s) activity of C. krusei biofilm was evaluated using Rhodamine 6G efflux assay with slight modifications of previous methods (Maesaki et al. 1999; Ells et al. 2013; Szczepaniak et al. 2017). Briefly, biofilms were formed as described above, in a black 96-well microtiter plate (Thermo Scientific, Denmark) for 6 h at 37oC. After biofilm formation, spent medium was removed from the wells, 200 μl of sterile PBS was dispensed into each well, and the plate was incubated at 37 °C for 1 h to de-energise the biofilm cells. Following incubation, PBS was removed from the wells, and 200 μl of 10 µM Rhodamine 6G (Rh6G) (Sigma-Aldrich, St. Louis, MO, USA) in sterile PBS (prepared from 10 mM Rh6G stock in DMSO) was dispensed into each well. The plate was incubated at 37oC, and the uptake of Rh6G was measured every 10 min for 1 h at an excitation wavelength of 530 nm and emission of 590 nm using Fluoroskan Ascent Fluorimeter (Thermo Scientific, China). Following the uptake step, leftover Rh6G was removed from the wells, and 200 μl of LA (0.1 or 1 mM), GLA (0.1 or 1 mM), AA (0.1 or 1 mM), FLC (32 μg/ml), FLC+LA, FLC+GLA, FLC+AA, or DE (control) in sterile PBS was subsequently dispensed into designated wells, and the plate was incubated at 37oC for another 1 h (Fourie 2020). Subsequently, the supernatant was removed from the wells, and 200 μl of 2 mM glucose (in sterile PBS) was dispensed into each well to induce Rh6G efflux from the treated biofilm cells. The plate was incubated at 37oC, and the efflux of Rh6G from the cells was measured extracellularly every 10 min for 1 h at an excitation wavelength of 530 nm and an emission wavelength of 590 nm using a Fluoroskan Ascent Fluorimeter (Thermo Scientific, China). Page | 71 2.3.11 In vivo evaluation of the potentiating effect of fatty acids on fluconazole activity 2.3.11.1 Nematode propagation and bacterial culture Caenorhabditis elegans AU37 (glp-4(bn2) I; sek-1(km4) X) nematode and non-pathogenic Escherichia coli OP50 (a uracil auxotroph) were obtained from Caenorhabditis Genetics Centre (University of Minnesota, USA). Prior to each experiment, glycerol stock of E. coli OP50 kept at -80°C was thawed on Luria-Bertani (LB) agar (10 g/l tryptone powder, 10 g/l yeast extract, 5 g/l sodium chloride, 17 g/l agar) and incubated at 37°C for 24 h. A loopful of resulting colonies from the LB agar plates was inoculated into fresh LB broth and incubated (37°C, 24 h). Accordingly, E. coli OP50 lawn was spotted on nematode growth medium (NGM) agar plates [2.5 g/l peptone powder, 3 g/l sodium chloride, 17 g/l agar, 1 ml/l cholesterol (5 mg/ml), 1 ml/l MgSO4 (1 M), 1 ml/l CaCl2 (1 M), 25 ml/l potassium phosphate buffer (1 M)] and incubated overnight at 37°C. The nematodes were then propagated on the resulting E. coli OP50-seeded NGM agar plates at 15°C for 96 h to reach L4 larvae or young adult stage prior to the infection assay (Brenner 1974). 2.3.11.2 Infection of C. elegans For infection with the least-susceptible strain (C. krusei UFS Y-0277), cells of this strain were inoculated into a 5 ml YPD broth (5 g/l peptone, 3 g/l yeast extract, 10 g/l glucose) and incubated overnight at 30°C. The resulting culture was standardised to OD600 of 0.8, and a 100 μl lawn was prepared on brain-heart infusion (BHI) agar (BHI 37g/l, Agar 17g/l) plates and incubated at 30°C for 24 h. Synchronized L4 or young adult nematodes were carefully harvested and washed twice with sterile M9 buffer (3 g/l KH2PO4, 6 g/l Na2PO4 and 1 mM MgSO4) at 4000 x g, 2 min. Thereafter, approximately 400 to 500 washed nematodes were deposited onto C. krusei lawn on BHI agar plates and incubated at 25°C for 4 h (infection stage). Plates with uninfected nematodes on E. coli OP50 were included as a control. 2.3.11.3 C. elegans treatment assay The ability of the combination treatments to enhance the survival of infected nematodes was assessed with a few modifications of previously described methods (Breger et al. 2007; Eldesouky et al. 2020). Briefly, following infection, infected nematodes were carefully harvested off the BHI agar plates with sterile M9 buffer and washed thrice (5000 x g, 5 min) to remove undigested yeast cells. Subsequently, 40 nematodes were treated with either FLC (32 μg/ml), LA (0.1 mM), GLA (0.1 M), FLC+LA, FLC+GLA, or DE (control) in 2 ml liquid medium (80% M9 buffer, 20% BHI, kanamycin 90 μg/ml/) in 6-well plates (Corning Incorporated, USA) and incubated at 25oC. The nematodes were monitored daily using a stereomicroscope (Olympus, Vietnam) and scored as either alive or dead. A nematode is considered dead if it shows no motility in response to mechanical stimulation with a sterile pipette tip. This assay Page | 72 was done in triplicate, with a total of 120 nematodes per treatment. The survival metrics, including Kaplan-Meier statistics, median survival time and log-rank test, were performed with online application for survival analysis 2 (OASIS 2) (Han et al. 2016). 2.3.11.4 Evaluation of fungal burden within C. elegans The combination treatments' propensity to reduce the fungal burden of infected nematodes was assessed with a few modifications of previously described methods (Breger et al. 2007; Eldesouky et al. 2018). Briefly, nematodes were infected as described above. Following infection, nematodes were carefully harvested off the BHI agar plates with sterile M9 buffer and washed thrice with M9 buffer to get rid of all undigested yeast cells. Subsequently, 60 nematodes (20 nematodes per replicate) were treated with either FLC (32 μg/ml), LA (0.1 mM), GLA (0.1 M), FLC+LA, FLC+GLA, or DE (control) in 2 ml liquid medium (80% M9 buffer, 20% BHI, 90 μg/ml kanamycin) in 6-well plates (Corning Incorporated, USA) and incubated at 25oC for 24 h. Following incubation, nematodes were washed twice with sterile M9 buffer and ingested C. krusei cells were released from the nematodes by a vigorous-vortex procedure with beads (150 mg in 3 ml M9 buffer) for 2 min (without affecting fungal viability). The resulting C. elegans homogenates were diluted hundred-fold, plated onto YPD agar (supplemented with ampicillin 100 μg/ml/ and kanamycin 90 μg/ml/ to preclude bacterial growth), and incubated at 30oC for 24 to 48 h. Accordingly, the percentage fungal burden per nematode relative to the untreated control (DE) was determined (Eldesouky et al. 2020). 2.3.12 Statistical analysis Unless stated otherwise, all experiments were conducted in triplicate. Averages and standard deviations were calculated using Excel 2013. Graphs were constructed with GraphPad Prism. The data of different groups, unless stated otherwise, were compared using one-way analysis of variance (ANOVA) complemented with Tukey’s multiple comparisons test. Unless stated otherwise, a p-value ≤ 0.05 was considered significant and statistical difference was indicated by different letters on the bars. 2.4 Results and Discussions 2.4.1 Biofilm formation and quantification Microbial biofilms have remarkable medical, veterinary, and environmental importance. Estimatedly, 65% of all hospital infections are associated with biofilm cells rather than planktonic cells, and this has redirected increased focus on biofilm communities when microbial pathogenicity and virulence are investigated (Mah and O’Toole 2001; Silva et al. 2010; Alnuaimi et al. 2013). Protection against host defences, virulence and increased antifungal resistance is afforded by biofilms (Baillie and Douglas 2000; Samaranayake et al. 2002). Many fungal species, including Candida spp., such as C. albicans and C. krusei, have Page | 73 the capacity to form a biofilm. Biofilm formation in Candida spp. reduces the effective drug penetration, which consequently confers decreased susceptibility to antifungal drugs, especially the azoles, polyenes, and nucleoside analogues (Ramage et al. 2012; Desai et al. 2014). Furthermore, it has been documented that biofilm structures and physiology are not only species-dependent but could also be strain-specific (Silva et al. 2009). Here, we briefly evaluate and compare the biofilm physiology of four C. krusei strains and a reference strain (C. albicans SC5314). The metabolic activity of biofilms of the strains was assessed using XTT reduction assay (Fig. 2A). This colorimetric assay depends on the reduction of XTT reagent to formazan by the dehydrogenases of metabolically active cells (Kuhn et al. 2002; Kuhn et al. 2003). While there was no significant difference in the metabolic activity of the four C. krusei strains (p > 0.05), their metabolic activity was in the hierarchy: UFS Y-0217 > UFS Y-0637 > UFS Y-0801 > UFS Y-0277. Moreover, the reference strain biofilm displayed a very high metabolic activity compared to any of the C. krusei strains (p < 0.05) (Fig. 2A). However, caution must be taken when comparing biofilms of different strains and species with XTT reduction assay, since the reduction of XTT to formazan is usually species and strain-dependent (Kuhn et al. 2003). Furthermore, we also quantified the biofilm biomass of strains using CV assay (Fig. 2B). Amongst the C. krusei strains, strain UFS Y-0801 had the highest biomass quantity, however, this was not significantly different from that of strain UFS Y-0217 (p > 0.05). The lowest biomass quantity was recorded for strain UFS Y-0277 (p < 0.05), and this is concordant with the earlier observed lower metabolic activity of the strain (Fig. 2A). In addition, similar to the XTT results, the reference strain had the highest biofilm biomass (p < 0.05). Notably, with the exception of C. krusei strain UFS Y-0277, the biomass quantities of all strains, including the reference strain, were more pronounced than corresponding metabolic activities (Fig. 2B). A similar observation has been reported by previous studies (Peeters et al. 2008; Xu et al. 2016). This is unsurprising since XTT reduction assay only measures the viability of the biofilm cells, while CV assay represents a good indicator of the amount of biofilm (i.e. viable cells, non- viable cells, and extracellular matrix) (Pitts et al. 2003; Kuhn et al. 2003). Rather than considering XTT and CV assays as two identical descriptors (i.e. providing the same information), it is more practical to use them complementarily. Moreover, a complementary use of both assays by Muthamil and co-workers (2020) highlighted the biofilm-reducing potential, without the inhibition of metabolic viability, of a plant-derived OA against Candida spp. As a result of these different types of information produced by XTT and CV assays, the use of XTT and CV ratio (XCR) has been attempted to compare the results produced by these two assays. However, this ratio (XCR) has poor performance and produces extremely high values when the CV value is relatively low. Thus, we resorted to using an index with a better Page | 74 performance and without low CV value-related problems, known as biofilm specific activity (BSA) (Corte et al. 2019). As shown in Figure 2C, there was no significant difference in the BSA values obtained for the four C. krusei strains (p > 0.05). However, the reference strain had a high BSA value (p < 0.05) and this observation is seemingly comparable to the XTT assay results. A B io film m e ta b o lic a c tiv ity B B io film b io m a s s 4 4 a 3 3 a 2 2 b b c b c 1 b b b 1 d 0 0 1 7 7 7 4 1 7 7 7 4 0 1 7 3 1 0 1 7 3 1 8 2 2 6 3 8 2 2 6 3 0 0 0 0 5 0 0 0 0 5 F F F F C F F F F C U U U U S U U U U S S tra in S tra in Fig. 2 Quantification of biofilms of Candida krusei C and Candida albicans. (A) Metabolic activity of biofilms measured by XTT reduction assay. (B) Biofilm’s biomass quantified by CV assay. (C) BSA index of biofilms determined using XTT and CV values. Values are means of three independent experiments, and the error bars indicate standard deviations. p-value ≤ 0.05 was considered significantly different and are indicated by different letters. UF0801: C. krusei UFS Y-0801, UF0217: C. krusei UFS Y-0217, UF0277: C. krusei UFS Y- 0277, UF0637: Pichia kudriavzevii UFS Y-0637, SC5314: C. albicans SC5314. Page | 75 A b s 4 9 2 n m A b s 5 9 5 n m 2.4.2 Determination of minimum biofilm inhibitory concentration of fluconazole While the inhibitory effect of FLC on the metabolic activity of all C. krusei isolates was concentration-dependent, the most pronounced reduction for all strains, including C. albicans SC5314, was recorded at the highest drug concentration (128 μg/ml) (Fig. 3). Additionally, the reference strain, C. albicans SC5314, was susceptible to all FLC concentrations tested with inhibition of >85%. 1 0 0 1 6  g /m l 8 0 3 2  g /m l 6 4  g /m l 6 0 1 2 8  g /m l 4 0 2 0 0 1 7 7 7 0 1 7 3 4 8 2 2 6 1 0 0 0 0 3 F F F F 5 U U U U CS S t r a in Fig. 3 The effect of various concentrations of fluconazole on the metabolic activity of biofilms of C. krusei strains and C. albicans SC5314 after incubation at 37oC for 48 h, using XTT reduction assay. Values are means of three independent experiments and the error bars indicate standard deviations. UF0801: C. krusei UFS Y-0801, UF0217: C. krusei UFS Y-0217, UF0277: C. krusei UFS Y-0277, UF0637: Pichia kudriavzevii UFS Y-0637, SC5314: C. albicans SC5314. The MBIC50 and SMBIC50 of FLC against the isolates were also determined (Table 1). The reference strain (C. albicans SC5314) biofilm was most susceptible to FLC with recorded MBIC50 value of <16 μg/ml (p < 0.05). However, the MBIC50 values of FLC ranged from 32 to 128 μg/ml for C. krusei, while their SMBIC50 values were between 16 and 64 μg/ml. The lowest MBIC50 value (MBIC50 = 32 μg/ml) was recorded against strains UFS Y-0217 and UFS Y-0637, and this might suggest them as the most susceptible C. krusei strains in this study. Notably, strain UFS Y-0217 (CBS573T) is the type strain of C. krusei, and a previous study has reported MBIC50 for FLC against this strain as 32 μg/ml (Douglass et al. 2018). In addition, biofilms of Page | 76 % In h ib it io n strain UFS Y-0801 was moderately resistant (MBIC50 = 64 μg/ml), while that of strain UFS Y- 0277 was the least susceptible (MBIC50 = 128 μg/ml). Overall, as expected, all tested C. krusei strains exhibited reduced sensitivity to FLC compared to the reference strain (C. albicans SC5314). Generally, about 97% of C. krusei isolates are considered inherently resistant to FLC (Whaley et al. 2017). The mechanism for this remains obscure; however, many studies have ascribed it to the unusually low affinity of Erg11p of C. krusei for FLC (Venkateswarlu et al. 1997; Orozco et al. 1998; Fukuoka et al. 2003). Moreover, a later study by Lamping and co-workers (2009) has attributed it to decreased susceptibility of Erg11p and constitutive expression of Abc1p (Cdr1p). Table 1 The MIC50 and SMIC50 of fluconazole against biofilms Strain Fluconazole (μg/ml) MBIC50 SMBIC50 UF0801 64 32 UF0217 32 16 UF0277 128 64 UF0637 32 16 SC5314 <16 <16 The MBIC50 denotes the concentration that resulted in at least 50% reduction in the biofilm metabolic activity (relative to the drug-free control), while the sub-inhibitory concentration (SMBIC50) is the concentration below the MBIC50. UF0801: C. krusei UFS Y-0801, UF0217: C. krusei UFS Y-0217, UF0277: C. krusei UFS Y-0277, UF0637: Pichia kudriavzevii UFS Y-0637, SC5314: C. albicans SC5314. 2.4.3 Determination of minimum biofilm inhibitory concentration of fatty acids In order to determine the most effective fatty acids and their accompanying non-inhibitory concentrations suitable for the combination treatment assay, the antifungal activity of varying doses of fatty acids was determined against the biofilms of all strains. This assay was done by treating the biofilms with serially ten-fold diluted concentrations (0.01, 0.1 and 1 mM) of unsaturated fatty acids (OA, LA, GLA, AA, and EPA) for 48 h, at 37oC and evaluating their metabolic activities. As shown in Figure 4, the unsaturated fatty acids tested exerted considerable anti-candidal activity. Notably, except for EPA – which exhibited an inverse dose- dependent effect against C. krusei UFS Y-0801, the inhibitory effect of all the tested fatty acids was dose-dependent, and the most pronounced effect was observed for GLA against the biofilms of all strains (Fig. 4C). This observation conforms with previous findings of Muthamil and co-workers (2020), which demonstrated a concentration-dependent biofilm inhibitory activity of OA against Candida spp. Similarly, a better anti-biofilm effect of some PUFAs [LA and alpha-linolenic acid (ALA)] against C. albicans and Staphylococcus aureus was observed Page | 77 at 100 μg/ml compared to 20 μg/ml (Kim et al. 2020). Moreover, AA has also been shown to exhibit bactericidal activity against Staphylococcus aureus in a dose-dependent manner (Beavers et al. 2019). Additionally, the inhibitory activity of the unsaturated fatty acids against all strains was also fatty acid-specific and strain-dependent. A O le ic a c id B L in o le ic a c id 1 0 0 1 0 0 0 .01 m M 0 .01 m M 8 0 0 .1 m M 8 0 0 .1 m M 6 0 1 m M 6 0 1 m M 4 0 2 0 4 0 0 -2 0 2 0 -4 0 0 -6 0 -8 0 -2 0 -1 0 0 1 7 7 7 4 0 1 7 3 1 1 7 7 7 4 8 2 2 6 3 0 1 7 3 1 0 0 0 0 5 8 2 2 6 3 F F F F C 0 0 0 0 5 U U U U F F F FS CU U U U S S tra in S tra in C G a m m a -lin o le n ic a c id D A ra c h id o n ic a c id 1 0 0 1 0 0 0 .01 m M 8 0 0 .01 m M 8 0 0 .1 m M 6 0 0 .1 m M 4 0 1 m M 1 m M 2 0 6 0 0 -2 0 4 0 -4 0 -6 0 2 0 -8 0 -1 0 0 0 -1 2 0 1 7 7 7 4 1 7 7 7 4 0 1 7 3 1 0 1 7 3 1 8 2 2 6 3 8 2 2 6 3 0 0 0 0 5 0 0 0 0 5 F F F F C F F F F C U U U U S U U U U S S tra in S tra in Fig. 4 Effect of varying concentrations of various E fatty acids (A- oleic acid, B- linoleic acid, C- gamma- linolenic acid, D- arachidonic acid, E- eicosapentaenoic) on the metabolic activity of C. krusei strains and C. albicans SC5314 biofilms after incubation at 37oC for 48 h, using XTT reduction assay. Values are means of three independent experiments and the error bars indicate standard deviations. UF0801: C. krusei UFS Y-0801, UF0217: C. krusei UFS Y-0217, UF0277: C. krusei UFS Y- 0277, UF0637: Pichia kudriavzevii UFS Y-0637, SC5314: C. albicans SC5314. Page | 78 % In h ib it io n % In h ib it io n % In h ib it io n % In h ib it io n The MBIC50 and SMBIC50 of the fatty acids against the biofilms of all strains were also determined. As shown in Table 2, while the reference strain (C. albicans SC5314) was the most susceptible to LA (MBIC50 <0.01 mM), it was the least sensitive to AA (MBIC50 >1 mM). Amongst C. krusei strains, the lowest MBIC50 (<0.01 mM) of OA was recorded against UFS Y-0801, and its highest MBIC50 (>1.0 mM) was observed against the other three C. krusei strains (UFS Y-0217, UFS Y-0277, UFS Y-0637). Similarly, this high MBIC50 was also seen for strain UFS Y-0277 in the cases of LA, GLA, and AA, while strain UFS Y-0801 also had the lowest MBIC50 (<0.01 mM) against AA. Additionally, the MBIC50 values of EPA against all C. krusei strains, except UFS Y-0801, were identical (1.0 mM). Table 2 MIC50 and SMIC50 of unsaturated fatty acids against C. krusei and C. albicans biofilms Strain OA (mM) LA (mM) GLA (mM) AA (mM) EPA (mM) MBIC50 SMBIC50 MBIC50 SMBIC50 MBIC50 SMBIC50 MBIC50 SMBIC50 MBIC50 SMBIC50 UF0801 <0.01 <0.01 0.1 0.01 1.0 0.1 <0.01 <0.01 * * UF0217 >1.0 1.0 0.1 0.01 1.0 0.1 >1.0 1.0 1.0 0.1 UF0277 >1.0 1.0 >1.0 1.0 >1.0 1.0 >1.0 1.0 1.0 0.1 UF0637 >1.0 1.0 0.1 0.01 1.0 0.1 0.1 0.01 1.0 0.1 SC5314 0.1 0.01 <0.01 <0.01 1.0 0.1 >1.0 1.0 0.1 0.01 The MBIC50 denotes the concentration that resulted in at least 50% reduction in the biofilm metabolic activity (compared to the drug-free control), while the sub-inhibitory concentration (SMBIC50) is the concentration below the MBIC50. OA: oleic acid, LA: linoleic acid, GLA: gamma-linolenic acid, AA: arachidonic acid, EPA: eicosapentaenoic acid. *: reverse dose-dependent activity observed. UF0801: C. krusei UFS Y-0801, UF0217: C. krusei UFS Y-0217, UF0277: C. krusei UFS Y-0277, UF0637: Pichia kudriavzevii UFS Y-0637, SC5314: C. albicans SC5314. Overall, similar to what was observed for FLC, C. krusei UFS Y-UF0277 appears least susceptible to the inhibitory effect of all fatty acids, and this prompted further analysis of its data. With the exception of AA, the biofilm inhibitory effect of fatty acids against this strain at the highest concentration tested (1 mM) directly correlates with their chain length and degree of unsaturation [EPA (5: double bonds) > GLA (3) > LA (2) > OA (1)] (Fig. 5A, 5B). This corresponds to an earlier observation that the cytotoxic effect of fatty acids on mammalian cell lines is not only dose-dependent but also relates to their chain length and level of unsaturation (Lima et al. 2002). In addition, the interaction of fatty acids with phospholipid membrane varies with their chain length and saturation level (Thibane et al. 2012a). Page | 79 A 8 0 B O A 6 0 LA G LA 4 0 AA E P A 2 0 0 -2 0 M M M m m m1 0 .1 .0. 0 0 1 C o n c e n tra t io n Fig. 5 The biofilm inhibitory activity and structures of unsaturated fatty acids. (A) Effect of varying concentrations of unsaturated fatty acids on metabolic activity of C. krusei UFS Y-0277 biofilms after incubation at 37oC for 48 h, using XTT reduction assay. Values are means of three independent experiments and the error bars indicate the standard deviation. (B) Structures of unsaturated fatty acids used in this study in ascending order of length and unsaturation. OA: oleic acid, LA: linoleic acid, GLA: gamma-linolenic acid, AA: arachidonic acid, EPA: eicosapentaenoic acid. Consistent with our study, the antifungal property of long-chain unsaturated fatty acids has been reported by previous studies. A study by Thibane and co-workers (2010) reported significant inhibitory effect of PUFAs, such as stearidonic acid (SDA), docosapentaenoic acid, and EPA, against C. albicans and C. dubliniensis biofilms. A more recent study by Kim and co-workers (2020) also highlighted the remarkable anti-biofilm activity of LA and ALA from centipede oil against C. albicans. Another study by Muthamil and co-workers (2020) reported the biofilm-reducing potential of plant-derived OA against C. albicans, C. glabrata, and C. tropicalis. Although the mechanism of action of unsaturated fatty acids in our present study is unknown, their mode of action in previous studies was suggested to be mainly through their incorporation into fungal membranes - which increases unsaturation index, membrane fluidity and permeability, and ultimately elicits membrane disorganisation and disruption of membrane proteins (Avis and Belanger 2001; Pohl et al. 2011; Mishra et al. 2014). Increased oxidative stress and membrane disruption, resulting from lipid peroxidation, have also been attributed to the insertion of PUFAs into fungal membranes (Cipak et al. 2006; Thibane et al. 2012a). Moreover, antifungal fatty acids also inhibited hyphal morphogenesis and biofilm formation in certain Candida spp. (Shareck et al. 2011; Manoharan et al. 2017). Similarly, studies have reported the antibacterial activity of unsaturated fatty acids such as OA, LA, and AA (Kabara et al. 1972; Greenway and Dyke 1979; Lee et al. 2017; Beavers et al. 2019). The chief mechanism of action of antibacterial fatty acids is also via their incorporation into cellular lipids and membranes, which consequently induces alterations in the cell membrane and disruption of cellular processes, such as electron transport chain and oxidative phosphorylation Page | 80 % In h ib it io n (Galbraith and Miller 1973; Yoon et al. 2018). Additionally, antibacterial fatty acids have also been reported to inhibit protein synthesis and influence virulence factors, such as biofilm formation (Yoon et al. 2018; Kim et al. 2019). 2.4.4 Polyunsaturated fatty acids potentiate the susceptibility of C. krusei biofilm to fluconazole Because of our interest in the least-susceptible strain, the potentiating effect of unsaturated fatty acids (LA or GLA) on FLC activity was evaluated against the biofilm of C. krusei strain UFS Y-0277. While EPA was excluded from this assay, due to its reverse dose-dependent activity observed against strain UFS Y-0801, only LA and GLA were included due to their greater efficacy against UFS Y-0277 biofilm compared to other fatty acids tested. As shown in Figure 6, when combined with FLC, either of the fatty acids increased the susceptibility of the tested strain biofilm to FLC (p < 0.05). While GLA afforded 96% increased susceptibility, a 55% increase was observed for LA. Our observation coheres with the findings of Mishra and co-workers (2014), who reported that AA increases the susceptibility of C. albicans, C. glabrata, C. parapsilosis and C. tropicalis biofilms to FLC via influence on the lipid saturation level and fluidity of yeasts cell membranes. In addition, a similar study by Ells and co-workers (2009) demonstrated that AA increases the antifungal susceptibility of C. albicans and C. dubliniensis to amphotericin B and clotrimazole. Furthermore, the synergism of SDA and amphotericin B against C. albicans and C. dubliniensis biofilms has also been reported (Thibane et al. 2012b). A 8 0 B 8 0 a 6 0 6 0 a b b 4 0 b 4 0 b 2 0 2 0 0 0 l) ) ) )M A l M A /m m L /m m Lg + µ .1 C g G L µ0 . 1 + 2 ( F 2 (0 C ( 3 A ( 3 L A F C L C L L L F F G Fig. 6 The effect of fluconazole in the presence or absence of LA (A) or GLA (B) on the metabolic activity of C. krusei UFS Y-0277 biofilm after incubation at 37oC for 48 h, using XTT reduction assay. Values are means of three independent experiments and the error bars indicate the standard deviation. p-value ≤ 0.05 was considered significantly different and indicated by different letters. FLC: fluconazole, LA: linoleic acid, GLA: Gamma-linolenic acid, FLC+LA: fluconazole and linoleic acid, FLC+GLA: fluconazole and gamma-linolenic acid. Page | 81 % In h ib it io n % In h ib it io n In the present study, the underlying mechanism behind the observed increased susceptibility afforded by LA and GLA is obscure; however, it could be via the disruption of membrane organisation, due to increased membrane fluidity and permeability facilitated by the incorporation of unsaturation fatty acids into the fungal membrane, which allowed enhanced uptake of FLC (McDonough et al. 2002; Ells et al. 2009). Additionally, it is possibly due to increased oxidative stress, induced by the incorporation of PUFAs into cellular lipids and membranes, which makes the combination-treated cells more susceptible to FLC (Thibane et al. 2012a). 2.4.5 Morphological examination of treated biofilms In order to delineate the underlying mechanism of the increased susceptibility observed following the combination treatments (Fig. 6), the morphology of C. krusei UFS Y-0277 biofilms treated with FLC in the presence or absence of a PUFA (LA or GLA) was examined using scanning electron microscopy (SEM). As shown in Figure 7.1, whilst LA-treated biofilm was the densest amongst the treated biofilms, all treated biofilms are sparser and have just one layer, compared to the untreated biofilm (Fig. 7.1A). Interestingly, hyphal or pseudohyphal cells were observed in all treatments with FLC, an observation that has not been previously reported for C. krusei grown in the presence of FLC. This was unexpected, as in addition to the inhibition of lanosterol 14α-demethylase (Erg11p), azoles exert antifungal activity in C. albicans via the inhibition of yeast-hyphae transition (Odds et al. 1985; Kabir and Ahmad 2013). However, in the presence of azoles, higher hyphal growth of azole-resistant C. albicans compared to the azole-susceptible isolates has been noted, presumably due to enough or increased ergosterol in the plasma membrane for hyphal production (Ha and White 1999; Costa et al. 2011; Sharma et al. 2019). Also, it is known that a defective azole target, Erg11p, responsible for ergosterol synthesis, limits hyphal growth (Lees et al. 1990). Hence, one possible explanation for the FLC-induced hyphal formation observed in this study is that it may be due to overproduction of ergosterol –which promotes hyphal formation since the upregulation of ERG11 has been demonstrated in Candida spp. (including C. krusei) grown in the presence of azoles, including FLC (Henry et al. 2000). Page | 82 A B C D E F Fig. 7.1 Scanning electron micrographs of C. krusei UFS Y-0277 biofilms under various treatment conditions after incubation at 37oC for 48 h. No treatment (A), 32 μg/ml FLC (B), 0.1 mM LA (C), 0.1 mM GLA (D), FLC+LA (E), FLC+GLA (F). Scale bar represents 10 μm. FLC: fluconazole, LA: linoleic acid, GLA: gamma-linolenic acid, FLC+LA: fluconazole and linoleic acid, FLC+GLA: fluconazole and gamma-linolenic acid. At higher magnifications (i.e. ×4000 and ×8000) the effect of these treatments on the ultrastructure of individual cells within the biofilm was observed. As shown in Figure 7.2, cells within both treated and untreated biofilms appear rough. However, extracellular vesicle (exosome or microvesicle)-like structures were visible on cells within biofilms exposed to the combination treatments. Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, are membranous vesicles released by healthy and dying cells across the three domains of life (Gill et al. 2019; Zhao et al. 2019; Battistelli and Falcieri 2020). Extracellular vesicles are thought to play critical roles in pathogenesis, virulence, antifungal resistance, intercellular communication, host-pathogen interaction, and extracellular transport of macromolecules, such as lipids, polysaccharide, and proteins (Oliveira et al. 2010; Samuel et al. 2015; Joffe et al. 2016). More specifically, in C. albicans, biofilm-associated EVs secretions critical for antifungal resistance have been identified (Zarnowski et al. 2018). Additionally, Zhao and co-workers (2019) have demonstrated that EVs could protect Saccharomyces cerevisiae cells from the cytotoxic effect of caspofungin through cell wall remodelling or acting as decoys to antifungal molecules. Studies have reported the release of EV-like structures after exposure of yeast cells to oxidative stress inducers, such as miconazole (Nollin and Borgers 1975), allyl alcohol (Lemar et al. 2005), and oxidised lipids (Leeuw 2010). Furthermore, similar structures, albeit identified as protuberances and fibrillar Page | 83 structures, were identified on cells within C. albicans and C. dubliniensis biofilms grown with marine PUFAs at 1 mM concentration (Thibane et al. 2010). Moreover, a follow-up study by the same authors concluded that these PUFAs exerted their antifungal effects via increased reactive oxygen species (ROS) production and induction of apoptosis (Thibane et al. 2012a). All this evidence suggests that the EVs-like vesicles observed in this study may be a response to increased oxidative stress induced by the combination treatments. Since FLC is a known inducer of oxidative stress (Arana et al. 2010; Peng et al. 2018; Dbouk et al. 2019), one would expect to see similar structures, presumably induced by increased oxidative stress, on cells treated with FLC only, however, this was not case. The possible explanation for this is that FLC alone had little or no effect on C. krusei biofilm, due to the yeast’s intrinsic resistance to FLC. Furthermore, as depicted below (Fig. 7.2E, F), it appeared that the combination treatments induced cell wall and plasma membrane stress, which ultimately caused cell rupture. However, this was not the case for cells treated with either of the fatty acids or FLC alone. Additionally, since unsaturated fatty acids are known inducers of oxidative stress, and are capable of promoting membrane permeability and disorganisation (Avis and Belanger 2001; Cipak et al. 2006; Thibane et al. 2010; Pohl et al. 2011). We speculated that the cell rupture observed with the combination treatments might be caused by a two-step process: an initial plasma membrane disorganisation and/or oxidative stress induced by the insertion of unsaturated fatty acid into the yeast membranes, followed by a further distortion of the plasma membrane and increased oxidative stress in the already vulnerable cells elicited by FLC. Furthermore, although yeast cell death can be via apoptosis or necrosis, the death of subpopulation of cells within the combination-treated biofilms is more likely due to necrosis than apoptosis, this is because of the observed cell rupture and loss of intracellular components. In contrast to apoptosis, necrosis is characterised by cell swelling, disruption of membrane integrity, cell rupture, and loss of intracellular contents (Červinka and Půža 1995; Lima et al. 2002; Eisenberg et al. 2010). Moreover, studies have shown that unsaturated fatty acids can induce cell death via either of the two processes – apoptosis at low doses, and necrosis at higher concentrations (Finstad et al. 1998; Mainou-Fowler et al. 2001). Other agents, such as acriflavine (Keyhani et al. 2009), acetic acid (Ludovico et al. 2001), H2O2 (Madeo et al. 1999), and antifungal agent, amphotericin B (Phillips et al. 2003), can also trigger yeast cell death via both apoptosis and necrosis. Page | 84 A B C D E F Fig. 7.2 Scanning electron micrographs of C. krusei UFS Y-0277 biofilms under various treatment conditions after incubation at 37oC for 48 h (×8000). No treatment (A), 32 μg/ml FLC (B), 0.1 mM LA (C), 0.1 mM GLA (D), FLC+LA (E), FLC+GLA (F). Small panel on bottom-left corner indicates micrograph with a lower magnification (x4000). White arrows depict extracellular vesicles, while red arrows indicate damaged cell wall and membrane and/or cell rupture. Scale bar represents 1 μm. FLC: fluconazole, LA: linoleic acid, GLA: Gamma-linolenic acid, FLC+LA: fluconazole and linoleic acid, FLC+GLA: fluconazole and gamma-linolenic acid. Page | 85 2.4.6 Influence of fatty acids on membrane integrity of C. krusei To investigate the extent of cell membrane damage induced by various treatment groups and to further substantiate our SEM results, propidium iodide assay was performed. Propidium iodide is a membrane-impermeant nucleic acid-binding fluorescent dye, which only penetrates cells with compromised plasma membranes and emits red fluorescence upon interaction with nucleic acids (Boulos et al. 1999; Rosenberg et al. 2019). This assay was done by incubating cells, harvested from C. krusei UFS Y-0277 biofilms exposed to varying treatment conditions, with propidium iodide stain and measuring the resultant fluorescence of stained cells at excitation and emission wavelengths of 485 nm and 635 nm, respectively. As expected, a significant increase (p < 0.05) in fluorescence was observed with cells treated with FLC+LA in comparison to cells treated with LA only. Unexpectedly, however, there was no significant difference (p > 0.05) between the fluorescence of cells exposed to FLC (32 μg/ml) monotherapy and FLC+LA combination treatment (Fig. 8A). Similarly, as anticipated, GLA- treated cells had a significantly lower fluorescence (p < 0.05) compared to cells exposed to the combination of FLC and GLA. In addition, no significant difference (p > 0.05) was observed in the fluorescence of FLC-treated and FLC+GLA-treated cells (Fig. 8B). The reason for this statistically similar fluorescence (p > 0.05) observed for cells treated with FLC monotherapy and those treated with the combinations is unclear. However, future studies will benefit from implementing other cell membrane integrity assays, such as 7-Aminoactinomycin D (7-AAD), Trypan Blue, and ToxiLight (Ogundeji et al. 2016), to further corroborate these findings. A 5 B 5 a 4 a 4 a a 3 b b 3 b b 2 2 1 1 0 0 E E CC A A A A L L L D L L L D FF + G G C + L C F LF T r e a t m e n t T r e a t m e n t Fig. 8 Fluorescence of C. krusei UFS Y-0277 cells stained with propidium iodide dye after exposure to various treatment conditions. Fluorescence of cells exposed to fluconazole in the presence or absence of LA (A) or GLA (B). The fluorescence corresponds to the quantity of dead cells and cells with damaged membranes. Values are means of four independent experiments, and error bars indicate standard deviation. p-value ≤ 0.05 was considered significantly different and are indicated by different letters. DE: DMSO+Ethanol (control), FLC: fluconazole, LA: linoleic acid, GLA: Gamma-linolenic acid. Page | 86 R e la t iv e F lu o r e s c e n c e U n i t R e la t iv e F lu o r e s c e n c e U n i t The fluorescence of C. krusei UFS Y-0277 cells stained with propidium iodide dye after exposure to various treatment conditions was also observed with fluorescence microscopy. As depicted in Figure 9, cells exposed to either of the combinations accumulated more propidium iodide stain than any of the corresponding mono-treatments (i.e. fatty acid or FLC alone). Fig. 9 Fluorescence micrographs of C. krusei UFS Y-0277 cells stained with propidium iodide dye after exposure to various DE treatment conditions [DE: DMSO+Ethanol (control), FLC: fluconazole only, LA: linoleic acid only, GLA: gamma linolenic FLC acid only, FLC+LA: combination of fluconazole and linoleic acid, FLC+GLA: combination of LA fluconazole and gamma- linolenic acid]. Left panel represents white light micrographs, Middle panel represents GLA superimposed white light and fluorescent light micrographs, Right panel represents fluorescent light micrographs. Scale FLC+LA bar represents 10 μm. FLC+GLA Page | 87 2.4.7 Antioxidants rescue biofilm from the toxicity of combination treatments Since we speculated induction of oxidative stress as a contributing factor responsible for the potentiating effect displayed by the combination treatments, it was important to examine the oxidative stress-inducing potential of the combination treatments in the presence and absence of known antioxidants. Our hypothesis was that if the increased susceptibility of the combination-treated biofilms was, in part, mediated by oxidative stress, the addition of antioxidants such as TPGS or BHT would offer biofilms some protective benefits. This was performed by exposing biofilms of the least-susceptible strain (C. krusei UFS Y-0277) to either of the combination treatments (FLC+LA or FLC+GLA) in the presence and absence of TPGS or BHT after which the biomass of the biofilms was quantified with CV assay. Notably, CV assay was used for this experiment instead of XTT assay since there is evidence that α- tocopherol (the active ingredient of TPGS) interferes with tetrazolium salt assays, such as XTT assay (Lim et al. 2015). As shown in Figure 10A, the addition of TPGS, but not BHT, was able to increase the biofilm biomass and rescue the biofilms from the oxidative stress effect induced by the combination of FLC and LA (p < 0.05). However, both TPGS and BHT rescued the biofilms from the toxic effect of the combination of FLC and GLA (p < 0.05) (Fig. 10B). A B 120 120 a a a 110 110 100 100 b b b 90 90 80 80 A T S A T S +L H G L H G C +B TP + G +B P L A + C LA + T F L A FL A+ L +G L LC + C + G F C L FL F FL C Treatment Treatment Fig. 10 Biomass of C. krusei UFS Y-0277 biofilms after exposure to the combination of FLC and LA (A) and FLC+GLA (B) with or without antioxidants (BHT or TPGS). Values are means of three independent experiments and error bars indicate standard deviation. p-value ≤ 0.05 was considered significantly different and are indicated by different letters. FLC+LA: fluconazole and linoleic acid, FLC+GLA: fluconazole and gamma-linolenic acid, BHT: butylated hydroxytoluene, TPGS: α-tocopherol polyethylene glycol succinate. Page | 88 % biomass relative to DE control % biomass relative to DE control The overall superior protection observed for TPGS compared to BHT may indicate that the combination treatments promote lipid peroxidation in the cell membrane since, unlike BHT (which is a general inhibitor of free radical-chain reactions), TPGS localises to the plasma membrane (Yin et al. 2011; Li et al. 2015; Yehye et al. 2015; Fourie 2020). Hence, this observation may suggest that the cell membrane damage observed earlier in this study is partly due to lipid peroxidation in the cell membrane. Taken together, these findings suggest oxidative stress as a contributing factor responsible for the potentiating effects of the combination treatments. 2.4.8 Influence of fatty acids on efflux pump activity of C. krusei Drug efflux pumps belong to either ATP-Binding Cassette (ABC) family (Lubelski et al. 2007), Major Facilitator Superfamily (MFS) (Pao et al. 1998), Multidrug resistance And Toxic compound Extrusion (MATE) family (Kuroda and Tsuchiya 2009), Small Multidrug Resistance (SMR) family (Jack et al. 2001), Resistance Nodulation Division (RND) superfamily (Nikaido and Takatsuka 2009), or Drug Metabolite Transporter (DMT) superfamily (Piddock 2006; Soto 2013) of transporters. Each of these proteins functions to pump out toxic compounds, including antimicrobial agents, and their overexpression results in multidrug-resistant phenotype in pathogenic microbes (Lamping et al. 2009). In Candida spp., only the ABC and MFS transporters have been characterised (Wirsching et al. 2001; White et al. 2002; Cannon et al. 2009). While the MFS transporters are secondary transporters, which are powered by electrochemical proton-motive force, ABC family members are primary transporters and rely on the hydrolysis of ATP for energy (Pao et al. 1998; Cannon et al. 2009; Rees et al. 2009; Redhu et al. 2016). Additionally, although C. krusei possesses ABC transporters, such as Abc1p, Abc2p, Abc11p, and Abc12p (Katiyar and Edlind 2001; Lamping et al. 2009; Lamping et al. 2017; Douglass et al. 2018), no MFS transporter has been characterised in this yeast. Evidently, efflux pumps play a role in C. krusei resistance to FLC (Lamping et al. 2009; Lamping et al. 2017). We, therefore, hypothesised that the increased susceptibility of combination-treated biofilms might be due to the disruption of efflux pump activity. To test this hypothesis, the influence of either of the unsaturated fatty acids (LA or GLA) alone or in combination with FLC on efflux pump activity of C. krusei UFS Y-0277 biofilm was evaluated using Rh6G efflux assay. The fluorescent dye, Rh6G, is a substrate of efflux pump (ABC) transporters (Nakamura et al. 2001; Mukherjee et al. 2003; Tsao et al. 2009). As depicted in Figure 11A, at a concentration of 0.1 mM, LA alone had a slight inhibitory influence on efflux pump activity, which was further enhanced by the combination. Interestingly, GLA (0.1 mM) alone could not inhibit the efflux of Rh6G; however, its combination with FLC significantly inhibited the activity of the efflux pumps (p < 0.05) (Fig. 11C). An unexpected, slightly reduced efflux of Rh6G was observed for treatment with FLC Page | 89 only (Fig. 11C). This might be a result of competitive inhibition since both compounds (i.e. FLC and Rh6G) are substrates for efflux pumps (Maesaki et al. 1999; Lamping et al. 2009; Prasad et al. 2015). Strikingly, a more pronounced inhibition of efflux pump activity was observed after treatment with a higher concentration (1 mM) of the fatty acids alone or in combination with FLC (Fig. 8B, D). A B 3 0 3 0 D E D E F L C F L C L A (0 .1 m M ) L A (1 m M ) 2 0 2 0 F L C + L A F L C + L A 1 0 1 0 0 0 0 1 0 2 0 3 0 4 0 5 0 6 0 0 1 0 2 0 3 0 4 0 5 0 6 0 T im e T im e C D 4 0 4 0 D E D E F L C F L C 3 0 3 0 G L A (0 .1 m M ) G L A (1 m M ) F L C + G L A F L C + G L A 2 0 2 0 1 0 1 0 0 0 0 1 0 2 0 3 0 4 0 5 0 6 0 0 1 0 2 0 3 0 4 0 5 0 6 0 T im e (m in ) T im e (m in ) Fig. 11 Rhodamine 6G efflux in C. krusei UFS Y-0277 biofilms after treatment with fluconazole in the presence or absence of 0.1 mM LA (A), 1 mM LA (B), 0.1 mM GLA (C) or 1 mM GLA (D). Values are means of three independent experiments and error bars indicate standard deviation. DE: DMSO+Ethanol (control), FLC: fluconazole, LA: linoleic acid, GLA: Gamma-linolenic acid. Since drug efflux pumps are localised to the cell membrane, and it is known that unsaturated fatty acids could elicit cell membrane disruption (Avis and Belanger 2001; Pohl et al. 2011; Mishra et al. 2014), the observed inhibition of efflux pump activity may be due to the mislocalisation of efflux pumps, following the disruption of membrane fluidity and membrane disorganisation. Additionally, since all the presently characterised efflux pumps in C. krusei are ABC transporters, which hydrolyse ATP for their efflux activities (Lamping et al. 2017; Douglass et al. 2018), another factor that might have contributed to the loss of activities of the efflux pumps could be ATP deficit. This may be because antimicrobial fatty acids have been Page | 90 R e la t iv e F lu o r e s c e n c e U n i t R e la t iv e F lu o r e s c e n c e U n i t R e la t iv e F lu o r e s c e n c e U n i t R e la t iv e F lu o r e s c e n c e U n i t reported to disrupt cellular processes, such as oxidative phosphorylation, which synthesise ATP essential for efflux pump activity (Galbraith and Miller 1973; Yoon et al. 2018). Similarly, a recent study of Kuloyo and co-workers (2020) has demonstrated that AA potentiates C. albicans biofilm's sensitivity to FLC via interference with ATP biosynthetic pathways. 2.4.9 Combination treatments prolong the lifespan of infected nematodes Undoubtedly, in vitro experimental findings do not always correlate with that of in vivo assays. Because of this, animal models are employed not to only assess the correlation between observations generated by in vitro and in vivo assays, but to also gain insights into the mechanisms of microbial pathogenesis in humans since some virulence traits and immune reactions are only induced in vivo (Brenner 1974; Marsh and May 2012). Hence, to further corroborate our in vitro findings, we evaluated the protective effect of the combination treatments in a C. elegans infection model. Caenorhabditis elegans has been described as a powerful system for many studies, including drug discovery because unlike mammalian models, it has simple growth conditions, great ease of cultivation, a rapid life cycle, it is tractable, and its use requires no ethical clearance (Brenner 1974; Pukkila-Worley et al. 2009; Corsi et al. 2015; Madende et al. 2020). Moreover, this model also has innate immunity which overlaps with that found in humans (Pukkila-Worley et al. 2011). The use of this nematode in this study instead of a mammalian model also ensures compliance with the Russell’s and Burch’s 3Rs (Replacement, Reduction, and Refinement) principles which recommend the replacement of “conscious” vertebrates with “non-sentient” or “less sentient” invertebrates such as C. elegans and Drosophila melanogaster (Russell and Burch 1959; Kretlow et al. 2010; Cheluvappa et al. 2017). For this study, Caenorhabditis elegans AU37 (glp-4(bn2) I; sek-1(km4) X) strain was selected because of its enhanced immune-deficiency which facilitates microbial infections due to mutation in SEK-1 which encodes a conserved mitogen-activated protein kinase (Kim et al. 2002). Moreover, this strain cannot propagate at a temperature of 25oC due to GLP-4 mutation (Miyata et al. 2008). Additionally, the ability of C. krusei to infect and kill this C. elegans strain has been previously demonstrated (Scorzoni et al. 2013). Thus, the in vivo treatment assay was done by infecting nematodes with the least-susceptible strain of C. krusei (C. krusei UFS Y-0277). Thereafter, the infected nematodes were exposed to various treatments, including the combination treatments, and were monitored daily for survival. The survival statistical analysis and differences (log-rank test) were determined using OASIS 2 (Han et al. 2016). As depicted in Figure 12, in contrast to uninfected nematodes fed with E. coli OP50 with a median lifespan of 8.37 ± 0.14 days, infection with C. krusei significantly shortened the median lifespan of infected nematodes (2.83 ± 0.11 days) and resulted in a 100% mortality within five days post-infection. Although both FLC and LA singly could slightly extend the median lifespan of Page | 91 infected nematodes for 3.56 days, superior protection and elongation of the lifespan of the infected nematode were observed upon treatment with FLC and LA (5.08 ± 0.19 days) (Fig. 12A). A previous study by Breger and co-workers (2007) has demonstrated that FLC (at 32 μg/ml) could prolong the survival of C. albicans and C. parapsilosis. However, in their study, it could not extend the survival of and had a toxic effect on nematodes exposed to a FLC- resistant strain of C. krusei. This is not the case in the present study, and a possible explanation for this is that its antifungal efficacy against the C. krusei strain used in this study outweighs its toxicity on C. elegans. Importantly, although GLA alone was also able to prolong the median lifespan of infected nematodes (4.06 ± 0.16 days), a more pronounced lifespan extension (median lifespan of 5.32 ± 0.20 days) was observed when C. elegans was exposed to the combination of FLC and GLA (Fig. 12B). The overall enhanced activity displayed by the combination treatments may be due to a direct antifungal effect on C. krusei, as observed in vitro. Additionally, it might be due to improved immunity and ultimate enhanced clearance of fungal infection afforded by the fatty acids, since there is evidence that endogenous and exogenous PUFAs, such as GLA and SDA, are required for C. elegans immunity (Nandakumar and Tan 2008). Although LA does not have a direct impact on C. elegans immunity, it might have been converted to GLA or SDA (following its conversion to ALA by fat- 1) by Δ6-desaturase fat-3 to facilitate the observed antifungal and immunoprotective effect (Watts and Browse 2002). Other studies have also reported possible links between fatty acid metabolism and immunity in this model (Lee et al. 2010; Ward et al. 2014; Anderson et al. 2019). Furthermore, a recent study by Lee and co-workers (2020) has demonstrated the ability of a medium-chain fatty acid, nonanoic acid, to prolong the survival of C. elegans infected with C. albicans. Page | 92 Fig. 12 Survival of infected Caenorhabditis elegans after treatment with linoleic acid (A) or gamma-linolenic acid (B) in the presence or absence of fluconazole. OP50 represents uninfected nematodes fed with Escherichia coli OP50 (uninfected group). DE represents infected nematodes treated with DMSO and ethanol (untreated group), FLC represents infected nematodes exposed to fluconazole (32 μg/ml), LA represents infected nematodes treated with linoleic acid (0.1 mM), GLA represents infected nematodes exposed to gamma-linolenic acid (0.1 mM), FLC+LA represents infected nematodes treated with fluconazole and linoleic acid, while FLC+GLA represents infected nematodes exposed to the combination of fluconazole and gamma-linolenic acid. The tables depict the median lifespan along with the standard error (S.E.) as well as the post-infection days to reach 50% and 100% mortality. Log-rank test was used to assess significant differences in survival. *Significantly different from the untreated control (DE). # indicates statistical difference between the combination treatments compared to either of the single treatments (FLC, LA, or GLA). FLC: fluconazole, LA: linoleic acid, GLA: Gamma-linolenic acid. Page | 93 2.4.10 Combination treatments reduce the fungal burden of infected nematodes To further strengthen our in vitro findings, the yeast burden within the intestine of infected C. elegans after exposure to the combination treatments was assessed. This was experimentally demonstrated by exposing nematodes infected with the least-susceptible strain of C. krusei (C. krusei UFS Y-0277) to various treatments, including the combination treatments, for 24 h after which the fungal burden of each nematode was determined. Consistent with the C. elegans treatment assay results, FLC, GLA, and LA were able to reduce the fungal burden of infected C. elegans by circa 32%, 13%, and 11%, respectively (Fig. 13). However, treatments with the combination of FLC and LA or GLA profoundly reduced the yeast burden by circa 66% and 71%, respectively (p < 0.05). A B 150 150 100 100 * * 50 * # 50 * # 0 0 E A E C A LC LA LA D F L L L D F + G +G LCF FL C Treatment Treatment Fig. 13 Fungal burden of infected Caenorhabditis elegans after treatment with linoleic acid (A) or gamma-linolenic acid (B) in the presence or absence of fluconazole. *Significantly different from the untreated group (DE). # indicates a statistical difference between the combination treatments compared to either of the monotherapies (FLC, LA, or GLA). DE: DMSO and ethanol (untreated group), FLC: fluconazole (32 μg/ml), LA: linoleic acid (0.1 mM), GLA: gamma-linolenic acid (0.1 mM), FLC+LA combination of fluconazole and linoleic acid, FLC+GLA: combination of fluconazole and gamma- linolenic acid. Taken together, our in vivo findings correlate with the earlier observed in vitro susceptibility profiles of the combination treatments. However, one possible explanation for the overall increased antifungal efficacy displayed by all the treatments in vivo is that the innate immunity of infected C. elegans assisted with infection clearance, a phenomenon that cannot be assayed in vitro (Mylonakis et al. 2007; Pukkila-Worley et al. 2011). Page | 94 % fungal burden per nematode % fungal burden per nematode 2.5 Conclusions Although C. albicans is the major cause of invasive candidiasis, the incidence of infections caused by NAC species, including Candida krusei, is on the rise. This epidemiological shift may be partly explained by the increasing resistance of NAC species to antifungal agents (Chi et al. 2011; da Silva et al. 2013; Sadeghi et al. 2018). For example, C. krusei, a potential multidrug-resistant NAC yeast, exhibits intrinsic resistance to FLC, with more than 97% isolates exhibiting resistance (Whaley et al. 2017), while also rapidly developing adaptive resistance to other antifungal drugs, including the echinocandins (Forastiero et al. 2015). The combination of antifungal agents with non-antimicrobial agents, including phytocompounds and fatty acids, has been receiving considerable attention as a potential infection control strategy, especially for drug-resistant fungal infections. Moreover, PUFAs, such as AA and SDA, have been shown to increase the sensitivity of C. albicans and C. dubliniensis biofilms to conventional antifungal drugs, including FLC, clotrimazole, and amphotericin B (Ells et al. 2009; Thibane et al. 2012b; Mishra et al. 2014). Several mechanisms, such as increased oxidative stress, disruption of membrane organisation, and inhibition of drug efflux pumps have been speculated to be involved in this phenomenon (McDonough et al. 2002; Ells et al. 2009; Thibane et al. 2012b). The results of this present study demonstrate that either of two unsaturated fatty acid (LA or GLA) potentiates the susceptibility of the biofilm of the least-susceptible strain of C. krusei to FLC in vitro as observed by XTT and SEM assays. The in vitro underlying mechanisms of action of these combination treatments were demonstrated to be through damage to the cell wall and membrane. Additionally, the inhibition of efflux pump activity, following the disruption of membrane organisation, and induction of oxidative stress are suspected as complementary mechanisms responsible for this potentiating activity. Furthermore, due to the observed cell rupture and loss of intracellular contents, death following exposure of cells to the combination treatments might be due to necrosis. However, the occurrence of apoptosis following oxidative damage amongst subpopulation of cells within the combination-treated biofilms cannot be dismissed, since such insult could be inflicted by both unsaturated fatty acids (Finstad et al. 1998; Mainou-Fowler et al. 2001; Thibane et al. 2012a) and FLC (Peng et al. 2018; Dbouk et al. 2019). Further, our in vivo findings in a C. elegans model suggest that the combination treatments could prolong the overall survival and reduce the intestinal fungal burden of nematodes infected with C. krusei. This study demonstrates, for the first time, the potentiating activity of unsaturated fatty acids (LA or GLA) with FLC against intrinsically-resistant C. krusei in vitro and in vivo in a C. elegans infection model. 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International Journal of Molecular Sciences 19:1114. https://doi.org/10.3390/ijms19041114 Zarnowski R, Sanchez H, Covelli AS, et al (2018) Candida albicans biofilm–induced vesicles confer drug resistance through matrix biogenesis. PLoS Biology 16:e2006872. https://doi.org/10.1371/journal.pbio.2006872 Zhao K, Bleackley M, Chisanga D, et al (2019) Extracellular vesicles secreted by Saccharomyces cerevisiae are involved in cell wall remodelling. Communications Biology 2:305 https://doi.org/10.1038/s42003-019-0538-8 Page | 111 CHAPTER 3 The development of a CRISPR-Cas9 genome editing system for Candida krusei Page | 112 3.1 Abstract The 2020 Nobel Prize in Chemistry was co-awarded to Emmanuelle Charpentier and Jennifer Doudna for the discovery of Clustered Regularly Interspaced Short Palindromic Repeats- Cas associated protein 9 (CRISPR-Cas9) system – a precise editing tool that can be used for gene- editing in virtually all domains of life. This editing tool has revolutionised genetic engineering because it is facile, economical, rapid, and has a better efficiency compared to other genome editing systems. Although functional CRISPR-Cas9 editing systems have been designed and harnessed for gene engineering in many yeasts, including Candida albicans and other non- albicans Candida (NAC) species, such as C. glabrata, C. parapsilosis, and C. auris. No such system is available for C. krusei. The absence of such a simple and precise tool has dramatically hampered the full molecular delineation of this recalcitrant yeast's resistance mechanisms. This chapter involves developing a CRISPR-Cas9 mediated gene editing system for use in C. krusei. This was done by adapting a previously designed, C. albicans-specific, CRISPR-Cas9 system (HIS-FLP type). Our newly adapted system consists of a CAS9 gene under the control of C. krusei ENO1 promoter; and a gRNA, under the control of SNR52 promoter, adaptable to target any locus within C. krusei genome. This system was designed to integrate at HIS1 locus in the genome of C. krusei, and successful integration allows selection of transformants on nourseothricin- containing plates. As proof of concept, its efficacy was validated by the successful deletion of two auxotrophic marker genes, URA3 and ADE2. Keywords: CRISPR-Cas9, Candida krusei, Gene engineering, Resistance, Auxotrophic marker Page | 113 3.2 Introduction In modern biotechnology, the most important tools for gene modification are endonucleases, including zinc-finger nucleases, transcription activator-like effector nucleases, and engineered meganucleases (Richardson et al. 2016; Tang et al. 2019). However, these systems have various limitations and drawbacks, such as low specificity, single-site targeting, occurrence of non-specific mutations, low efficiency, huge complexity, high time-consumption and cost (Wang et al. 2013; Zhou et al. 2014; Abdallah et al. 2015; Adli 2018; Waryah et al. 2018). An adaptive immune system known as CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein) found in many bacteria and most archaea helps protect bacterial and archaeal genomes from invading phages and plasmids (Doudna and Charpentier 2014; Lander 2016; Hale et al. 2009; Khadempar et al. 2019). This system relies upon a dual RNA-Cas complex, formed by the association of a guide RNA (gRNA) [composed of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA)] with Cas nuclease, to target and cleave foreign DNA with sites complementary to the protospacer sequence within the gRNA. A protospacer adjacent motif (PAM) site positioned next to the protospacer sequence is recognised by the Cas nuclease, and it is particularly important for this cleavage as it helps the Cas protein to discriminate between self-and invading DNA (Brouns et al. 2008; Deltcheva et al. 2011; Jinek et al. 2012; Nishimasu et al. 2014; Jiang et al. 2016). The discovery and subsequent elaborate study of this bacterial and archaeal adaptive immunity led to the development of a CRISPR-Cas9-mediated genome editing system. This editing tool has revolutionised genetic engineering because not only is it economical, versatile, and rapid, it is also simple to design and offers high-efficiency gene- editing compared to other genome editing systems (Khadempar et al. 2019; Saha et al. 2019). Moreover, the discovery of this revolutionary gene-editing tool has earned its discoverers, Emmanuelle Charpentier and Jennifer Doudna, the 2020 Nobel Prize in Chemistry (https://www.nobelprize.org/prizes/chemistry/2020/press-release/). The CRISPR-Cas9 gene-editing tool uses a programmable Cas9 nuclease guided by a synthetic gRNA to introduce double-strand breaks (DSBs) at specific sites within the genome (Nguyen et al. 2017). These DSBs are usually lethal and require repair (Ranjha et al. 2018). Two repair mechanisms: non-homologous end-joining (NHEJ) and homology-directed repair (HDR) are involved in repairing these breaks. The NHEJ is the main repair pathway found in eukaryotes, and it repairs DSBs by ligating the broken ends of DNA without the need for a homologous DNA template (Lieber et al. 2003; Hefferin and Tomkinson 2005). Although this pathway has a high incidence rate (i.e. can occur and repair DSBs during all phases of the cell cycle), it has low fidelity (introduces indels), and could lead to frameshift mutations (Bernheim et al. 2017). Conversely, HDR has high fidelity and requires homologous DNA Page | 114 sequences either from a sister chromosome (in case of a diploid cell) or a foreign donor DNA (dDNA) (Branzei and Foiani 2008; Arnoult et al. 2017; Saha et al. 2019). Notably, scientists have harnessed this pathway to generate precise gene-editing and modification by introducing desired exogenous dDNA fragments at target loci (Lin et al. 2014) (Fig. 1). Fig. 1 Mechanisms used to repair double-strand breaks (Obtained from Saha et al. 2019). The first implementation of a CRISPR-Cas9 mediated gene-editing in a yeast was demonstrated by DiCarlo and co-workers (2013) in the model yeast, Saccharomyces cerevisiae. The system was constructed using a codon-optimised CAS9 gene under the control of the Gal-L promoter and CYC1 terminator; and a transient guide RNA, under the control of the SNR52 promoter and SUP4 terminator, designed to target CAN1 gene, a cell membrane arginine permease, in S. cerevisiae. The gRNA cassette and dDNA fragment was co-transformed into S. cerevisiae cells constitutively expressing Cas9. The Cas9 guided by the gRNA induced DSBs at the CAN1 locus. This was followed by homologous recombination with the supplied dDNA, and selection of transformed cells with disrupted CAN1 on media containing canavanine, a toxic analogue of arginine transported only by a functional Can1p. Several factors, such as diploidy, absence of plasmid systems, alternative codon usage, lack of selectable markers and a known meiotic phase, impede genetic engineering in many Candida species (Vyas et al. 2015; Nguyen et al. 2017; Román et al. 2019). Despite these hurdles, Vyas and co-workers (2015) successfully demonstrated the first use of a CRISPR- mediated genome editing system in Candida albicans. This system was constructed with a C. albicans codon-optimised version of CAS9 (CaCAS9) fused at the 3' end with SV40 nuclear localisation signal and FLAG-tag sequences. The CaCas9 cassette was integrated into C. albicans SC5314 at the ENO1 locus and was expressed by the constitutive ENO1 promoter. The gRNA cassette (containing a 20 bp protospacer specific to ADE2 gene) under an SNR52 promoter integrated at RP10 locus (duet system) or at ENO1 locus (solo system) directs CaCas9 to create DSBs at the ADE2 locus. These breaks induce selective pressure for the Page | 115 ultimate integration of an unmarked dDNA at the target locus via HDR. The system (duet system) was highly efficient and generated up to 80% ade2Δ/Δ transformants. Additionally, after successful transformation, the nourseothricin N-acetyltransferase (NAT) marker and gRNA fragment can be removed with the expression of flippase (FLP) recombinase. However, one allele of the ENO1 gene is left permanently disrupted with this system because of the non- recyclable Cas9 fragment. This might affect the downstream use of the generated mutants since Eno1 protein plays essential roles in the morphogenesis, virulence, cell growth, and osmotic protection of C. albicans (Ko et al. 2013; Leu et al. 2020). A similar system developed by Min and co-workers (2016) addressed this limitation by using transient Cas9 and gRNA cassettes that are not integrated into the C. albicans genome. Homozygous mutants generated using this system could be selected using marked dDNA fragments integrated at the target locus following the creation of site-specific DSBs by the RNA-guided Cas9. However, one limitation of this system is that it uses a non-recyclable marker – the dDNA does not get excised from the target locus. This roadblock was addressed with a revised system designed by Huang and Mitchell (2017), which allows the excision of the dDNA marker. However, this system does not fully represent a markerless genome editing tool since it relies upon two distinct selectable markers to achieve the CRISPR-Cas9-induced marker excision (Nguyen et al. 2017). A new CRISPR system that supports markerless and rapid genome engineering was designed for gene-editing in C. albicans. This system not only eludes the need for lengthy gRNA cloning procedures, but also allows the removal of gRNA/Cas9 and dDNA cassettes, and enables homozygous restoration of complete wildtype open reading frame (ORF) at the native loci – i.e. allows the generation of complementary (addback) strains (Nguyen et al. 2017). Furthermore, CRISPR-Cas9 systems have been developed for gene-editing in several common non-albicans Candida (NAC) species including C. glabrata (Enkler et al. 2016), C. parapsilosis (Lombardi et al. 2017), C. tropicalis (Zhang et al. 2019), C. auris (Grahl et al. 2017), and non-common NAC species, such as C. lusitaniae (Norton et al. 2017), C. orthopsillosis (Zoppo et al. 2019), and C. aaseri (Ibrahim et al. 2020). However, no such system is available for genetic engineering in C. krusei. The lack of such facile and precise tool for gene-editing in C. krusei has greatly impeded the molecular understanding of this yeast's resistance mechanisms. Hence, this chapter aimed at developing a CRISPR-Cas9 system specific for genome engineering in C. krusei by adapting a previously designed, C. albicans-specific, HIS-FLP type CRISPR-Cas9 system of Nguyen and co-workers (2017). Page | 116 3.3 Materials and Methods 3.3.1 Strains used The type strain of Candida krusei, C. krusei UFS Y-0217 (CBS573T), was used in this study. This strain is diploid (2n) and was specifically chosen for this study because of the availability of its annotated genome on the database of National Center for Biotechnology Information (Accession: PRJNA434433) (Douglass et al. 2018). This strain was obtained from the Yeast Culture Collection of the University of the Free State, Bloemfontein, South Africa, and was revived on Yeast Malt extract (YM) agar plates (10 g/l glucose, 3 g/l yeast extract, 3 g/l malt extract, 5 g/l peptone, 17 g/l agar) at 30oC for 24 h. Glycerol stock (15%, v/v) was prepared and stored at -80oC for future use. The XL-10 Gold competent Escherichia coli (Agilent Technologies) was used for bacterial transformation and cloning. 3.3.2 In silico analyses Construction of primers, fragments and constructs, simulation of polymerase chain reaction, cloning, and restriction analyses, sequence alignments, and identification of CRISPR sites were done in silico with Geneious® 11.1.4 prior to all in vitro and in vivo assays. 3.3.3 Plasmids and primers used This study involves constructing a CRISPR-Cas9 system for use in C. krusei by adapting a previously developed C. albicans-specific CRISPR-Cas9 (HIS-FLP) system. The CRISPR- Cas9 plasmids used for this purpose, previously designed by Prof. Aaron Hernday's research lab at the University of California Merced, Merced, California, USA (Nguyen et al. 2017), were obtained from Addgene and are shown in Table 1. All primers used in this study were purchased from Integrated DNA Technologies (IDT) and are listed in Table 2. Each primer was diluted to a concentration of 10 μM with Tris-EDTA (Ethylenediaminetetraacetic acid) (TE) buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) before use. Table 1 Description of HIS-FLP plasmids constructed by Nguyen and co-workers (2017) Plasmid Addgene ID Major component Function pADH99 #90979 Candida albicans 5'-HIS1 region + C. Cas9 cassette albicans ENO1 promoter + CAS9 gene + ½ NAT gene pADH110 #90982 2/2 NAT gene + C. albicans SNR52 promoter ½ gRNA cassette pADH147 #90991 gRNA scaffold + C. albicans 3'-HIS1 region 2/2 gRNA cassette Page | 117 Table 2 Primers used in this study Primer Sequence (5' to 3') Tm (oC) Reference pADH99-5' CK HIS1 overlap-F CGTTTAAACCGCCTCAAGCAGCACACAATTTCATGATTAATGGT 65.6 This study CK-5' HIS1-R CCGGATAATATCAAAACCCCTCT 54.3 This study pADH99::CK HIS1::FRT overlap TGTGAGAGGGGTTTTGATATTATCCGGGAAGTTCCTATACTTTCTAGAGAA 64.9 This study pADH99::CK FRT::ENO1 overlap GGGATGCCACGTGGTAATGAAACAGAAGTTCCTATTCTCTAGAAAGTAT 65.1 This study CK ENO1-F TGTTTCATTACCACGTGGCA 54.9 This study pADH99-3'-CK CAS9 overlap CAATACTATACTTTTTATCCATCCCTGTTTGGTTGGAGGGGGTTA 64.0 This study CK-3' HIS1-overlap-1F AGAGAATAGGAACTTCCCAATGTCACAAAACTCAAACAGGA 63.6 This study CK- 3' HIS1-overlap-1R CTGGGGTTTAAACACCGTAACTAGACAAGCGAGTTTGCA 65.5 This study pADH147-1F TACGGTGTTTAAACCCCAGC 55.2 This study pADH147-1R TGGGAAGTTCCTATTCTCTAGAAAGTA 54.7 This study CK-3' HIS1-F ATGTCACAAAACTCAAACAGGA 52.7 This study CK-3' HIS1-R ACTAGACAAGCGAGTTTGCA 54.3 This study AHO1096-ver2 GACGGCACGGCCACGCGTTTAAAC 65.1 Modified from Nguyen et al. 2017 AHO1098-ver2 CAAATTAAAAATAGTTTACGCAAGTCTCG 53.8 Modified from Nguyen et al. 2017 AHO1097 CCCGCCAGGCGCTGGGGTTTAAACACCG 70.2 Nguyen et al. 2017 AHO1237 AGGTGATGCTGAAGCTATTGAAG 55.0 Nguyen et al. 2017 URA3-CRISPR-1 CGTAAACTATTTTTAATTTGATTGCGCAACACGATATGGGGTTTTAGAGCTAGAAATAGC 65.2 This study URA3-2F GCCTTTGTTAAACAACTTTTTCT 50.6 This study URA3-2R ATGGCGTCATGCTGGTTGGAATGCTTATTT 62.5 This study URA3-3F TCCAACCAGCATGACGCCATCCTTGACAAA 65.1 This study URA3-3R CGCCTTGAAATGAAAATGCTG 53.3 This study ADE2-CRISPR-2 CGTAAACTATTTTTAATTTGTTAGGGTCTGATGTGCCAAAGTTTTAGAGCTAGAAATAGC 64.4 This study ADE2-2F TAGAAGGGCCAGAGTCAGAG 55.5 This study ADE2-2R TTCCATTTCAACCGATAGTTTTCGAGTCCA 59.8 This study ADE2-3F AACTATCGGTTGAAATGGAAACGTACATGAATT 58.7 This study ADE2-3R_new AGAACGCTATTTTAAACGCTAATA 50.7 This study NB: Target-specific CRISPR site is underlined Page | 118 3.3.4 Polymerase chain reaction (PCR) amplification In this study, PCR amplification of genes, fragments, and constructs was done using either KAPA Taq PCR kit (KAPA Biosystems), KAPA HiFi PCR kit (KAPA Biosystems), or KOD Hot Start DNA polymerase kit (Novagen®). The reaction components used for each kit are shown in Tables 3, 4, and 5. The concentration and volume of these components were scaled appropriately for each experiment. The PCR conditions (program) used for each kit are also depicted in Tables 6, 7, and 8. These conditions were modified as required to suit the purpose of each experiment. Additionally, the specific kit used for each amplification is indicated, where necessary. Table 3 Reaction mixture for KAPA Taq PCR kit (KAPA Biosystems) Component Reaction mixture Nuclease-free water Up to 25 μl 10X KAPA Taq Buffer 2.5 μl dNTP Mix (10 mM) 0.5 μl Forward primer (10 μM) 1.0 μl Reverse primer (10 μM) 1.0 μl KAPA Taq DNA polymerase (5 U/μl) 0.1 μl DNA template (less complex DNA)* ≤25 ng *tenfold excess for genomic DNA Table 4 Reaction mixture for KAPA HiFi PCR kit (KAPA Biosystems) Component Reaction mixture Nuclease-free water Up to 25 μl 5X KAPA HiFi Buffer 5.0 μl KAPA dNTP Mix (10 mM) 0.75 μl Forward primer (10 μM) 0.75 μl Reverse primer (10 μM) 0.75 μl KAPA HiFi Hot Start DNA polymerase (1 U/μl) 0.1 μl Template DNA (less complex DNA)* 1 ng *hundred-fold excess for genomic DNA Page | 119 Table 5 Reaction mixture for KOD Hot Start DNA polymerase kit (Novagen®) Component Reaction mixture Nuclease-free water Up to 25 μl 10X Buffer for KOD Hot Start DNA Polymerase 2.5 μl 25 mM MgSO4 1.5 μl dNTP Mix (2 mM) 2.5 μl Forward primer (10 μM) 0.75 μl Reverse primer (10 μM) 0.75 μl KAPA Taq DNA polymerase (5 U/μl) 0.5 μl Template DNA (less complex DNA)* ≤10 ng * tenfold excess for genomic DNA Table 6 PCR condition for KAPA Taq PCR kit (KAPA Biosystems) Step Temperature Time Cycle Initial denaturation 94oC 5 min 1 Denaturation 94oC 30 sec Annealing Lowest primer Tm - 5oC 30 sec 25 Extension 72oC 1 min Final extension 72oC 5 min 1 Hold 4oC ∞ Table 7 PCR condition for KAPA HiFi PCR kit (KAPA Biosystems) Step Temperature Time Cycle Initial denaturation 95oC 3 min 1 Denaturation 98oC 20 sec Annealing Lowest primer Tm - 1oC 15 sec 15 – 35 Extension 72oC 1 min /kb Final extension 72oC 1 min/kb 1 Hold 4oC ∞ Table 8 PCR condition for KOD Hot Start DNA polymerase kit (Novagen®) Step Target size Cycle < 500 bp 500 – 1000 bp 1000 – 3000 bp > 3000 bp Polymerase activation 95oC, 2 min 95oC, 2 min 95oC, 2 min 95oC, 2 min 1 Denaturation 95oC, 20 95oC, 20 sec 95oC, 20 sec 95oC, 20 sec 30 sec Annealing Lowest Primer Tm oC, 10 sec Extension 70oC, 10 70oC, 15 sec/kb 70oC, 20 sec/kb 70oC, 25 1 sec/kb sec/kb Page | 120 3.3.5 Genomic DNA extraction In this study, extraction of genomic DNA from both wildtype and transformed yeast cells was done using either the Zymo Research Quick-DNATM Fungal/Bacterial Miniprep kit or the manual method of Labuschagne and Albertyn (2007). 3.3.5.1 DNA extraction with Zymo Research kit Genomic DNA (gDNA) was extracted using the Zymo Research Quick-DNATM Fungal/Bacterial Miniprep kit in accordance with the manufacturer's instructions. Briefly, pelleted cells of C. krusei was re-suspended in ZR Bashing BeadTM lysis tube containing 200 μl Phosphate Buffered Saline (PBS) [10 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM sodium chloride (pH 7.4)]. A volume of 750 μl of the Bashing BeadTM buffer was dispensed into the lysis tube; the tube was secured in a bead beater [Lasec South Africa (Pty) Ltd] and processed at a maximum speed (400 x g for 3 min) to lyse the yeast cells. The tube was centrifuged (Eppendorf, Germany) at 10,000 x g for 1 min, after which 400 μl of the supernatant was transferred into a Zymo-SpinTM III-F filter (in a collection tube) and was centrifuged at 8,000 x g for 1 min. Subsequently, 1200 μl genomic lysis buffer was added to the filtrate obtained and 800 μl of the mixture (of the filtrate and genomic lysis buffer) was dispensed into a Zymo-SpinTM IIC column (in a new collection tube) and was centrifuged at 10,000 x g for 1 min. The flow-through was discarded, the remaining 800 μl volume of the previous mixture was dispensed into the Zymo-SpinTM IIC column (in the same collection tube) and was centrifuged again at 10,000 x g for 1 min. The Zymo-SpinTM IIC column was transferred into a new collection tube, 200 μl of DNA pre-wash buffer was added to the column, and centrifuged at 10,000 x g for 1 min. Following this step, the column was washed with 500 μl gDNA wash buffer at 10,000 x g for 1 min. The Zymo-SpinTM IIC column was subsequently transferred to a clean 1.5 ml microcentrifuge tube and the gDNA was eluted (10,000 x g, 30 sec) from the column with 100 μl DNA elution buffer (10 mM Tris-HCl, pH 8.5). The concentration of the eluted gDNA was determined with a Nanodrop (NanoDrop Technologies, USA) and the DNA was stored at -20oC prior to further use. 3.3.5.2 DNA extraction with a manual method A slightly modified protocol of Labuschagne and Albertyn (2007) was followed for the manual isolation of DNA from yeast cells. This manual method was utilised for the preliminary extraction of DNA from transformants. Briefly, pelleted cells of C. krusei was re-suspended in 500 μl lysis solution (10 mM Tris-HCl pH 8.0, 50 mM EDTA pH 8.0, 1% SDS) containing 200 μl glass beads. The mixture was vortexed for 5 min (with a 60-second incubation on ice after each minute vortexing), and 275 μl ammonium acetate (2.3 M, pH 7) was added. This was followed by incubating the mixture at 65oC for 5 min, on ice for another 5 min, and centrifugation (20,000 x g, 4oC, 2 min) after the addition of 500 μl chloroform. The resulting Page | 121 supernatant was transferred to a new 1.5 ml Eppendorf tube, the DNA was precipitated at room temperature (5 min) with 1 volume isopropanol and centrifuged at 20,000 x g for 2 min at 4oC. Subsequently, the supernatant was discarded, the pellet was washed with ice-cold 70% ethanol, dried with SpeedVac (Eppendorf, Germany), reconstituted in 100 μl TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing 5 μl of 0.5 mg/ml RNase, and stored at - 20oC prior to further use. 3.3.6 Agarose gel electrophoresis Unless stated otherwise, successful extraction and amplification of DNA were confirmed using agarose gel [Whitehead Scientific (Pty) Ltd] at a concentration of 0.8% (w/v). The gel was prepared with 1X Tris Acetate EDTA (TAE) electrophoresis buffer (40 mM Tris base, 20 mM acetic acid, 1 mM EDTA) and stained with ethidium bromide (Science Lab) or SYBR® Safe DNA Gel Stain (Invitrogen) (in case of gel extraction) at 1:10,000 volume. DNA samples with loading dye were loaded into the wells of the solidified gel, and a 10 kb O'GeneRuler DNA ladder mix (Thermo Fisher Scientific) was used as a DNA marker for size comparison. The DNA samples were separated at 90 volts for 30 min, and the gel was visualised under UV light in a Gel DocTM XR+ (Bio-Rad, Canada) (Lee et al. 2012). 3.3.7 Gel extraction Following gel electrophoresis, DNA samples which needed to be purified prior to further downstream applications were purified using the Thermo Scientific GeneJET Gel Extraction kit according to the manufacturer's instructions. Briefly, agarose gel containing the desired DNA sample was excised from the gel and transferred to a 1.5 ml Eppendorf tube. The excised gel was incubated at 60oC with a 1:1 volume of binding buffer until it completely dissolved. Subsequently, 1 volume isopropanol (100%) was added to the solubilised gel, and 800 μl of this mixture was transferred to a GeneJET purification column (in a collection tube). The column was centrifuged at 16,000 x g (Eppendorf, Germany) for 1 min, and the flow-through was discarded. Following this step, the column was washed (16,000 x g, 1 min) with 700 μl wash buffer and transferred to a new 1.5 ml Eppendorf tube. The ultrapure DNA was eluted (16,000 x g, 1 min) from the column with 50 μl DNA elution buffer (10 mM Tris-HCl, pH 8.5), its concentration was determined with a NanoDrop (NanoDrop Technologies, USA) and was stored at -20oC until further use. 3.3.8 Restriction digest Restriction digestion of DNA was done using the component depicted in Table 9. In the case of double digests, 1 μl of each restriction enzyme was included, and the reaction scaled appropriately. Following every digestion reaction, the enzyme(s) was inactivated at appropriate conditions. Page | 122 Table 9 Reaction mixture for digestion reaction Component Reaction mixture Nuclease-free water Up to 25 μl DNA As required Buffer (restriction-enzyme specific) 2.0 μl Restriction enzyme 0.5 – 1.0 μl 3.3.9 DNA assembly using NEBuilder® All primers, including the overlapping primers, used for the amplification of fragments and/or plasmids expected to be assembled with the NEBuilder® HiFi DNA Assembly kit (New England Biolabs®Inc.) were designed as shown in Figure 2. Each overlapping primer was designed with an overlap sequence of about 15 – 25 nucleotides (with Tm >50oC). The reaction components used for assembly are indicated in Table 10. Following assembly reaction at 50oC (myBlockTM Mini Dry Bath, Benchmark Scientific) for 60 min, the assembled product was stored at -20oC until further use. An illustration of the assembly reaction is shown in Figure 3. Fig. 2 An illustration of the procedure followed for primer designs for the assembly of fragments with NEBuilder® HiFi DNA Assembly kit (https://international.neb.com/). Page | 123 Table 10 Reaction component for NEBuilder assembly Component Reaction mixture Nuclease-free water Up to 20 μl Fragments* As required NEBuilder HiFi DNA assembly master mix 10.0 μl *Two-fold excess of insert(s) was used (i.e. when using 50 ng of vector mass, 100 ng of insert(s) was used). Fig. 3 A schematic representation of the NEBuilder® HiFi DNA Assembly reaction (https://international.neb.com/). 3.3.10 Bacterial transformation The bacterial transformation was done using a modified NEBuilder® HiFi DNA Assembly protocol. Briefly, competent E. coli cells removed from -80oC were thawed on ice. The cells were mixed with 2 μl DNA (e.g. assembled product), incubated on ice for 30 min, heat-shocked at 42oC (myBlockTM Mini Dry Bath, Benchmark Scientific) for 30 sec and returned on ice for 2 min. Subsequently, 1 ml Luria Bertani (LB) broth (10 g/l tryptone powder, 10 g/l yeast extract, 5 g/l sodium chloride) was added and cells were grown by gentle shaking at 37oC for 60 min. A 100 μl volume of the transformed cells was plated on LB agar plates (10 g/l tryptone powder, 10 g/l yeast extract, 5 g/l sodium chloride, 17 g/l agar) supplemented with 100 μg/ml ampicillin Page | 124 (Roche®), and incubated overnight at 37oC. Untransformed E. coli cells were used as a negative control. 3.3.11 Plasmid extraction and purification 3.3.11.1 Miniprep – lysis by boiling method After every bacterial transformation step, plasmid DNA was extracted using a modified lysis by boiling protocol of Holmes and Quigley (1981). Briefly, an overnight broth culture of transformed E. coli cells was centrifuged (10,000 × g, 2 min) (Eppendorf, Germany), the pelleted cells were re-suspended in 350 μl STET buffer [8% (w/v) sucrose, 5% (v/v) Triton X- 100, 50 mM EDTA, 50 mM Tris-HCl, pH 8.0] in a 1.5 ml Eppendorf tube and was vortexed to allow complete mixing. A 5 μl volume of 50 mg/ml lysozyme was added, the mixture was mixed gently by inversion (7×, 35 sec) and was boiled in boiling water for 40 sec. Subsequently, the mixture was vortexed and allowed to precipitate at room temperature following the addition of 40 μl sodium acetate (2.5 M, pH 5.2) and 420 μl isopropanol. The resulting mixture was centrifuged (7800 × g, 4oC, 5 min) and the supernatant was removed without disturbing the pellet. The pellet was washed with 1 ml of ethanol (70%) at 7800 × g for 2 min at 4oC, and the remaining ethanol was removed. Following the washing step, the pellet was dried in a SpeedVac (Eppendorf, Germany) for 5 min and dissolved with 50 μl TE buffer (10 mM Tris- HCl, 1 mM EDTA, pH 8.0). After the extraction procedure, extracted plasmids were digested with an appropriate restriction enzyme(s), and their profiles were confirmed with gel electrophoresis. Plasmids confirmed to contain the inserted fragments in the correct orientation were subsequently purified using the Thermo Scientific GeneJET Plasmid Miniprep kit. 3.3.11.2 Plasmid purification The purification of plasmid DNA was done according to the protocol of Thermo Scientific GeneJET Plasmid Miniprep kit. Briefly, transformed E. coli cells (already verified to contain recombinant plasmids) were grown overnight at 37oC in LB broth (supplemented with 100 μg/ml ampicillin). The overnight culture was centrifuged (10,000 x g, 2 min) (Eppendorf, Germany), and the resulting pelleted cells were re-suspended in 250 μl resuspension solution in a 1.5 ml Eppendorf tube. A 250 μl volume of lysis solution was added to the mixture and was thoroughly mixed by inversion until it became viscous. Following this step, a 350 μl neutralisation solution was added, and the mixture was again thoroughly mixed by inversion until it became cloudy. The resulting mixture was centrifuged (16,000 x g, 5 min) to pellet the chromosomal DNA and cell debris. The supernatant was pipetted into a GeneJET spin column (in a collection tube), the flow-through was discarded following centrifugation (16,000 x g, 1 min) and the column was returned to the collection tube. The column was washed twice with 500 μl wash solution at 16,000 x g for 60 sec, and this was followed by the elution of the Page | 125 plasmid from the column into a new 1.5 ml Eppendorf tube with 50 μl pre-warmed elution buffer (pre-warmed at 70oC for better recovery) at 16,000 x g for 2 min. Expected profiles (i.e. insertion of inserts in the correct orientation) of the purified plasmids were again confirmed using gel electrophoresis after a digestion reaction. 3.3.12 Minimum fungicidal concentration (MFC) for nourseothricin Since the CRISPR-Cas9 system used in this study utilises a dominant NAT marker, it was important to determine the concentration of nourseothricin (NTC) appropriate for the selection of transformed C. krusei cells. The MFC of NTC suitable for the selection of transformed cells of C. krusei was determined with a few modifications of a previous protocol (du Plooy 2019). Briefly, a loopful of C. krusei UFS Y-0217 cells from a YM agar plate was inoculated into 5 ml Yeast extract Peptone Dextrose (YPD) broth (10 g/l yeast extract, 20 g/l peptone, 20 g/l glucose) and was incubated overnight at 30oC in a shaking incubator. Following incubation, 100 μl of this culture was transferred onto YPD agar plates (10 g/l yeast extract, 20 g/l peptone, 20 g/l glucose, 17 g/l agar) supplemented with varying concentrations (600 μg/ml, 500 μg/ml, 400 μg/ml, 300 μg/ml, 200 μg/ml) of NTC (Jena Bioscience). These plates were subsequently incubated at 30oC for 2 to 3 days, after which the MFC of NTC for C. krusei UFS Y-0217 strain was determined. The MFC was defined as the lowest concentration of NTC that prevented any visible growth of the yeast. The yeast was unable to grow at a concentration of 400 μg/ml and higher; hence 400 μg/ml was used as a starting concentration for the selection of transformed cells. 3.3.13 Construction of a HIS-FLP type CRISPR-Cas9 system for C. krusei In order to develop a working CRISPR-Cas9 system for use in C. krusei, a HIS-FLP type CRISPR-Cas9 system designed by Nguyen and co-workers (2017) for gene-editing in C. albicans was adapted. Plasmids required for this purpose were obtained from Addgene (Table 1), and were adapted for use in C. krusei. 3.3.13.1 Adaptation of pADH99 plasmid The pADH99 plasmid (#90979) was linearised by double digestion (Fig. 4) with NcoI and SmaI restriction enzymes (Thermo Scientific) (see section 3.3.8), and subsequently gel purified. Next, the 5’-HIS1 region was amplified from the genome of C. krusei, using the reaction mixture and PCR condition of KAPA HiFi PCR kit (Tables 4, 7), at an annealing temperature of 53oC, with primer pair pADH99-5' CK HIS1 overlap-F and CK-5' HIS1-R (Table 2). A flippase recognition target (FRT) fragment was amplified with overlapping oligonucleotides, pADH99::CK HIS1::FRT overlap and pADH99::CK FRT::ENO1 overlap, using the reaction mixture and program of KAPA Taq PCR kit (Tables 3, 6) at an annealing temperature of 50oC. The third round of PCR involved amplifying the ENO1 promoter from the genome of C. krusei, with primer pair CK ENO1-F and pADH99-3'-CK CAS9 overlap, using the reaction mixture and Page | 126 condition of KAPA HiFi PCR kit at an annealing temperature of 54oC. The oligonucleotides pADH99::CK HIS1::FRT overlap and pADH99::CK FRT::ENO1 overlap were designed to overlap with CK 5'-HIS1 and CK ENO1p fragments, respectively, to aid the assembly of these fragments. Also, the primers pADH99-5' CK HIS1 overlap-F and pADH99-3'-CK CAS9 overlap contain sequences that are complementary to pADH99 backbone and CAS9 gene, respectively, to aid cloning into the plasmid. The successful amplification of these fragments, except for the FRT fragment (which was visualised on a 2% agarose gel) was confirmed with a 0.8% agarose gel (see section 3.3.6), the fragments were then gel extracted and purified using the Thermo Scientific GeneJET Gel Extraction kit (see section 3.3.7) prior to assembly. Accordingly, the purified CK 5’-HIS1, FRT and CK ENO1p fragments were linked to the linearised pADH99 (Fig. 4) using the NEBuilder® HiFi DNA Assembly kit (see section 3.3.9) and transformed into competent E. coli cells (see section 3.3.10). Following transformation, plasmids were extracted from selected colonies using lysis by boiling method (see subsection 3.3.11.1), and were screened for anticipated inserts by restriction digestion with XbaI (Thermo Scientific). Subsequently, plasmids were extracted and purified from selected colonies (suspected to be containing the anticipated plasmid based on the profile obtained after restriction digest with Xbal) but this time with the Thermo Scientific GeneJET Plasmid Miniprep kit (see section 3.3.11.1). The newly purified plasmids' profiles were checked using gel electrophoresis following a digestion reaction with BamHI to confirm the correct insertion and orientation of the inserted fragments. Following this, the concentration of the purified plasmid CK pADH99 was determined, and the plasmid was stored at -20oC prior to future use. Fig. 4 A schematic summary of the steps involved in the construction of CK pADH99 plasmid. First, pADH99 was double digested with NcoI and SmaI to linearise it and release the HIS1-ENO1p-FRT region. Next, an intact CK pADH99 plasmid is prepared by ligating amplified (overlapping) C. krusei- specific fragments into the linearised pADH99 plasmid. NB: Shapes not drawn to scale. Page | 127 3.3.13.2 Propagation of pADH110 More copies of pADH110 plasmid (#90982), containing an overlapping portion of NAT marker gene and a C. albicans SNR52 promoter, were generated by transforming the plasmid into competent E. coli cells (see section 3.3.10). Following transformation, the plasmid was purified using the Thermo Scientific GeneJET Plasmid Miniprep kit (see section 3.3.11.1) and was stored at -20oC until future use. 3.3.13.3 Adaptation of pADH147 Plasmid pADH147 (#90991) was linearised with primer pair pADH147-1F and pADH147-1R (Table 2) using the reaction mixture and program of the KAPA HiFi PCR kit supplied in Tables 4 and 7, at an annealing temperature of 54oC. Following confirmation of the successful amplification with agarose gel electrophoresis (see section 3.3.6), the PCR product was digested with DpnI (New England Biolabs®Inc.), and subsequently, gel purified using the Thermo Scientific GeneJET Gel Extraction kit (see section 3.3.7). Next, the 3’-HIS1 region was amplified from the genome of C. krusei, using the same kit as above, at an annealing temperature of 62.6oC, with primer pair CK-3' HIS1-overlap-1F and CK-3' HIS1-overlap-1R. These two primers were designed to overlap with pADH147 plasmid to allow cloning of amplified 3’-HIS1 region into the plasmid. The successful amplification of the CK 3’-HIS1 fragment was confirmed using agarose gel electrophoresis; the amplicon was subsequently gel extracted and purified. The resulting purified amplicon was cloned into the linearised pADH147 fragment (Fig. 5) using the NEBuilder® HiFi DNA Assembly kit (see section 3.3.9), and transformed into competent E. coli cells (see section 3.3.10). Following transformation, plasmids were extracted from selected colonies using lysis by boiling protocol (see subsection 3.3.11.1), and were screened for anticipated inserts by double digests with Bgll and HindIII (Fermentas). Subsequently, anticipated plasmids were extracted, and purified from another group of selected colonies (suspected to be containing the anticipated plasmid based on the profile obtained after restriction digests with Bgll and HindIII) but this time with the Thermo Scientific GeneJET Plasmid Miniprep kit (see section 3.3.11.1). The purified plasmids were double-digested with Bgll and HindIII (Fermentas), and their profiles reconfirmed using gel electrophoresis (A reaction with no plasmid and one with the original pADH147 plasmid were included as negative and positive controls, respectively). Following this, the concentration of the purified plasmid CK pADH147 was checked, and the plasmid was stored at -20oC until future use. Page | 128 Fig. 5 A workflow for the preparation of CK pADH147 plasmid. First, the plasmid was amplified with two primers to linearise it and release C. albicans 3’-HIS1 region. Next, the linearised plasmid is cloned with amplified CK 5’-HIS1 fragment to create an intact CK pADH147 plasmid. NB: Shapes not drawn to scale. 3.3.14 Validation of the system Following the adaptation of the CRISPR-Cas9 plasmids for C. krusei, the efficacy of the system for gene-editing in this yeast was verified by targeting two auxotrophic marker genes, URA3 and ADE2. 3.3.14.1 Deletion of URA3 gene 3.3.14.1.1 Construction of CRISPR-Cas9 cassettes for the deletion of URA3 gene The Cas9 cassette (Fig. 6A) was liberated from CK pADH99 by digesting 2 μg concentration of the plasmid with restriction enzyme MssI (Thermo Scientific) (see section 3.3.8). Successful digestion was confirmed with agarose gel electrophoresis (see section 3.3.6). The first component (Fragment A) of the gRNA cassette was amplified from pADH110, with primer pair AHO1096-ver2 and AHO1098-ver2 (Table 2), at an annealing temperature of 53.8oC, using the reaction mixture and PCR program of KOD Hot Start DNA polymerase kit (Novagen®) (Tables 5, 8) (Fig. 6B). The second part (Fragment B, URA3-specific) of the gRNA cassette was amplified from CK pADH147 with oligonucleotide URA3-CRISPR-1 and primer AHO1097 with the same kit using touch down PCR (Fig. 6B). The URA3-CRISPR-1 oligo (alias URA3- specific gRNA oligo) contains a unique 20 bp CRISPR site or target sequence (5’- ATTGCGCAACACGATATGGG-3') complementary to a site present only within URA3 gene in the entire genome of C. krusei UFS Y-0217 (Douglass et al. 2018). The CRISPR site was Page | 129 identified with Geneious® 11.1.4, and was selected because it contains a protospacer adjacent motif (PAM) site, has high on-site activity score of 0.787 (Doench et al. 2014) and off-target activity score of 100% (Hsu et al. 2013). The 20 bp CRISPR site (without the PAM site) within the oligo is flanked by 5'-CGTAAACTATTTTTAATTTG-3' and 5'- GTTTTAGAGCTAGAAATAGC-3' complementary to 3' end of SNR52 promoter (within pADH110) and 5' end of gRNA scaffold (within CK pADH147), respectively, to enable the generation of the full URA3-gRNA cassette. Using stitching PCR, Fragments A and B were fused with primer pair AHO1237 and CK-3' HIS1-R, at an annealing temperature of 54.3oC to generate a complete gRNA cassette (Fragment C) (Fig. 6C). Successful amplification of these fragments was confirmed with agarose gel electrophoresis. Page | 130 Fig. 6 A flow chart of the steps followed to construct Cas9 and gRNA expression cassettes. (A) CK pADH99 plasmid is digested with restriction enzyme MssI to generate an intact Cas9 cassette (B) The 5' (Fragment A) and 3' (Fragment B) regions of the gRNA cassette are prepared from pADH110 and CK pADH147, respectively, by PCR with appropriate primers and oligonucleotide. Note that the gRNA oligo is customised for each target. (C) The complete gRNA expression cassette is generated via ligation of Fragments A and B with stitching PCR. Page | 131 3.3.14.1.2 Design and synthesis of URA3 donor DNA A full URA3 dDNA was designed as described in Figure 7. The first part (Fragment 1) of URA3 dDNA was amplified from the genome of C. krusei with primer pair URA3-2F and URA3-2R at an annealing temperature of 50.6oC using the reaction mixture and PCR program of KOD Hot Start DNA polymerase kit supplied in Tables 5 and 8. The second fragment (Fragment 2) of the dDNA was amplified with primers URA3-3F and URA3-3R, at an annealing temperature of 53.3oC with the same kit. Primers URA3-2R and URA3-3F share a 20 bp overlap sequence to aid ligation of the amplified fragments. Further, the full URA3 dDNA was synthesised by fusing the two overlapping fragments in a stitching PCR with primer pair URA3-2F and URA3- 3R, at an annealing temperature of 50.6oC. Correct amplification of all amplicons was checked with agarose gel electrophoresis. Fig. 7 A schematic representation of the steps followed to design a donor DNA. First, fragment 1 of the dDNA is amplified from the genome of Candida krusei with FP-1 (forward primer-1) and RP-1 (reverse primer-1). Next, fragment 2 is synthesised with FP-2 (forward primer- 2) and RP-2 (reverse primer-2). Finally, the two overlapping fragments are stitched with FP-1 and RP-2 to generate a full donor DNA. 3.3.14.1.3 Transformation of URA3-specific fragments into C. krusei The co-transformation of URA3 dDNA fragment, Cas9 cassette, and URA3-specific gRNA cassette into C. krusei was done following a modified protocol of Nguyen and co-workers (2017). Briefly, an overnight 5 ml YPD culture of C. krusei UFS Y-0217 was diluted in a fresh YPD broth at 1:50, incubated at 30oC with shaking and allowed to reach OD600 between 0.5 to 0.8. The cells were washed twice with milliQ water and re-suspended in 1/100 of the original volume. A 50 μl volume each of re-suspended cells, Cas9 cassette, full URA3-specific gRNA Page | 132 cassette, and complete URA3 dDNA fragment was mixed by gentle flicking with 1 ml plate mix [875 μl 50% PEG 3350 (Sigma-Aldrich), 100 μl 10X TE buffer, 25 μl 1 M Lithium acetate (adjusted to pH 7 with acetic acid; Sigma-Aldrich)] and incubated overnight at 30°C without shaking. In the following day, cells were heat-shocked (15 min, 44.6oC), washed with sterile YPD, allowed to recover (5 h at 30oC with shaking), plated onto YPD agar plate supplemented with 400 μg/ml NTC and incubated at 30oC for 2 to 3 days for colonies formation. 3.3.14.1.4 Selection and confirmation of ura3Δ/Δ mutant Following transformation, visible colonies were selected, and replica plated on a new YPD plate (+ 600 μg/ml NTC) and a minimal medium lacking uracil plate [1.7 g/l YNB w/o amino acids and ammonium sulphate, glucose 20 g/l, 0.6 g/l Complete Supplement Mixture (CSM) w/o His-Leu-Trp-Ura, 5 g/l ammonium sulphate, 0.2 g/l histidine, 1 g/l leucine, 0.2 g/l tryptophan, 17 g/l agar], and incubated at 30oC for 24 h (wildtype was included as control). Following incubation, colonies that showed no growth on the uracil-deficient medium upon comparison with the YPD plate were regarded as putative ura3Δ/Δ mutants. The genotype of these mutants was confirmed with PCR genotyping by amplifying the URA3 dDNA. Genomic DNA of the mutants was extracted (see subsection 3.3.5.2) and used as template. URA3 dDNA was amplified with primer pair URA3-2F and URA3-3R, using the PCR program and reaction component of KAPA Taq PCR kit. Further, the morphological properties of the mutant and wildtype colonies were compared. The microscopic differences between the mutant and wildtype cells were also analysed with light microscopy. 3.3.14.2 Deletion of ADE2 gene 3.3.14.2.1 Construction of CRISPR-Cas9 cassettes for the deletion of ADE2 gene The Cas9 cassette (Fig. 6A) was removed from plasmid CK pADH99 as earlier described (see 3.3.14.1.1). The first component (Fragment A) of the gRNA cassette was also prepared as previously described (Fig. 6B). However, this time, the second part (Fragment B) of the gRNA cassette was amplified from CK pADH147 with oligonucleotide ADE2-CRISPR-2 and primer AHO1097 (Fig. 6B). The ADE2-CRISPR-2 oligo (alias ADE2-specific gRNA oligo) contains a unique 20 bp target sequence (5'-TTAGGGTCTGATGTGCCAAA-3') complementary to a sequence within ADE2 in the genome of this yeast (Douglass et al. 2018). This CRISPR site was identified as described earlier and had on-target activity and off-target scores of 0.864 and 100%, respectively. Further, an intact gRNA cassette (Fragment C) was generated by ligating Fragments A and B with primer pair AHO1237 and CK-3' HIS1-R using stitching PCR (Fig. 6C). 3.3.14.2.2 Design and synthesis of ADE2 donor DNA A complete ADE2 dDNA was designed as illustrated in Figure 7. Briefly, the first part (Fragment 1) of ADE2 dDNA was amplified from the genome of C. krusei with primer pair Page | 133 ADE2-2F and ADE2-2R at an annealing temperature of 55.5oC using the reaction mixture and PCR condition of KOD Hot Start DNA polymerase kit. Using the same kit, the second fragment (Fragment 2) of the dDNA was amplified with primers ADE2-3F and ADE2-3R_new at an annealing temperature of 50.7oC. Primer pair ADE2-2R and ADE2-3F share a 20 bp overlap to aid fusion of the amplified fragments. Subsequently, an intact ADE2 dDNA was synthesised by ligating the two overlapping fragments in a stitching PCR with primer pair ADE2-2F and ADE2-3R_new at an annealing temperature of 50.7oC. Correct amplification of all amplicons was confirmed with agarose gel electrophoresis. 3.3.14.2.3 Transformation of ADE2-specific fragments into C. krusei The co-transformation of C. krusei with ADE2-specific gRNA cassette, ADE2 dDNA fragment, and Cas9 cassette was done as described earlier. Briefly, an overnight culture of C. krusei UFS Y-0217 diluted at 1:50 was incubated at 30oC with shaking, allowed to reach OD600 between 0.5 to 0.8, washed twice with milliQ water and re-suspended in 1/100 of the original volume. A 50 μl volume each of the re-suspended cells, Cas9 cassette, intact ADE2-specific gRNA cassette, and complete ADE2 dDNA fragment was gently mixed with 1 ml plate mix and incubated overnight at 30°C without shaking. Following this, cells were heat-shocked at 44.6oC for 15 min, washed with sterile YPD, allowed to recover at 30oC for 5 h with shaking, plated onto YPD agar plate supplemented with 400 μg/ml NTC and incubated at 30oC for 2 to 3 days for colonies formation. 3.3.14.2.4 Selection and confirmation of ade2Δ/Δ mutant Following transformation, visible colonies were selected and replica plated on a new YPD plate (+ 600 μg/ml NTC) and a minimal medium lacking adenine plate [1.7 g/l YNB w/o amino acids and ammonium sulphate, glucose 20 g/l, 5 g/l ammonium sulphate, 0.3 g/l isoleucine, 1.5 g/l valine, 0.2 g/l arginine, 0.3 g/l lysine, 0.2 g/l methionine, 0.5 g/l phenylalanine, 2 g/l threonine, 0.3 g/l tyrosine, 0.2 g/l uracil, 0.2 g/l histidine, 1 g/l leucine, 0.2 g/l tryptophan, 17 g/l agar] and incubated at 30oC for 24 h (wildtype was included as control). Following incubation, colony displaying reddish/pinkish phenotype on the adenine-deficient medium was considered ade2Δ/Δ mutant. PCR genotyping via the amplification of ADE2 dDNA with primers ADE2-2F and ADE2-3R was implemented to confirm the genotype of the mutant. Further, the macroscopic and microscopic properties of the mutant and wildtype colonies were juxtaposed with unaided eyes and light microscopy, respectively. 3.3.15 Removal of CRISPR-Cas9 cassette The CRISPR-Cas9 cassette was removed with growth in a maltose-supplemented media. Briefly, mutant cells were inoculated into a yeast extract peptone maltose (YPM) broth (10 g/l yeast extract, 20 g/l peptone, 20 g/l maltose) and were incubated at 30oC for 24 h with shaking. The resulting culture was diluted to 10-5 and plated onto YPD agar plates (30oC, 1 - 2 days). Page | 134 Colonies obtained were re-streaked onto YPD plates (+ 600 μg/ml NTC) and incubated for 24 h at 30oC to confirm the successful removal of the cassette. 3.4 Results and Discussions 3.4.1 Constructing a HIS-FLP type CRISPR-Cas9 system for C. krusei Due to its high precision, low cost, simplicity, high efficiency, and great adaptability relative to other gene-engineering tools, the CRISPR-Cas9 system has been harnessed at an explosive rate for gene-editing in virtually all cell types, including many fungi since its evolution as a genome-editing tool (Adli 2018; Román et al. 2019). However, this system has not been customised for use in C. krusei. The absence of a simple, precise, and efficient tool like CRISPR technology for gene-editing in this recalcitrant yeast has greatly hampered the full molecular understanding of its resistance mechanisms, knowledge which is important for the preservation of the current antifungal arsenal and development of novel therapeutic interventions. A functional CRISPR-Cas9 system was developed for C. krusei by adapting a system previously developed for use in C. albicans. In their study, Nguyen and co-workers (2017) designed two types of CRISPR systems, LEUpOUT and HIS-FLP. Both types utilise a recyclable CRISPR-Cas9 cassette and NAT marker and support homozygous genome editing in any strain of C. albicans that is NTC-sensitive. One bottleneck of the LEUpOUT system is that it can only be utilised in strains that are heterozygous for the LEU2 gene (i.e. LEU2/leu2Δ mutant in case of a diploid strain). Due to this limitation, and because of our intention to perform gene-editing in a diploid C. krusei strain (2n), we resorted to using the HIS-FLP system, which is suitable for any strain. This system relies upon the FLP/FRT recombination system and was designed to integrate at the HIS1 locus within the genome of C. albicans. The HIS-FLP system takes advantage of two cassettes; the Cas9 and gRNA cassettes. The Cas9 cassette is contained within the pADH99 plasmid and consists of the ENO1 promoter linked to the Cas9 gene. The gRNA cassette is contained within pADH110 and pADH147. The co- transformation of the Cas9 and gRNA cassettes generates an intact CRISPR-Cas9 cassette with a full NAT marker gene that integrates at the HIS1 locus of C. albicans by the 5’-HIS1 and 3’-HIS1 (integration) regions that flank the cassette. Following gene-editing by the RNA- guided Cas9 nuclease, the cassette is easily removed upon induction of the FLP recombinase with growth on a maltose-containing medium. However, to optimise and utilise this system for genome editing in C. krusei, it was necessary to replace some of its components with homologous fragments from this yeast. 3.4.1.1 Adapting pADH99 plasmid The pADH99 plasmid contains a C. albicans 5’-HIS1 integration region which, together with the 3’-HIS1 region, is necessary for the integration of the CRISPR-Cas9 cassette at the HIS1 Page | 135 locus of C. albicans; a Streptococcus pyogenes CAS9 gene under the control of a C. albicans ENO1 promoter; a part of the NAT marker gene, necessary for NTC-resistance (NTCR) together with an overlapping portion on pADH110; and an FRT sequence required for the removal of the cassette at a later stage (Fig. 8). Fig. 8 A plasmid map of pADH99 showing components such as C. albicans 5’-HIS1 region, flippase recognition target (FRT) region, CAS9 gene under the control of C. albicans ENO1 promoter, and an overlapping portion of the nourseothricin N-acetyltransferase (NAT) marker gene. The plasmid map also indicates the NcoI and SmaI restriction sites used to remove the C. albicans HIS1-FRT-ENO1 region. The customisation of the plasmid for use in C. krusei entailed replacing the C. albicans 5'- HIS1 and ENO1 promoter regions with 5'-HIS1 and ENO1 promoter regions from C. krusei. Substitution with C. krusei 5'-HIS1 region was necessary to allow the integration of the CRISPR system into the genome of C. krusei. Further, the ENO1 promoter of C. krusei was used to express the CAS9 gene since species-specific promoters have been demonstrated to optimise the activity of the Cas9 nuclease (Lombardi et al. 2017; Norton et al. 2017). The adaptation process started by double digesting the pADH99 with NcoI and SmaI restriction enzymes. This process linearised the plasmid (~9877 bp) and liberated the C. albicans HIS1-FRT-ENO1p region (~1245 bp) within it (Fig. 9). Page | 136 Fig. 9 Profile of pADH99 plasmid digested with NcoI and SmaI restriction enzymes. (A) Profile obtained after an in silico digestion analysis of the plasmid (Lane 1). (B) Profile obtained after digesting the plasmid in vitro (Lane 2). Expected band sizes of 9877 bp and 1245 bp that represent plasmid backbone and removed HIS1-FRT-ENO1 region, respectively, were obtained. M represents the DNA ladder. Lane 1 in the in vitro gel is empty. In order to replace the removed HIS1-FRT-ENO1p region, the 5'-HIS1 region was first amplified from the genome of C. krusei with primers pADH99-5' CK HIS1 overlap-F and CK- 5' HIS1-R. As depicted in Figure 10, the expected band size of ~522 bp was obtained. Moreover, the amplified product shares about 19 bp overlap sequence with pADH99 to aid its ligation into the plasmid. Next, the ENO1 promoter was amplified from the yeast’s genome with primer pair CK ENO1-F and pADH99-3'-CK CAS9 overlap. The expected band size of ~806 bp was obtained, as shown in Figure 11. Additionally, the amplicon generated shares a 25 bp overlap sequence with CAS9 gene to streamline cloning into the linearised pADH99 plasmid. Page | 137 Fig. 10 Amplification of 5'-HIS1 region of Candida krusei. (A) A map depicting the 5'-HIS1 region and primers used for its amplification. (B) A gel profile indicating successful amplification of the 5'-HIS1 region (~522 bp; Lanes 1 and 2). Fig. 11 Amplification of ENO1 promoter region of Candida krusei. (A) A map depicting the ENO1 promoter region and primers used for its amplification. (B) A gel profile showing successful amplification of the ENO1 promoter region (~806 bp; Lanes 1 and 2). Page | 138 Further, two overlapping oligonucleotides, pADH99::CK HIS1::FRT overlap and pADH99::CK FRT::ENO1 overlap were used to synthesise the FRT fragment of approximately 85 bp (Fig. 12). The synthesised fragment is flanked by regions homologous to the 3' region of CK 5’- HIS1 and 5' region of CK ENO1p fragments, respectively, necessary to link these two fragments. Fig. 12 Synthesis of flippase recognition target (FRT) fragment. (A) Schematic depicting the FRT region and oligonucleotides used for its polymerisation. Oligonucleotides, pADH99::CK HIS1::FRT overlap and pADH99::CK FRT::ENO1 overlap, overlap with CK 5’-HIS1 and CK ENO1p fragments, respectively (B) Gel profile indicating successful amplification of the FRT fragment (~85 bp; Lanes 1 and 3). After confirming the successful amplification of these fragments with agarose gel electrophoresis, they were purified and integrated into the linearised pADH99 with NEBuilder® HiFi DNA Assembly kit in a single reaction. Gel purification prior to assembly was essential for improved assembly and transformation efficiency. The complete construct was transformed into competent E. coli cells. Transformed colonies were screened for anticipated plasmid with restriction digest using XbaI and BamHI enzymes in two separate reactions. As depicted in Figures 13 and 14, plasmid profiles obtained after restriction digests revealed successful assembly and cloning, as well as the successful adaptation of pADH99 for use in C. krusei. The new construct was purified and denominated as CK pADH99. Page | 139 Fig. 13 Screening of transformants for CK pADH99 plasmid with XbaI restriction enzyme (A) Physical map of CK pADH99 depicting cut sites of XbaI restriction enzyme (not drawn to scale). (B) Restriction digest profile obtained in silico. (C) Profile obtained after in vitro restriction digest assay. Expected band sizes of 6757 bp, 2981 bp, and 1457 bp were obtained. M represents the DNA ladder. Fig. 14 Screening of transformants for CK pADH99 plasmid with BamHI restriction enzyme (A) Physical map of CK pADH99 depicting the cut sites of BamHI restriction enzyme (not drawn to scale). (B) Restriction digest profile obtained in silico. (C) Profile obtained after in vitro restriction digest assay. Expected band sizes of 3976 bp, 3132 bp, 3004 bp, and 783 bp were obtained. M represents the DNA ladder. Page | 140 3.4.1.2 Propagating pADH110 The pADH110 plasmid (Fig. 15) contains an overlapping portion of the NAT marker gene and a C. albicans SNR52 promoter that regulates the expression of gRNA. Although the RNA pol III SNR52 promoter of C. albicans is well characterised, identifying the uncharacterised SNR52 promoter of any organism is difficult, especially because it does not get translated into protein. This process could be partly simplified with the availability of transcriptome data to guide the promoter's search (Morio et al. 2020); however, such data is currently unavailable for C. krusei. Further, there is evidence that supports the usage of slightly-related species promoter to drive the expression of gRNA (Fuller et al. 2015; Matsu-ura et al. 2015). Because of this, we reasoned that the CRISPR system should work in C. krusei with the C. albicans SNR52 promoter; hence the promoter was not replaced. Instead, we propagated the pADH110 plasmid in competent E. coli cells to obtain enough concentration for further downstream experiments. Fig. 15 A map of pADH110 plasmid depicting an overlapping portion of the nourseothricin N- acetyltransferase (NAT) marker gene and SNR52 promoter (of gRNA). 3.4.1.3 Adapting pADH147 The pADH147 (Fig. 16) contains the gRNA scaffold, FRT sequence, and C. albicans 3’-HIS1 region which constitute the other half of a full gRNA cassette. As stated earlier, both the 5’- HIS1 region located on pADH99 plasmid and the 3’-HIS1 region are indispensable for the integration of the HIS-FLP system at the HIS1 locus. Because of this, we adapted the plasmid for use in C. krusei by replacing the 3’-HIS1 region of C. albicans with a homologous region from the HIS1 locus of C. krusei. Page | 141 Fig. 16 A map of pADH147 plasmid showing gRNA scaffold, flippase recognition target (FRT) region, and 3’- HIS1 region of C. albicans. Plasmid pADH147 was amplified with primer pair pADH147-1F and pADH147-1R, this linearised the plasmid, and also removed the C. albicans 3’-HIS1 region. The gel profile depicted in Figure 17B showing a band of approximately 2383 bp (Lane 2) confirms the plasmid's successful linearisation. The generated amplicon was subsequently treated with DpnI enzyme to remove plasmid template, and was gel purified for increased assembly and cloning efficiency. To replace the 3'-HIS1 fragment, a 3'-HIS1 region was amplified from the genome of C. krusei with primer pair CK-3' HIS1-overlap-1F and CK- 3' HIS1-overlap-1R. As shown in Figure 18B, an expected band size of approximately 551 bp was obtained. The generated amplicon shares overlaps with pADH147 to aid its cloning into the plasmid. Page | 142 Fig. 17 Linearisation of pADH147 (A) Physical map of pADH147 depicting the primers used for its linearisation (not drawn to scale). (B) Gel profile depicting successful linearisation of the plasmid (Lane 2; ~2383 bp). Lanes M and 1 represent the DNA ladder and negative control, respectively. Fig. 18 Amplification of 3’- HIS1 region of Candida krusei. (A) A map depicting the 3’- HIS1 region and primers used for its amplification. (B) A gel profile indicating successful amplification of the 3’-HIS1 region (~551 bp; Lanes 2 and 3). M represents the DNA ladder. Page | 143 The purified C. krusei 3'-HIS1 region was thereafter cloned into the linearised pADH147 plasmid with NEBuilder® HiFi DNA Assembly kit and transformed into competent E. coli cells. Transformed colonies were screened for anticipated plasmid by double digestion with Bgll and HindIII restriction enzymes. As depicted in Figure 19, band sizes of 1326 bp, 1081 bp, and 489 bp were obtained for lanes 3, 4, 5, and 6 which confirmed the success of the assembly and cloning procedures as well as the successful adaptation of the pADH147 for C. krusei. The new construct was purified and named as CK pADH147. Fig. 19 Screening of transformants for CK pADH147 plasmid with Bgll and HindIII restriction enzymes (A) Physical map of CK pADH147 depicting the cut sites of Bgll and HindIII restriction enzymes (not drawn to scale). (B) Restriction digest profile obtained in silico. (C) Profile obtained after in vitro restriction digest assay. Expected band sizes of 1326 bp, 1081 bp, and 489 bp were obtained (Lanes 3, 4, 5, and 6). M represents the DNA ladder. Lanes 1 and 2 represent negative and positive controls, respectively. 3.4.2 Validating the adapted system After successfully adapting the HIS-FLP plasmids for C. krusei, it was important to validate the efficacy of the system for genetic engineering in this yeast. This was done by harnessing auxotrophy. Auxotrophy is a technique that has been widely used for recombinant DNA research in molecular genetics. By definition, an auxotrophic marker gene is essential for growth, and its disruption or deletion would result in the inability of an auxotrophic mutant to grow in a medium lacking an essential nutrient (Pronk 2002; Yuan 2011). This consequently allows easy identification of mutants upon replica plating (Lederberg and Lederberg 1952). The deletion of an auxotrophic marker, such as the ADE2 gene, also produces a distinctive Page | 144 phenotype in the absence of an essential nutrient. Hence, the system's efficacy was tested by targeting two auxotrophic marker genes, namely URA3 and ADE2, for deletion. The deletion of any gene using the CRISPR-Cas9 system requires two main components, the Cas9 and gRNA. In our case, the CAS9 gene (encoding Cas9) is harboured within the Cas9 cassette located on the CK pADH99 plasmid, and the gRNA cassette is shared between pADH110 and CK pADH147. These two cassettes are co-transformed to generate a complete CRISPR-Cas9 cassette. Following the creation of double-strand breaks (DSBs) by the gRNA- guided CAS9 nuclease, the breaks are repaired via NHEJ or HDR upon provision of a dDNA. 3.4.2.1 Deleting URA3 gene A first approach to validate the efficacy of the adapted system involved targeting the URA3 gene for deletion. The URA3 gene encodes an enzyme known as orotidine 5'-monophosphate (OMP) decarboxylase, which plays a role in de novo pyrimidine ribonucleotide biosynthetic pathway – converts OMP to uridine monophosphate (UMP) (Fig. 20) (Losberger and Ernst 1989; Lay et al. 1998). The deletion or disruption of this gene consequently makes ura3 mutants auxotrophic for uracil (uridine) biosynthesis (Lacroute 1968). Fig. 20 A representation of de novo pyrimidine ribonucleotide biosynthetic pathway. The deletion or mutation of URA3 gene makes mutants become auxotrophic for uracil (uridine) biosynthesis (de Gontijo et al. 2014). 3.4.2.1.1 Constructing CRISPR-Cas9 cassettes for deleting URA3 gene To delete the URA3 gene, it was essential to first construct the CRISPR cassettes with respective plasmids. The Cas9 cassette (~8970 bp) was obtained from CK pADH99 by digesting the plasmid with MssI restriction enzyme (Fig. 21). Using the pADH110 plasmid as a template, the first component (Fragment A) of the gRNA cassette, containing the SNR52 promoter, was amplified with primer pair AHO1096-ver2 and AHO1098-ver2. As depicted in Figure 22, a band size of approximately 1066 bp obtained confirms this fragment's successful amplification. Page | 145 Fig. 21 Construction of Cas9 cassette (A) A schematic of Cas9 cassette digested from CK pADH99 with MssI restriction enzyme (B) Restriction digest profile obtained in silico. (C) Profile obtained after in vitro restriction digest assay. The expected band size of ~8970 bp (red box) represent the Cas9 cassette. Fig. 22 Construction of the first component (Fragment A) of gRNA cassette (A) A schematic of Fragment A amplified from pADH110 (B) Amplified product of Fragment A from pADH110. The expected band size of approximately 1066 bp was obtained. Next, the second component (Fragment B, ~722 bp) of the gRNA cassette which contains the gRNA scaffold was amplified from CK pADH147 with a target-specific oligonucleotide URA3- CRISPR-1 and primer AHO1097. The URA3-CRISPR-1 oligo contains a 20 bp URA3-specific CRISPR sequence (5'-ATTGCGCAACACGATATGGG-3') which corresponds to a site within Page | 146 the URA3 gene, which is targeted and nicked by the Cas9 nuclease (Fig. 23). This CRISPR sequence within the oligo is flanked by regions complementary to the 3' end of SNR52 promoter and 5' end of gRNA scaffold to enable ligation of Fragments A and B. Fragments A and B's successful ligation with stitching PCR generated an intact URA3-gRNA cassette (Fragment C) with a band size of approximately 1788 bp (Fig. 24). Fig. 23 Construction of the second component (Fragment B) of gRNA cassette specific for URA3 (A) A schematic of Fragment B amplified from CK pADH147. Grey box indicates CRISPR site specific for URA3 and overlapping regions with SNR52 promoter and gRNA (B) Amplified product of Fragment B from CK pADH147. Expected band size of approximately 722 bp was obtained (Red box). Fig. 24 Construction of complete URA3- specific gRNA cassette (A) A schematic representing the stitching of Fragments A and B to generate full URA3-gRNA cassette (B) Gel profile depicting full URA3-gRNA cassette of approximately 1788 bp. Page | 147 3.4.2.1.2 Designing and synthesising URA3 donor DNA For the repair of DSBs with the HDR pathway, a dDNA is required to replace the targeted gene. The study by Vyas and co-workers (2015) with C. albicans demonstrated that the provision of a dDNA is important for genome engineering with CRISPR-Cas9 after obtaining targeting efficiencies of 0% and up to 80% in the absence and presence of a dDNA, respectively. A dDNA with homology arms of about 150 to 500 bp complementary to the targeted gene's flanking regions was used to replace nearly the entire ORF of the gene. The dDNA was constructed by amplifying two fragments (150 – 500 bp) that flank the gene of interest and fusing them to generate a full dDNA (Fig. 7). Specifically, the first part (Fragment 1) of URA3 dDNA was amplified from the genome of C. krusei with primer pair URA3-2F and URA3-2R (overlap). An expected band size of approximately 209 bp was obtained (Fig. 25A). The second fragment (Fragment 2) of the dDNA was amplified with primer pair URA3-3F (overlap) and URA3-3R. An obtained band size of approximately 193 bp confirms this fragment's successful amplification (Fig. 25A). These two amplified fragments (Fragment 1 and 2) share an overlap sequence of 20 bp, when stitched with primers URA3-2F and URA3- 3R a full URA3 dDNA of approximately 382 bp was generated (Fig. 25B). Fig. 25 Synthesis of an intact URA3 donor DNA (dDNA) (A) Gel profile showing expected band sizes of 209 bp (red box) and 193 bp (yellow box) for Fragment 1 and Fragment 2, respectively, of URA3 dDNA (B) Gel profile depicting full URA3 dDNA of an expected band size of ~382 bp (Lanes 1 and 2). Page | 148 3.4.2.1.3 Transforming, selecting, and confirming ura3Δ/Δ mutant Upon co-transformation, into the yeast, the Cas9 cassette and the URA3-specific gRNA cassettes generate a complete CRISPR-Cas9 cassette which integrates at the HIS1 locus. Successful transformants are NTC-resistant (NTCR) accomplished by an intact NAT maker gene within the complete CRISPR-Cas9 cassette and were selected on YPD plates containing NTC. Out of 49 transformants obtained, eight were unable to grow on a minimal medium lacking uracil and were regarded as putative ura3Δ/Δ mutants (Fig. 26). Fig. 26 Uracil-deficient minimal medium plate with the transformed and wildtype colonies. Putative ura3Δ/Δ mutants showed no growth (black dotted boxes). Wildtype (green indication) and transformants with intact URA3 gene grew on the plate. PCR genotyping was performed, using the primers URA3-2F and URA3-3R, to confirm the genotype of the putative ura3Δ/Δ mutants obtained. As depicted in Figure 27, an expected band size of approximately 382 bp was obtained which confirms successful homozygous deletion of URA3 gene and replacement of the gene with the supplied URA3 dDNA in all the ura3Δ/Δ mutants. Homozygous URA3 mutants demonstrated a NTCR/URA3‒ phenotype as they were able to grow on plates containing NTC but unable on media lacking uracil. Page | 149 Fig. 27 Gel profile of representative ura3Δ/Δ mutants. All putative mutants that were unable to grow on uracil-deficient media show expected band size of approximately 382 bp (corresponding to the size of URA3 donor DNA, Yellow box). An expected band size of 1125 bp which correspond to the size of an intact URA3 gene was obtained for the wildtype (red box). Upon comparing the colonial morphology of the wildtype (C. krusei UFS Y-0217) and the mutant strains, we noticed that ura3Δ/Δ mutant appear smoother and less wrinkled compared to the wildtype strain (Fig. 28). This may suggest that the deletion or disruption of URA3 may have a detrimental effect on filamentation in this yeast. This observation was supported by fewer pseudohyphae observed microscopically for the mutant strain (Fig. 29). Interestingly, literature has also attributed reduced virulence and adherence to C. albicans mutants with disrupted URA3 gene (Kirsch and Whitney 1991; Bain et al. 2001; Staab and Sundstrom 2003). It is reasonable to suggest that the reduced virulence of C. albicans ura3 mutants might be due to distorted filamentation since hyphal formation is a known virulence factor (Lo et al. 1997; Berman and Sudbery 2002; Pukkila-Worley et al. 2009). However, virulence studies are needed before such a conclusion could be made for C. krusei ura3Δ/Δ mutant. Page | 150 Fig. 28 Comparison of the colonial morphology of ura3Δ/Δ mutant and wildtype strain. ura3Δ/Δ mutant appears smoother and less wrinkled compared to the wildtype strain. The yeast extract peptone dextrose (YPD) plate was inoculated with 10 μl of serially diluted culture of respective strain, 10-1 to 10-5 (top to bottom). Fig. 29 Microscopic comparison of the phenotype of ura3Δ/Δ mutant (B) and wildtype strain (A). A ura3Δ/Δ mutant appears more in yeast form compared to the wildtype strain, which displays more pseudohyphae. Light microscopy at 40X magnification. Page | 151 3.4.2.2 Deleting ADE2 gene As a second approach to validate the efficacy of our adapted system, ADE2 gene was targeted for deletion. This gene encodes P-ribosyl aminoimidazole carboxylase, which plays a role in de novo purine biosynthetic pathway. This enzyme converts P-ribosyl aminoimidazole (AIR) to P-ribosyl aminoimidazole carboxylate (CAIR) (Fig. 30) (Tsang et al. 1997). Mutation in the ADE2 gene results in the accumulation of AIR which turns red/pink upon oxidation thus allowing distinctive reddish phenotype of ADE2 mutants when grown in the absence of adenine (Poulter and Rikkerink 1983). Fig. 30 A representation of de novo purine biosynthetic pathway. The deletion or mutation of ADE2 gene results in the accumulation of P-ribosyl aminoimidazole, which turns pink upon oxidation (https://www.phys.ksu.edu/gene/genefaq.html). 3.4.2.2.1 Constructing CRISPR-Cas9 cassettes for deleting ADE2 gene The Cas9 cassette and the first component (Fragment A) of gRNA cassette are non-specific for the deletion of any gene using this system. However, the second part (Fragment B) of the gRNA cassette, which harbours the CRISPR site is specific for each gene and must be streamlined for each deletion. The Cas9 cassette was obtained by digesting CK pADH99 plasmid with MssI restriction enzymes (Fig. 21). The first component (Fragment A) of the gRNA cassette (~1066 bp) was obtained upon amplifying pADH110 with primers AHO1096- ver2 and AHO1098-ver2 (Fig. 22). The target-specific part (Fragment B) of the gRNA was amplified from CK pADH147 with oligonucleotide ADE2-CRISPR-2 and primer AHO1097. The ADE2-CRISPR-2 oligo contains a 20 bp ADE2-specific target sequence (5’- TTAGGGTCTGATGTGCCAAA-3') (Fig. 31). This sequence is flanked by regions complementary to the 3' and 5' ends of SNR52 promoter and gRNA scaffold, respectively, to Page | 152 aid ligation of Fragments A and B. Upon ligation in a stitching PCR with primer pair AHO1237 and AHO1236, a full ADE2-gRNA cassette (Fragment C) with a band size of approximately 1788 bp was obtained (Fig. 32). Fig. 31 Construction of the second component (Fragment B) of gRNA cassette specific for ADE2 gene (A) A representation of Fragment B amplified from CK pADH147. The grey box indicates ADE2-specific CRISPR site and overlapping regions with SNR52 promoter and gRNA (B) Amplified product of Fragment B from CK pADH147. Expected band size of approximately 722 bp was obtained. Fig. 32 Construction of complete ADE2-specific gRNA cassette (A) A schematic depicting the stitching of Fragments A and B to generate a full ADE2-gRNA cassette (B) Gel profile depicting full ADE2-gRNA cassette of approximately 1788 bp. Page | 153 3.4.2.2.2 Designing and synthesising ADE2 donor DNA An intact ADE2 dDNA was synthesised in a similar way to that of URA3, but with a different set of primers (Fig. 7). The first component (Fragment 1) of ADE2 dDNA was amplified from the yeast’s genome with primers ADE2-2F and ADE2-2R (overlap). As shown in Figure 33A, a band size of approximately 183 bp was obtained, which confirms the successful amplification of Fragment 1. Again, with the gDNA as a template, the second part (Fragment 2) of ADE2 dDNA with a size of 214 bp was generated, with primer pair ADE2-3F (overlap) and ADE2- 3R_new (Fig. 33A). The two generated overlapping fragments were stitched together with primers ADE2-2F and ADE2-3R_new, and an intact ADE2 dDNA with an expected size of approximately 377 bp was obtained (Fig. 33B). Fig. 33 Synthesis of intact ADE2 donor DNA (dDNA) (A) Gel profile showing expected band sizes of 183 bp (red box) and 214 bp (yellow box) for Fragment 1 and Fragment 2, respectively, of ADE2 dDNA (B) Gel profile depicting complete ADE2 dDNA of an expected band size of ~377 bp (Lanes 1 and 2). 3.4.2.2.3 Transforming, selecting and confirming ade2Δ/Δ mutant Upon co-transformation into C. krusei, the Cas9 cassette and the ADE2-specific gRNA cassettes generate a full CRISPR-Cas9 cassette – with an intact NAT marker gene, which integrates at the HIS1 locus in this yeast’s genome. Transformants were selected on YPD plates containing NTC. Out of 10 transformed colonies obtained, only one was an ade2Δ/Δ mutant evident of a distinctive red phenotype it displays on a minimal medium lacking adenine (Fig. 34). The homozygous ADE2 mutant demonstrated a NTCR/ADE- phenotype as it was able to grow on a plate supplemented with NTC and accumulated pink/reddish colour on plates lacking adenine. Page | 154 Fig. 34 Adenine-deficient minimal medium plate showing growth of ade2Δ/Δ mutant. Mutant displays a distinctive reddish/pinkish colony. Labels 1 and 2 represent ade2Δ/Δ mutant without and with CRISPR-Cas9 cassette, respectively. Labels 3 and 4 represent transformant with intact ADE2 gene and wildtype, respectively. PCR genotyping was also performed, using the primers ADE2-2F and ADE2-3R_new, to confirm the genotype of the mutant. As shown in Figure 35, the band size obtained for the mutant strain is comparable to that of a white transformant and a wildtype. This observation indicates that, in the case of the ADE2 gene, the DSBs created by the Cas9 nuclease were repaired via NHEJ instead of HDR. The reason why the organism chose NHEJ over HDR pathway to repair the nicked ADE2 gene is unclear since a dDNA was co-transformed into the yeast alongside the CRISPR-Cas9 cassettes. This could be circumvented by inactivating genes, such as KU70 and LIG4, which are essential for the NHEJ pathway (Norton et al. 2017; Weninger et al. 2017). However, care must be taken when performing pathogenesis and virulence studies since strains with defective NHEJ may exhibit reduced virulence (Wang et al. 2016). Page | 155 Fig. 35 Gel profile of ade2Δ/Δ mutant (Lane 2), wildtype (Lane 1), and white transformant (Lane 3). An ade2Δ/Δ mutant display an unexpected band size of 1963 bp which corresponds to that of the wildtype and white transformant. This indicates that the double- strand break induced by Cas9 was repaired via non-homologous end joining (NHEJ) instead of homology-directed repair (HDR) pathway. Furthermore, in addition to the red phenotype exhibited by the ade2Δ/Δ mutant, its colony morphology also appears smoother and shows less filamentation (Fig. 36), an observation which is similar to what was observed for ura3Δ/Δ mutant. Microscopic analysis also revealed that most cells of the ade2Δ/Δ mutant are in the yeast form (Fig. 37). Altogether, these observations suggest that the disruption of ADE2 disruption may affect filamentation in this yeast. Additionally, previous studies with C. albicans have reported a reduced virulence of ade2Δ/Δ mutants (Kirsch and Whitney 1991; Donovan et al. 2001), which may possibly be attributed to diminished filamentation since hypha is an important virulence factor (Lo et al. 1997; Berman and Sudbery 2002; Pukkila-Worley et al. 2009). Page | 156 Fig. 36 Comparison of the colonial morphology of ade2Δ/Δ mutant and wildtype strain. ade2Δ/Δ mutant appears round, less wrinkled, and smoother compared to the wildtype strain. The yeast extract peptone dextrose (YPD) plate was inoculated with 10 μl of serially diluted culture of respective strain, 10-1 to 10-5 (top to bottom). Fig. 37 Microscopic comparison of the phenotype of ade2Δ/Δ mutant (B) and wildtype strain (A). ade2Δ/Δ mutant appear predominantly in yeast form. Light microscopy at 40X magnification. Page | 157 3.4.3 Removing CRISPR-Cas9 cassette Following confirmation of the genotypes of the mutants, the CRISPR-Cas9 cassette with the NAT marker was removed by inducing the FLP recombinase with growth in maltose- supplemented media. Mutants that have released the CRISPR-Cas9 cassette from their genomes consequently displayed a NTCS phenotype, and were unable to grow on NTC- supplemented media (Fig. 38). Fig. 38 Successful excision of CRISPR-Cas9 cassette from the mutants’ genome. After cassette removal, mutants displayed a nourseothricin- sensitive (NTCS) phenotype and could not grow on media containing nourseothricin. Page | 158 3.4.4 The complete system This system consists of a CAS9 gene (from S. pyogenes) – under the control of C. krusei ENO1 promoter, which is carried on a Cas9 cassette; and a gRNA (under the control of SNR52 promoter) – harboured within a gRNA cassette and adaptable to target any locus within C. krusei genome. Upon co-transformation, these two cassettes generate a complete CRISPR- Cas9 cassette, accomplished by homologous recombination between the overlapping NAT marker elements, integrated at the HIS1 locus (Fig. 39A). Upon expression of the CAS9 and gRNA, the Cas9 nuclease guided by the target-specific gRNA generates DSBs at the desired locus within the genome of C. krusei. The breaks are repaired via either NHEJ or HDR with a customised donor DNA (Fig. 39B). Successful transformants exhibit a NTCR phenotype fulfilled by the full NAT maker gene present on the intact CRISPR-Cas9 cassette. Following gene-editing, the CRISPR-Cas9 cassette (with NAT marker) is removed by growth on maltose- containing media, accomplished by the expression of flippase (FLP) recombinase gene and recombination between the FLP recognition target (FRT) sequences (Fig. 39C). As proof of principle, this system's efficacy for gene-editing in C. krusei was validated by the homozygous deletion of two auxotrophic marker genes, URA3 and ADE2. One advantage of the adapted HIS-FLP system for C. krusei is that; in contrast to that of Nguyen and co-workers (2017) which integrates at, and replaces one allele of the HIS1 ORF of C. albicans – which might consequently affect histidine biosynthesis, it was designed to integrate downstream of the HIS1 ORF in the genome of C. krusei to avoid displacing the histidine biosynthetic gene. Page | 159 Fig. 39 Schematic representation of the complete CRISPR-Cas9 system used for gene-editing in Candida krusei. (A) Upon co-transformation of the Cas9 and gRNA cassettes, an intact CRISPR-Cas9 cassette is obtained which integrate at the HIS1 locus. (B) Upon expression of the gRNA and Cas9 protein, the gRNA- guided Cas9 generates a double- strand break at the target locus within the genome of C. krusei which is repaired via homology-directed repair with the supplied dDNA. (C) Following gene-editing, the CRISPR system is excised from the genome of the yeast via induction of the FLP- recombinase with growth in a maltose supplemented medium (Adapted from Fourie 2020). Page | 160 3.5 Conclusions With intrinsic resistance to FLC and increased non-susceptibility to other antifungal drugs, C. krusei represents a potential multidrug-resistant yeast (Whaley et al. 2017; Jamiu et al. 2020). However, the absence of an efficient, facile, fast, and precise tool like CRISPR technology for gene-editing in this yeast has greatly precluded full molecular insights into its mechanisms of resistance. Knowledge which is vital to preserve the current antifungal drugs and inspire the development of novel therapeutic strategies. In this chapter, we have demonstrated the development of a CRISPR-Cas9 system for genome engineering in C. krusei by adapting a C. albicans-specific system designed by Nguyen and co-workers (2017). As proof of concept, this system's efficacy was validated by successfully deleting URA3 and ADE2 auxotrophic marker genes. However, the system's engineering efficiency was not very high as expected – 16% and 10% for URA3 and ADE2 deletion, respectively. This might be because the CAS9 gene was not codon-optimised for expression in C. krusei (Weninger et al. 2016; Raschmanová et al. 2018). 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Hence, duplication of some information could not be entirely avoided. Page | 170 4.1 Abstract With innate resistance to fluconazole (FLC) and rapid acquired resistance to other antifungal drugs, Candida krusei represents a potential multidrug-resistant pathogen. The mechanism of intrinsic FLC resistance in this yeast has been chiefly attributed to decreased susceptibility of FLC target, lanosterol 14α-demethylase (Erg11p), however, the role of efflux pump transporters remains controversial, and requires further investigation. Furthermore, although the overexpression of these transporters, including Abc1p, results in a multidrug-resistance phenotype, their inhibitors are limited. There is currently no class of antifungal that specifically targets these transporters. Polyunsaturated fatty acids (PUFAs), including arachidonic acid (AA), which are known disruptors of the cellular membranes might function well as effective efflux pump inhibitors; however, this needs to be investigated. Hence, this study attempted to examine the influence of AA and FLC on the expression, localisation, and activity of a representative ABC transporter, Abc1p, in C. krusei. This was carried out by attempting to construct a Green Fluorescent Protein (GFP) fusion of Abc1 and exposing C. krusei biofilms to varying concentrations of AA and FLC alone or in combination; and determining Abc1p expression and function using western blot analysis and Rhodamine 6G efflux assay, respectively. Although the Abc1-GFP fusion construction was unsuccessful, our results demonstrate that Abc1p is overexpressed following exposure to FLC alone, but not in any treatments with AA. Further, Abc1p exhibited increased functionality in the presence of FLC; however, this was diminished upon exposure to 1 mM AA, either alone or in combination with FLC. These findings demonstrate AA as a potential inhibitor of Abc1p expression and subsequent activity and lent credence to the importance of this transporter in FLC resistance. Keywords: Candida krusei, fluconazole, resistance, arachidonic acid, efflux pumps, Abc1 transporter Page | 171 4.2 Introduction The yeast Candida krusei is a potential multidrug-resistant pathogen with intrinsic resistance to FLC and rapid adaptive resistance to other antifungal drugs, including the echinocandins (Forastiero et al. 2015; Whaley et al. 2017; Jamiu et al. 2020). The key mechanisms of antifungal resistance in this yeast and other pathogenic Candida spp. include overexpression of target proteins (e.g. Erg11p); alteration of drug targets (e.g. Fks1p); and reduction in intracellular drug concentration, due to cell membrane modification, biofilm formation, or enhanced efflux pump activity (Mukhopadhyay et al. 2002; Sanglard and Odds 2002; Mansfield et al. 2010; Pappas et al. 2016; Robbins et al. 2017). The mechanism of intrinsic resistance to FLC in C. krusei has been chiefly attributed to reduced sensitivity of Erg11p towards FLC (Venkateswarlu et al. 1997; Orozco et al. 1998; Fukuoka et al. 2003). However, the role of efflux pump transporters, including Abc1p (the homolog of Cdr1p in C. albicans), in this inherent resistance cannot be dismissed. The Cdr (Candida drug resistance) transporters are members of the ABC transporter family and are capable of conferring resistance to all azole drugs (Prasad et al. 1995). Amongst these transporters, Cdr1 and Cdr2 are the most clinically relevant (Perea et al. 2001; Prasad et al. 2015). The homologs of these proteins in C. krusei are Abc1p and Abc2p, respectively, and their overexpression has been attributed to resistance to azole antifungals in this yeast. Specifically, the resistance mechanism of C. krusei to itraconazole and voriconazole has been partly attributed to the overexpression of Abc1 and Abc2 transporters (Tavakoli et al. 2010; Ricardo et al. 2014; He et al. 2015). However, these transporters’ role, particularly Abc1, in the intrinsic mechanism of C. krusei remains unclear. A study by Katiyar and Edlind (2001) demonstrated the upregulation of ABC1 in the presence of other azoles, such as miconazole and clotrimazole, but not in the presence of FLC. Similarly, another report suggested that no ABC transporters are involved in the intrinsic resistance of C. krusei (Guinea et al. 2006). However, a later study by Lamping and co- workers (2009) demonstrated that the constitutive expression of Abc1p and low affinity of Erg11p are responsible for FLC resistance in C. krusei. Hence, these conflicting reports warrant further studies. Furthermore, despite their crucial roles in antifungal resistance, there is currently no antifungal class available that specifically targets the efflux pumps. One approach to discovering novel efflux pump inhibitors is through the characterisation of efflux pump-inhibiting potential of natural compounds with known antimicrobial properties, including polyunsaturated fatty acids (PUFAs). Recent studies in our group have shown that a PUFA, arachidonic acid (AA), induces the upregulation of the CDR1 gene and increases Cdr1 protein production in C. albicans. However, the activity of this transporter was severely diminished in the presence of Page | 172 AA in the same yeast (Fourie 2020; Kuloyo 2020). The observed dissipated activity of Cdr1p was attributed to various factors, including possible mislocalisation of the Cdr1 transporter, reduced mitochondrial activity, and competitive inhibition. However, it is unknown if these phenomena are conserved amongst other Candida spp., including C. krusei. On this background, this study was conceptualised to determine the influence of AA and FLC on the localisation, expression, and activity of Abc1p in C. krusei to better understand the role of this efflux pump in antifungal resistance. 4.3 Materials and Methods 4.3.1 Strains used Two strains of Candida krusei, C. krusei UFS Y-0217 (CBS573T) and C. krusei UFS Y-0277 were used in this study. These strains were obtained from the Yeast Culture Collection of the University of the Free State, Bloemfontein, revived on Yeast Malt extract (YM) agar plates (10 g/l glucose, 3 g/l yeast extract, 3 g/l malt extract, 5 g/l peptone, 17 g/l agar) at 30oC for 24 h, and their stocks were stored at -80oC for future use. 4.3.2 Drug and fatty acids Fluconazole (FLC) was obtained from Sigma-Aldrich (St. Louis, MO, USA), a stock of 5 mg/ml was prepared in dimethyl sulfoxide (DMSO) and stored at -20oC. Arachidonic Acid (AA) (20:4) was also obtained from Sigma-Aldrich (St. Louis, MO, USA) and a 10 mM stock was prepared in ethanol and stored at -20oC. 4.3.3 Construction of ABC1-GFP mutant with CRISPR-Cas9 system The CRISPR-Cas9 system adapted for genome engineering in C. krusei in Chapter 3 was used to attempt the insertion of Green Fluorescent Protein (GFP) sequence at the 3’ end of ABC1 gene to construct an ABC1-GFP mutant of C. krusei, in order to assess its fluorescence (localisation and expression) under various treatment conditions. The description of the adapted plasmids used for this purpose is depicted in Table 1. Design of primers and fragments, simulation of polymerase chain reaction (PCR), and identification of CRISPR sites were done in silico with Geneious® 11.1.5 (www.geneious.com) prior to all in vitro and in vivo assays. Unless stated otherwise, PCR was done with KOD Hot Start DNA polymerase kit (Novagen®) using the reaction mixture and PCR condition supplied in Tables 2 and 3, respectively. All primers used were purchased from Integrated DNA Technologies and are listed in Table 4. Page | 173 Table 1 Plasmids used in this study Plasmid Major component Function CK pADH99 5' CK HIS1 locus + CK ENO1 promoter + CAS9 gene + Cas9 cassette ½ NAT gene pADH110 2/2 NAT gene + Ca SNR52 promoter ½ gRNA cassette CK pADH147 gRNA scaffold + 3' CK HIS1 locus 2/2 gRNA cassette Table 2 Reaction mixture for KOD Hot Start DNA polymerase kit (Novagen®) Component Reaction mixture Nuclease-free water Up to 25 μl 10X Buffer for KOD Hot Start DNA Polymerase 2.5 μl 25 mM MgSO4 1.5 μl dNTP Mix (2 mM) 2.5 μl Forward primer (10 μM) 0.75 μl Reverse primer (10 μM) 0.75 μl KAPA Taq DNA polymerase (5 U/μl) 0.5 μl Template DNA (less complex DNA)* ≤10 ng * tenfold excess for genomic DNA Table 3 PCR condition for KOD Hot Start DNA polymerase kit (Novagen®) Step Target size Cycle < 500 bp 500 – 1000 bp 1000 – 3000 bp > 3000 bp Polymerase 95oC, 2 min 95oC, 2 min 95oC, 2 min 95oC, 2 min 1 activation Denaturation 95oC, 20 sec 95oC, 20 sec 95oC, 20 sec 95oC, 20 sec 30 Annealing Lowest Primer Tm oC, 10 sec Extension 70oC, 10 sec/kb 70oC, 15 sec/kb 70oC, 20 sec/kb 70oC, 25 sec/kb 1 Page | 174 Table 4 Primers used in this study Primer Sequence (5' to 3') Tm (oC) Description Reference AHO1096-ver2 GACGGCACGGCCACGCGTTTAAAC 65.1 Forward primer for the amplification of Modified fragment A (5’ region) of gRNA cassette from Nguyen et al. 2017 AHO1098-ver2 CAAATTAAAAATAGTTTACGCAAGTCTCG 53.8 Reverse primer for the amplification of Modified fragment A (5’ region) of gRNA cassette from Nguyen et al. 2017 CK-ABC1- CGTAAACTATTTTTAATTTGATATATCTGTGTTACCAAAAGTTTTAGAGCTAGAAATAGC 61.8 ABC1 specific oligo and forward primer for This study CRISPR-1 the amplification of fragment B (3’ region) of gRNA cassette AHO1097 CCCGCCAGGCGCTGGGGTTTAAACACCG 70.2 Reverse primer for the amplification of Nguyen et fragment B (3’ region) of gRNA cassette al. 2017 AHO1237 AGGTGATGCTGAAGCTATTGAAG 55.0 Forward primer for stitching fragments A Nguyen et and B to obtain full gRNA cassette al. 2017 CK-3' HIS1-R ACTAGACAAGCGAGTTTGCA 54.3 Reverse primer for stitching fragments A This study and B to obtain full gRNA cassette CK-ABC1-1F GGAGAAAACTGCCAGACGTT 55.4 Forward primer for the amplification of This study fragment 1a of ABC1-GFP donor DNA (dDNA) CK-ABC1-1R TGCTGGAGCACTCTTTTGGTAACACAGATA 61.1 Reverse primer for the amplification of This study fragment 1a of ABC1-GFP dDNA (Has overlap sequence with CK-ABC1-2F) CK-ABC1-2F ACCAAAAGAGTGCTCCAGCAGTACTATCG 61.4 Forward primer for the amplification of This study fragment 1b of ABC1-GFP dDNA (Has overlap sequence with CK-ABC1-1R) Page | 175 CK-ABC1-2R TGCTTCAGAAGTAGTTTGTGG 52.7 Reverse primer for the amplification of This study fragment 1b of ABC1-GFP dDNA GFP-1-F ATGTCTAAAGGTGAAGAATTATTCA 50.4 Forward primer for the amplification of Fourie 2020 fragment 2a of ABC1-GFP dDNA CK-ABC1-GFP- TTATTTGTACAATTCATCCATACC 56.3 Reverse primer for the amplification of This study overlap-R fragment 2a of ABC1-GFP dDNA (Has overlap sequence with CK-GFP-ABC1- overlap-F) CK-GFP-ABC1- GTACAAATAAGAAGTTGCTTTTTTGACTAT 53.2 Forward primer for the amplification of This study overlap-F fragment 2b of ABC1-GFP dDNA (Has overlap sequence with CK-ABC1-GFP- overlap-R) CK-ABC1-3R CTGAACTCTTGGATCATTTCAAC 51.9 Reverse primer for the amplification of This study fragment 2b of ABC1-GFP dDNA CK- TCCACAAACTACTTCTGAAGCATCTGGTGCCGGTGCCGGTGCCGGTGCCGGTGCCATTTTG 74.6 Forward primer for the polymerisation of This study ABC1::SPACER linker region (fragment 3) between ABC1 and GFP sequence (overlaps with CK- ABC1-2R) SPACER-GFP-1R TGAATAATTCTTCACCTTTAGACATCAAAATGGCACCGGCACCGGCACCGGCACCGGCACCAGA 73.4 Reverse primer for the polymerisation of Fourie 2020 linker region (fragment 3) between ABC1 and GFP sequence (overlap with GFP-1-F) NB: Target-specific CRISPR site is underlined Page | 176 4.3.3.1 CRISPR-Cas9 cassettes for ABC1-GFP fusion The first component (Fragment A) of the gRNA cassette, containing the SNR52 promoter, was amplified from pADH110 with primer pair AHO1096-ver2 and AHO1098-ver2 (Table 4), at an annealing temperature of 53.8oC, using the reaction mixture and PCR program of KOD Hot Start DNA polymerase kit (Novagen®) (Tables 2, 3). To introduce a CRISPR site specific for ABC1 gene into the gRNA cassette, ABC1 sequence with about 500 bp upstream and downstream were extracted from the genome of C. krusei UFS Y-0217 (CBS573T) (Douglass et al. 2018). ABC1 gene exists as an ABC11-ABC1 tandem with ABC11 in this strain’s genome and shares about 99% nucleotide identity with this gene. A CRISPR site specific for ABC1 was only identified following the alignment of the nucleotide sequences of the two genes (Fig. 1). The CRISPR site (5'-ATATATCTGTGTTACCAAAAGGG-3') was retrieved with Geneious® 11.1.5, and was selected because it contains a protospacer adjacent motif (PAM) site, has high on-site activity score of 0.648 (Doench et al. 2014) and off-target activity score of 100% (Hsu et al. 2013). The flanking sequences 5'-CGTAAACTATTTTTAATTTG-3' and 5'- GTTTTAGAGCTAGAAATAGC-3', complementary to the 3' end of SNR52 promoter (on pADH100) and 5' end of gRNA scaffold (on CK pADH147), respectively, were added to the 5' and 3' regions of the 20 bp CRISPR site without the PAM site (5'- ATATATCTGTGTTACCAAAA-3') to obtain CK-ABC1-CRISPR-1 oligonucleotide (Table 4). This oligo was used alongside primer AHO1097 to obtain the 3' region (Fragment B) of gRNA cassette by amplifying CK pADH147 (Chapter 3, Fig. 6B). Using stitching PCR, Fragments A and B were fused with primer pair AHO1237 and CK-3' HIS1-R, at an annealing temperature of 54.3oC, to generate an intact gRNA cassette (Fragment C) (Chapter 3, Fig. 6C). Lastly, the Cas9 cassette was liberated from CK pADH99 by digesting 2 μg concentration of this plasmid with restriction enzyme MssI (Thermo Scientific) (Chapter 3, Fig. 6A). Successful amplification and restriction digest of all fragments were confirmed with agarose gel electrophoresis. Page | 177 Fig. 1 Nucleotide sequence alignment of selected regions of C. krusei ABC1 and ABC11 genes. Using Clustal Omega tool (www.ebi.ac.uk) ABC1 nucleotide sequence was aligned with that of ABC11. The two sequences share up to 99% identity. Highlighted sequence indicate ABC1-specific CRISPR site. 4.3.3.2 Design of ABC1-GFP fusion donor DNA The ABC1-GFP donor DNA (dDNA) was designed to consist of GFP sequence flanked by regions complementary to the ABC1 gene (Fig. 2). This fragment (dDNA) was constructed with three components (fragments 1, 2, and 3). The first fragment (fragment 1) is a 1290 bp sequence from the 3’ end of ABC1 gene (without the stop codon); the 5' region (fragment 1a) of this fragment (representing the 5' end of the dDNA) was amplified from the genome of C. krusei with primers CK-ABC1-1F and CK-ABC1-1R, at an annealing temperature of 55.4oC. The 3' region (fragment 1b) of this fragment modified to remove the CRISPR site was also amplified from this yeast’s genome with primer pair CK-ABC1-2F and CK-ABC1-2R, at an annealing temperature of 52.7oC. The two fragments (fragments 1a and 1b) were stitched together with primers CK-ABC1-1F and CK-ABC1-2R, at an annealing temperature of 52.7oC, to obtain an intact fragment 1. The second component (fragment 2) of the dDNA is made up of GFP sequence (with stop codon) at its 5' end and a 231 bp sequence downstream the ABC1 gene (representing the 3’ end of the dDNA) at its 3’ end. The GFP region of this fragment Page | 178 (fragment 2a) was amplified from Clp10-URA3-PMA1-GFP construct (Fourie 2020) with primer pair GFP-1-F and CK-ABC1-GFP-overlap at an annealing temperature of 50.4oC, while its 3' region (fragment 2b) was amplified from the genome of C. krusei with primers CK-GFP- ABC1-overlap-F and CK-ABC1-3R, at an annealing temperature of 51.9oC. The primers CK- ABC1-GFP-overlap and CK-GFP-ABC1-overlap-F contain a 20 bp overlap sequence to aid stitching. The two fragments (fragments 2a and 2b) were subsequently stitched with primers GFP-1-F and CK-ABC1-3R at an annealing temperature of 50.4oC to obtain an intact fragment 2. The last component of the dDNA, fragment 3, of approximately 86 bp was polymerised using two overlapping oligonucleotides, CK-ABC1::SPACER and SPACER-GFP-R. This fragment consists of a 39 bp sequence, for 13 amino acid linker (SGAGAGAGAGAIL) (Janke et al. 2004), flanked by regions complementary to the 3' and 5' ends of fragments 1 and 2, respectively, to enable the fusion of ABC1 and GFP, and facilitate proper folding of GFP (Janke et al. 2004). Lastly, fragments 1, 2, and 3 were ligated using the NEBuilder® HiFi DNA Assembly kit to generate the full ABC1-GFP dDNA. Following assembly reaction, primers CK- ABC1-1F and CK-ABC1-3R were used to confirm the correct ligation of these fragments. Fig. 2 A section of a vector map depicting the components of ABC1-GFP donor DNA. The donor DNA is flanked by sites homologous to regions within the ABC1 locus (Vector map generated using Geneious® 11.1.5). Page | 179 4.3.3.3 Transformation This was done following a modified protocol of Nguyen and co-workers (2017). Briefly, an overnight 5 ml YPD culture of C. krusei UFS Y-0217 was diluted in a fresh YPD broth at 1:50, incubated at 30oC with shaking, allowed to reach OD600 of 0.5 to 0.8, washed twice with milliQ water, and re-suspended in 1/100 of the original volume. A 50 μl volume of the re-suspended cells, Cas9 cassette, intact gRNA cassette, and complete dDNA fragment was mixed by gentle flicking with 1 ml plate mix [875 μl 50% PEG 3350 (Sigma-Aldrich), 100 μl 10X TE buffer, 25 μl 1 M Lithium acetate (adjusted to pH 7 with acetic acid; Sigma-Aldrich)], and incubated overnight at 30°C without shaking. In the following day, cells were heat-shocked (15 min, 44.6oC), washed with sterile YPD, allowed to recover (5 h at 30oC with shaking), plated onto YPD agar plate supplemented with 400 μg/ml nourseothricin (NTC), and incubated at 30oC for 2 to 3 days for colonies formation. Colony PCR was performed to confirm the integration of ABC1-GFP dDNA at the ABC1 loci with primers CK-ABC1-1F and CK-ABC1-3R. 4.3.4 Influence of arachidonic acid and fluconazole on Abc1p expression The influence of AA and FLC on the expression of Abc1 protein was assessed using SDS- PAGE and western blot analyses. 4.3.4.1 Biofilm formation Biofilm of C. krusei UFS Y-0277 strain exposed to various treatments was formed with a few modifications of previous methods (Ramage et al. 2001; Mishra et al. 2014). Briefly, a loopful of cells of this yeast was inoculated into a 5 ml sterile Yeast Nitrogen Base (YNB) broth (10 g/l glucose, 6.7 g/l YNB), and incubated for 24 h at 30oC. Cells were harvested and washed twice with sterile Phosphate Buffered Saline [PBS; 10 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM sodium chloride (pH 7.4) (Sigma-Aldrich, St. Louis, MO, USA)] by centrifugation at 3000 x g for 5 min (Eppendorf, Germany). Accordingly, cells were re- suspended in a 5 ml sterile PBS, counted with a haemocytometer and standardised to a final concentration of 1.0 x 107 cells/ml in a 5 ml filter sterilised YNB broth. A volume of 2 ml of the standardised cell suspension was dispensed into the wells of 6-well plates (Thermo Scientific, Denmark), and incubated at 37oC for 90 min to facilitate cell adhesion. Following this step, the media (containing non-adherent cells) was aspirated, then 3 ml of YNB broth supplemented with AA (at 0.1 mM or 1 mM concentration) with and without FLC (32 μg/ml) was dispensed into designated wells, and incubated at 37oC for 6 h to allow biofilm formation. Each treatment group had three replicates. 4.3.4.2 Protein extraction and visualisation of Abc1p on SDS-PAGE Following biofilm formation, protein extraction from the biofilm cells was done following a previously described method (Szczepaniak et al. 2015). Briefly, biofilm was scrapped off and re-suspended in 1 ml ice cold milli-Q H2O. Biofilm cells were lysed with a 150 μl volume of Page | 180 1.85 M NaOH-7.5% mercaptoethanol solution and incubated for 10 min on ice. Next, a 150 μl volume of 50% trichloroacetic acid was added and incubated for 10 min on ice to precipitate proteins. Accordingly, the solution was centrifuged (10,000 x g for 5 min, 4oC), washed twice with a 1 ml volume of Tris-HCl (1 M), resulting proteins were re-suspended in 50 μl sample buffer (40 mM Tris-HCl, 0.1 mM EDTA, 5% SDS, 8 M urea, 1% β-mercaptoethanol, 0.1 mg/ml bromophenol blue) and incubated at 37oC for 30 min. A 10 μl volume of the protein sample was loaded onto a 7.5% acrylamide/bis-acrylamide SDS-PAGE, and proteins were allowed to separate for 60 min at 125 V. The gel was subsequently stained with Coomassie blue, de- stained with acetic acid (Fairbanks et al. 1971) and visualised under UV light in a Gel DocTM XR+ (Bio-Rad, Canada) (Lee et al. 2012). 4.3.4.3 Western blot analysis and Immunodetection of Abc1p After the separation of proteins by SDS-PAGE analysis, proteins from the unstained SDS- PAGE gel were transferred onto a polyvinylidene fluoride (PVDF) membrane with a Trans-Blot Turbo Transfer System (Bio-Rad, USA). Accordingly, the immunodetection of Abc1p was performed with 1:2,500 anti-Cdr1p antibody (a generous gift from Prof. Dominique Sanglard, Lausanne, Switzerland) and horseradish peroxidase (HRP) conjugated secondary antibody (1:50,000) (Thermo Fischer Scientific, USA) following the manufacturers’ specifications. Visualisation was achieved by viewing with a ChemiDocumentation™ MP imaging system (Bio-Rad). 4.3.5 Influence of arachidonic acid and fluconazole on the activity of Abc1p The influence of AA on the function of Abc1p was evaluated using Rhodamine 6G efflux assay with slight modifications of previous methods (Maesaki et al. 1999; Ells et al. 2013; Szczepaniak et al. 2017). Briefly, biofilms were formed as described above in a black 96-well microtiter plate (Thermo Scientific, Denmark) for 6 h at 37oC. After biofilm formation, the spent medium was removed, sterile PBS was dispensed into each well, and the plate was incubated at 37°C for 1 h to de-energise the biofilm cells. Following incubation, PBS was removed, and 200 μl of 10 µM Rhodamine 6G (Rh6G) (Sigma-Aldrich, St. Louis, MO, USA) in sterile PBS (prepared from 10 mM Rh6G stock in DMSO) was dispensed into each well. The plate was incubated at 37oC, and the uptake of Rh6G was measured every 10 min for 1 h at an excitation wavelength of 530 nm and emission of 590 nm using Fluoroskan Ascent Fluorimeter (Thermo Scientific, China). Following the uptake step, leftover Rh6G was removed, and AA (0.1 or 1 mM), FLC (32 μg/ml), or DMSO and EtOH (DE, control) in sterile PBS was dispensed into designated wells, and the plate was incubated at 37oC for another 1 h (Fourie 2020). Accordingly, the supernatant was removed from the wells, and 2 mM glucose (in sterile PBS) was dispensed into each well to induce efflux of Rh6G. The plate was incubated at 37oC, and Rh6G efflux from the cells was measured extracellularly every 10 min for 1 h at an excitation Page | 181 wavelength of 530 nm and emission wavelength of 590 nm using a Fluoroskan Ascent Fluorimeter (Thermo Scientific, China). 4.3.6 Statistical analysis The data of different groups, unless stated otherwise, were compared using one-way analysis of variance (ANOVA) complemented with Tukey’s multiple comparisons test and a p-value ≤ 0.05 was considered significant. 4.4 Results and Discussions 4.4.1 Constructing an ABC1-GFP mutant with CRISPR-Cas9 system This chapter's main objective was to examine the influence of AA and FLC on the localisation, expression, and activity of Abc1p in C. krusei. To achieve this, we attempted to tag Abc1p with GFP using the adapted CRISPR-Cas9 system created in Chapter 3. This will then allow its fluorescence (localisation and expression) to be assessed under various treatments. This was done by first preparing relevant CRISPR cassettes and donor DNA, and then co-transforming these fragments into C. krusei to enable the creation of DSBs and knock-in of ABC1-GFP dDNA at the 3’ end of ABC1 gene. 4.4.1.1 CRISPR-Cas9 cassettes for ABC1-GFP fusion Firstly, the first component (fragment A) of the gRNA cassette was obtained from pADH110 with primer pair AHO1096-ver2 and AHO1098-ver2. A band size of approximately 1066 bp obtained confirms this fragment’s successful amplification (Fig. 3A). Next, using CK pADH147 as a template, CK-ABC1-CRISPR-1 oligo and primer AHO1097 was used to amplify and introduce a CRISPR site (5’-ATATATCTGTGTTACCAAAA-3’) specific for ABC1 gene into the second component (fragment B) of the gRNA cassette. As depicted in Figure 3A, an expected band size of approximately 722 bp obtained confirms this fragment’s successful amplification. As previously mentioned, the CK-ABC1-CRISPR-1 oligo is flanked by sequences complementary to the 3' end of SNR52 promoter (on fragment A) and 5' end of gRNA scaffold (on fragment B), and this allowed the ligation of these two fragments to generate an intact gRNA cassette (fragment C) of approximately 1788 bp (Fig. 3B). Accordingly, the Cas9 cassette was liberated from CK pADH99 by digesting the plasmid with MssI restriction enzyme. As depicted in Figure 3B, an expected size of approximately 8970 bp representing the Cas9 cassette was obtained. Page | 182 Fig. 3 Construction of the components of CRISPR-Cas9 cassette. (A) An agarose gel profile depicting successful amplification of fragment A (red box) and fragment B (yellow box) of gRNA cassette. (B) A gel profile showing successful generation of an intact gRNA (orange box) following stitching of fragments A and B, and successful digestion of Cas9 cassette from CK pADH99 (green box). M represents 10 kb O’GeneRuler DNA ladder (Thermo Fisher Scientific). 4.4.1.2 Designing ABC1-GFP fusion donor DNA The ABC1-GFP donor DNA (dDNA) consists of a GFP sequence flanked by sequences homologous to regions within ABC1 gene to enable its incorporation at the 3' region of this gene (via homology-directed repair) following the creation of DSBs by Cas9 protein (Fig. 2). Its successful incorporation at this region would result in the creation of ABC1-GFP mutant of C. krusei. This fragment (dDNA) was constructed with three components (fragments 1, 2, and 3). Specifically, the first fragment (fragment 1) consisting of a 1290 bp sequence from the 3’ end of ABC1 gene (without the stop codon) was obtained by stitching fragments 1a and 1b (Fig. 4A). The second fragment (fragment 2) of approximately 948 bp consisting of GFP sequence (with stop codon) and a 231 bp sequence downstream the ABC1 gene was synthesised by stitching fragments 2a and 2b (Fig. 4B). These two fragments were ligated with a third fragment which consists of a 39 bp sequence (for 13 amino acid linker: SGAGAGAGAGAIL) (Janke et al. 2004), flanked by regions complementary to the 3' and 5' ends of fragments 1 and 2, respectively, to generate the full ABC1-GFP dDNA of approximately 2277 bp (Fig. 5). The intact dDNA also consists of homology arms of approximately 305 bp and 241 bp. Page | 183 Fig. 4 Synthesis of components of ABC1-GFP donor DNA (A) Gel profile showing expected band size of 1290 bp of fragment 1. (B) Gel profile depicting fragment 2 of an expected band size of ~948 bp. M represents DNA ladder. Fig. 5 Gel profile showing successful synthesis and amplification of intact ABC1-GFP donor DNA (~2277 bp). M represents DNA ladder. Page | 184 4.4.1.3 Transformation Following co-transformation, the Cas9 and gRNA cassettes generate a complete CRISPR- Cas9 cassette which integrates at the HIS1 locus (Chapter 3, Fig. 39). Transformants are NTC-resistant (NTCR) due to an intact NAT marker present within the complete CRISPR-Cas9 cassette and were selected on YPD plates containing NTC. However, none of the transformants incorporated the ABC1-GFP dDNA (Fig. 6), even after several attempts. The reason why the organism failed to incorporate the dDNA was unclear; however, future attempts would consider including longer homology arms into the dDNA (Wang et al. 2016; Norton et al. 2017). Fig. 6 Gel profile depicting unsuccessful incorporation of ABC1-GFP donor DNA into the genome of the transformants. A band size of ~1389 bp representing a region within the unmodified ABC1 loci was obtained (lanes 2 – 6), this corresponds to what was obtained for the wildtype strain (lane 1). M represents the DNA ladder. 4.4.2 Influence of arachidonic acid and fluconazole on Abc1p expression Although the mechanism of intrinsic resistance to FLC in C. krusei has been chiefly attributed to alterations in FLC target, Erg11p, which consequently result in the decreased susceptibility of this target towards FLC (Venkateswarlu et al. 1997; Orozco et al. 1998; Fukuoka et al. 2003), the role of efflux pump transporters, including Abc1p, remains controversial. Efflux pumps are transport proteins that pump toxic compounds, including antimicrobial agents, out of the cell, and their overexpression results in a multidrug-resistance phenotype in pathogenic microbes (Webber and Piddock 2003; Lamping et al. 2009). The ATP-Binding Cassette (ABC) and Major Facilitator Superfamily (MFS) are the main transporter families in fungal species (Wirsching et al. 2001; White et al. 2002). The hydrolysis of ATP powers the ABC transporters' members; however, the MFS transporters rely on energy from the proton-motive force (Pao et al. 1998; Cannon et al. 2009; Rees et al. 2009; Redhu et al. 2016). Although four ABC transporters (Abc1p, Abc2p, Abc11p, Abc12p) have been identified in C. krusei (Lamping et Page | 185 al. 2017; Douglass et al. 2018), Abc1p (Cdr1p) has proven to be the main transporter, and its overexpression results in resistance to antifungal drugs, including the azoles (Tsao et al. 2009; Lamping et al. 2009). Inhibitors of this efflux pump are limited, and PUFAs (e.g. AA) known to cause membrane disorganisation may represent potential inhibitors of this transporter (Avis and Belanger 2001; Pohl et al. 2011; Mishra et al. 2014). However, since we were unable to obtain an ABC1-GFP mutant, we sought to assess the influence of AA and FLC on the expression of this transporter using SDS-PAGE and western blot analyses. This was performed by exposing biofilms of C. krusei UFS Y-0277 to AA (0.1 mM or 1 mM) and FLC (32 μg/ml) alone, and in combination, extracting total protein from these biofilms, and then assessing the expression of Abc1p using an anti-Cdr1 antibody. As shown in Figure 7, when compared to the control (DE), there was no apparent difference in the protein profile obtained when C. krusei biofilm was exposed to either AA or FLC singly, or in combination. Moreover, a protein with a size of almost 180 kDa, which may represent Abc1p (172 kDa), was identified in all treatments, including the DE control. Fig. 7 SDS-PAGE profile of proteins from Candida krusei biofilms following exposure to various treatments. 1: DE (DMSO and ethanol) control, 2: 32 μg/ml fluconazole (FLC), 3: 0.1 mM Arachidonic acid (AA), 4: combination of 0.1 mM AA and FLC, 5: 1 mM AA, 6: combination of 1 mM AA and FLC. M represents SDS-PAGE Pre-stained Protein Ladder (3.5 to 245 kDa). Arrow indicates a band corresponding to expected protein size of 172 kDa. Page | 186 Additionally, western blot analysis was done using an anti-Cdr1 antibody to confirm this protein’s identity as Abc1p and assess its expression levels. As depicted in Figure 8, a band which may represent Abc1p and suggest its expression was identified only in the presence of DE (control) and FLC. This may mean that Abc1p is constitutively expressed in this particular strain (Lamping et al. 2009), and overexpressed in the presence of FLC only. Its expression was however abrogated in all treatments with AA regardless of the concentration. However, these results are not entirely definitive, and future research would consider corroborating it with other analyses, such as quantitative western blot analysis and enzyme-linked immunosorbent assay, to measure the expression levels of Abc1p (Taylor and Posch 2014; Finger et al. 2018). Fig. 8 Confirmation of Abc1p protein and assessment of its expression level with western blot analysis following exposure to various treatments. 1: DE (DMSO and ethanol) control, 2: 32 μg/ml fluconazole (FLC), 3: 0.1 mM Arachidonic acid (AA), 4: combination of 0.1 mM AA and FLC, 5: 1 mM AA, 6: combination of 1 mM AA and FLC. Lanes 1 and 2 show expected band. M represents SDS-PAGE Pre-stained Protein Ladder (3.5 to 245 kDa). 4.4.3 Abc1p activity is increased by fluconazole but extenuated by arachidonic acid in a dose-dependent manner We also evaluated the influence of AA and FLC on the functionality of Abc1p using Rhodamine 6G efflux assay. Rhodamine 6G is a substrate of ABC transporters, and its efflux is mediated by these transporters (Nakamura et al. 2001; Mukherjee et al. 2003; Tsao et al. 2009). Amongst these transporters, Cdr1p (Abc1p) is the primary transporter; hence Rh6G efflux was used to monitor its activity. To examine the influence of AA and FLC on the activity of Abc1p, Rh6G was allowed to passively diffuse into de-energised biofilms, the biofilms were then exposed to either AA (0.1 mM or 1 mM) or FLC alone, or in combination, after which the biofilms were re-energised with glucose, and the efflux of Rh6G was assessed. Page | 187 As shown in Figure 9A, at a concentration of 0.1 mM, AA alone and in combination with FLC, could not inhibit the efflux of Rh6G. Remarkably, the activity of Abc1p was significantly extenuated in the presence of a higher concentration of AA (1 mM), either alone or in combination with FLC (Fig. 9B). This observation correlates with an earlier reported finding of dissipated activity of C. albicans efflux pump (Cdr1) in the presence of a high concentration (1 mM) of AA (Fourie 2020, Kuloyo 2020). Further, the diminished functionality of Abc1p observed in the presence of 1 mM AA may be due to the mislocalisation of Abc1 transporter from the cellular membranes (Fourie 2020). A similar study by Shareck and co-workers (2011) also demonstrated the mislocalisation of a membrane-bound protein, Ras1p, by conjugated linoleic acid. Furthermore, interference with ATP synthesis (ATP is essential for Abc1p activity) and/or mitochondrial dysfunction may also contribute to the reduced activity (Fourie 2020). Notably, the functionality of Abc1p was enhanced in the presence of FLC alone compared to the negative control (DE), and this may suggest that this transporter plays a role in FLC resistance in this strain; however, this needs to be further investigated (Fig. 9). Conclusively, we observed the overexpression and increased functionality of Abc1p in the presence of FLC, and its diminished activity in the presence of 1 mM AA (but not 0.1 mM AA), either alone or in combination with FLC. Taken together, these findings suggest that AA influences the activity of Abc1 transporter in a dose-dependent manner and that this transporter plays a role in FLC resistance in this strain. A B 4 0 4 0 D E D E F L C F L C 3 0 3 0 A A (0 .1 m M ) A A (1 m M ) F L C + A A F L C + A A 2 0 2 0 1 0 1 0 0 0 0 1 0 2 0 3 0 4 0 5 0 6 0 0 1 0 2 0 3 0 4 0 5 0 6 0 T im e (m in ) T im e (m in ) Fig. 9 Rhodamine 6G efflux in C. krusei UFS Y-0277 biofilms after treatment with 0.1 mM arachidonic acid (A) and 1 mM arachidonic acid (B) in the presence or absence of fluconazole (32 μg/ml). Values are means of three independent experiments and error bars indicate standard deviation. DE: DMSO+Ethanol (control), FLC: fluconazole, LA: linoleic acid, GLA: gamma-linolenic acid. Page | 188 R e la t iv e F lu o r e s c e n c e U n i t R e la t iv e F lu o r e s c e n c e U n i t 4.5 Conclusions This chapter's main objective was to assess the influence of AA and FLC on the localisation, expression, and activity of Abc1p in C. krusei. An initial attempt to fulfil this objective involved tagging Abc1p with GFP using CRISPR-Cas9 system, allowing its fluorescence (expression and localisation) to be assessed under various treatments; however, this was unsuccessful after several attempts. The influence of AA and FLC on the expression and activity of Abc1p was instead examined using western blot analysis and Rh6G efflux assay, respectively. Although Abc1 transporter was overexpressed in the presence of FLC, its expression was abrogated and undetected in all treatments with AA. Additionally, the functionality of Abc1p was enhanced in the presence of FLC; however, this was severely extenuated upon exposure to 1 mM AA, either alone or in combination with FLC. 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Antimicrobial Agents and Chemotherapy 45:3416–3421. https://doi.org/10.1128/aac.45.12.3416-3421.2001 Page | 194 CHAPTER 5 General discussion, conclusions, and future recommendations Page | 195 5.1 Influence of polyunsaturated fatty acids on in vitro fluconazole susceptibility of C. krusei The epidemiology of invasive candidiasis is changing, with an increasing number of infections being attributed to non-albicans Candida (NAC) species with reduced antifungal susceptibility (Chi et al. 2011; da Silva et al. 2013; Sadeghi et al. 2018). However, the number of antifungal agents available to treat these infections remains limited. This is, in part, due to the eukaryotic nature of both fungi and humans (Campoy and Adrio 2017). For example, the clinical use of polyenes, which target fungal ergosterol, is hindered by nephrotoxicity because of the slight affinity of these drugs for cholesterol, an ergosterol homolog found in humans (Paterson et al. 2003; Lemke et al. 2005). Fluconazole (FLC) remains the drug of choice for the treatment of many fungal infections, especially in resource-limited settings, due to its affordability, amongst other factors. However, NAC species are generally less susceptible to this drug, with 35% of C. glabrata and 10-25% of C. tropicalis isolates exhibiting either a primary or secondary resistance (Whaley et al. 2017). More worrisome, up to 97% of C. krusei isolates are considered inherently resistant to FLC (Whaley et al. 2017). This yeast also rapidly displays adaptive resistance to other antifungals, including the echinocandins (Forastiero et al. 2015). Consequently, other therapeutic approaches are being explored, including combination therapy, which has been harnessed against infectious agents in various forms, including the combination of conventional drugs with appropriate non-antimicrobial compounds (e.g. fatty acids, phytochemicals) (Ells et al. 2009; Jia et al. 2019). Combination therapy may provide greater benefits, including increased microbicidal activity, reduced toxicity, and decreased rate of resistance development (Chang et al. 2017; Prasad et al. 2017). The antifungal property of unsaturated fatty acids against certain Candida spp. has been reported by previous studies (Thibane et al. 2010; Kim et al. 2020; Muthamil et al. 2020). This was confirmed in the present study. The antifungal effect of the tested polyunsaturated fatty acids (PUFAs), including oleic acid (OA), linoleic (LA), gamma-linolenic acid (GLA), arachidonic acid (AA), and eicosapentaenoic (EPA), on C. krusei biofilms was dependent on the strain, as well as on the chain length and dose of the fatty acid. The mechanism of action of antifungal unsaturated fatty acids has previously been found to be mainly through their incorporation into fungal membranes – which consequently increases membrane fluidity and permeability, and ultimately elicits membrane disorganisation (Avis and Belanger 2001; Pohl et al. 2011; Mishra et al. 2014). The disruption of hyphal morphogenesis by antifungal PUFAs has also been noted in certain Candida spp. (Shareck et al. 2011). Notably, in the present study, the most resistant strain to the action of PUFAs was also the least susceptible to FLC, possibly indicating a shared mechanism of action. Page | 196 Previous studies, using Candida spp. without intrinsic FLC resistance (i.e. C. albicans and C. dubliniensis), found that PUFAs, such as AA and stearidonic acid (SDA), increase the antifungal susceptibility of these yeasts to amphotericin B and clotrimazole (Ells et al. 2009; Thibane et al. 2012). However, it was unknown whether the same would apply to a pathogenic yeast with intrinsic resistance to an antifungal drug, as in the case of C. krusei. We found that either of the two PUFAs (LA or GLA) overcomes the intrinsic FLC resistance of C. krusei and potentiates the action of FLC. An initial attempt to dissect the mechanism responsible for this potentiating effect using scanning electron microscopy revealed that the combination treatments elicit the production of extracellular vesicles (EVs), cell membrane damage, and cell rupture. Biofilm-associated EV secretions, critical for antifungal resistance have been identified previously (Zarnowski et al. 2018). Moreover, these vesicles may act as decoys to protect yeast cells from the cytotoxic effect of antifungal agents (Zhao et al. 2019). These structures are also released upon exposure of yeast cells to known oxidative stress inducers (Nollin and Borgers 1975; Lemar et al. 2005), suggesting the induction of oxidative stress a possible mechanism of the combination treatments. Similar structures were found on biofilms cells exposed to a high concentration of marine PUFAs (Thibane et al. 2010; Thibane et al. 2012). A membrane integrity assay with propidium iodide dye further confirmed the detrimental effect of the combination treatments on the cell membrane; this effect is possibly a direct influence of PUFA insertion into fungal membranes or an aftermath of increased reactive oxygen species (ROS) and lipid peroxidation. Additionally, the induction of cell rupture and loss of intracellular contents (hallmarks of necrosis) observed might have been a consequence of a two-step process: an initial cell membrane disorganisation, induced by the insertion of PUFA into the yeast membranes, followed by an enhanced plasma membrane distortion and increased oxidative stress within the already vulnerable cells facilitated by FLC. This ultimately results in cell rupture via necrosis. Further findings in the present study showed that the induction of oxidative stress contributes to the potentiating effects of the combination treatments, as known antioxidants [i.e. butylated hydroxytoluene and α-tocopherol polyethylene glycol succinate (TPGS)], could rescue the biofilms from the cytotoxic effects of these treatments. Our results also suggest that the increased oxidative stress might be due to lipid peroxidation in the cell membrane since a better antioxidative effect was observed for TPGS which localises to the plasma membrane (Li et al. 2015; Yehye et al. 2015). Furthermore, it was necessary to assess the influence of these treatments on the functionality of efflux pumps due to their indispensable role in extruding antimicrobial agents and increasing antifungal resistance (Lamping et al. 2009). Since drug efflux pumps are localised in the cell membrane and unsaturated fatty acids have been reported to elicit membrane disruption (Avis and Belanger 2001; Pohl et al. 2011; Mishra et al. 2014), the observed inhibition of efflux pump activity by the combination treatments may be due to the mislocalisation of efflux pumps, following Page | 197 membrane disorganisation. Additionally, all the presently characterised efflux pumps in C. krusei are ATP-binding cassette (ABC) transporters which are dependent on the hydrolysis of ATP to function (Lamping et al. 2017; Douglass et al. 2018). Hence, the abrogated functionality of the efflux pumps might have been facilitated by ATP deficit, since antimicrobial fatty acids could disrupt oxidative phosphorylation (Yoon et al. 2018). In summary, PUFAs show potential as antifungal-potentiating agents against an intrinsically- resistant yeast. Based on the aforementioned and above-discussed mechanisms of action, the possible sequence of events responsible for this effect could be “direct” or “indirect”. The direct effect involves the incorporation of PUFA into cellular lipids and membranes. Such insertion increases the unsaturation index, directly alters membrane fluidity and stability, and ultimately results in membrane perturbation (Avis and Belanger 2001; Pohl et al. 2011; Mishra et al. 2014). As for the indirect influence, ROS production is enhanced following the incorporation of an exogenous unsaturated fatty acid (due to the presence of carbon-carbon double bonds) (Ayala et al. 2014). Next, oxidative degradation of lipids (lipid peroxidation) occurs due to the actions of free radicals (ROS) (Fuchs et al. 2014). At this point, lipid peroxidation results in either cell membrane disruption, oxidative stress, or a combination thereof. When the “membrane disruption” pathway is followed, the disruption of membrane protein functions occurs, and this pathway ultimately results in regulated and/or uncontrolled cell deaths (Yang and Stockwell 2016; Gaschler and Stockwell 2017). On the other hand, the pathway involving “oxidative stress” in most cases directly results in oxidative damage, which ultimately results in cell death via apoptosis, necrosis, or a combination thereof (Repetto et al. 2012; Pizzino et al. 2017). Notably, although both of these pathways could induce regulated (apoptosis) and uncontrolled (necrosis) cell death, the occurrence of necrotic cell death was more likely in this study due to the identification of some of its hallmarks, including cell rupture and loss of intracellular contents, in cells within the combination-treated biofilms (Eisenberg et al. 2010). 5.2 Influence of polyunsaturated fatty acids on the survival and fungal burden of infected C. elegans In drug discovery, the in vitro antimicrobial activity of lead compounds does not always translate to a corresponding in vivo efficacy. In addition to providing valuable information on the correlation between findings of in vitro and in vivo assays, animal models are also employed to gain better insights into the progression and characterisation of diseases in humans, since many virulence traits and immune reactions are only induced in vivo (Marsh and May 2012). Because of this, we took a step further by evaluating the potentiating effects of the combination treatments in a Caenorhabditis elegans infection model. This model organism has been used for genetic analysis (Watts and Browse 2002), lipid and fatty acid metabolism studies (Bouyanfif et al. 2019; Mokoena et al. 2020), and certain drug discovery research (Madende et al. 2020). Moreover, the ability of Page | 198 C. krusei to infect and kill this particular strain has been documented (Scorzoni et al. 2013). A study by Breger and co-workers (2007), demonstrated that FLC (at 32 μg/ml) could not prolong the survival of nematodes exposed to a FLC-resistant strain of C. krusei, and had a toxic effect on the nematodes. This was not the case in the present study, where it was able to slightly extend the median lifespan of C. elegans, one explanation for this is that its antifungal efficacy against the C. krusei strain used in this study outweighs its toxicity, if any, on infected nematodes. Our findings further demonstrated that each of the fatty acids (LA or GLA) alone was able to slightly extend the survival and reduce the intestinal fungal burden of infected nematodes. However, a superior overall activity was noted with the combination treatments. The mechanism for this in vivo potentiating effect was not investigated. However, it may be due to a direct effect on C. krusei, as found in vitro, or possibly due to improved immunity of C. elegans in the presence of these PUFAs, since there is evidence that endogenous and exogenous PUFAs, such as GLA and SDA, are required for C. elegans immunity (Nandakumar and Tan 2008). This, however, needs to be investigated. Conclusively, our in vivo findings correlate with the observed in vitro susceptibility profiles of the combination treatments; however, future studies should consider exploiting higher model animals. 5.3 Establishment of a CRISPR-Cas9 genome editing tool for C. krusei The serendipitous discovery of and elaborate research on a bacterial and archaeal adaptive system known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) led to the development of a CRISPR-Cas9 mediated genome editing tool. This tool is superior to the earlier gene-editing endonucleases, including zinc-finger nucleases and transcription activator- like effector nucleases, owing to its affordability, simplicity – simple to design, but effective for answering complex genomic questions, flexibility, versatility, and high targeting and editing efficiency (Khadempar et al. 2019; Saha et al. 2019). A functional CRISPR-Cas9 editing system has been designed and utilised for gene-editing in Candida albicans (Vyas et al. 2015). Moreover, similar systems are also available for recalcitrant non-albicans Candida (NAC) species, such as C. glabrata and C. auris (Grahl et al. 2017; Lombardi et al. 2017). Indeed, the use of this tool in medical mycology has improved the understanding of the roles of various genes in virulence and resistance. However, no such system is available for C. krusei. The lack of such a facile and precise tool for genome engineering in this yeast has hindered molecular understanding of its resistance mechanisms. In the present study, a CRISPR-Cas9 system was developed for this C. krusei by adapting a HIS-FLP type C. albicans-specific system of Nguyen and co-workers (2017). Plasmids for this system were obtained from Addgene. Relevant fragments were replaced with corresponding homologs from C. krusei genome. The resulting optimised system is made up of a CAS9 gene (which encodes the molecular scissor- Cas9 protein), placed under the control of C. krusei ENO1 promoter; and a gRNA (under an SNR52 promoter), which is adaptable to target Page | 199 and edit any gene within this yeast’s genome. The full CRISPR system integrates at the HIS1 locus and possesses an intact (dominant) nourseothricin N-acetyltransferase (NAT) marker gene, which allows transformants to exhibit a nourseothricin-resistance phenotype. This system is recyclable and upon gene-editing, it is removed from the genome via growth on a maltose- containing medium accomplished by flippase (FLP) recombinase. In literature, the efficacy of gene-editing tools is often assessed by targeting auxotrophic marker genes, since their disruption (or deletion) either prevents the growth of an auxotrophic mutant (in the case of URA3 gene) or results in the production of a distinctive phenotype in the absence of an essential nutrient (red colonies in the case of ADE2 gene) (Lacroute 1968; Poulter and Rikkerink 1983). By using our optimised system with relevant donor DNA (dDNA), we successfully targeted and deleted URA3 and ADE2, to generate ura3Δ/Δ and ade2Δ/Δ mutants, respectively. The engineering efficiency of our system for URA3 and ADE2 deletion was between 10% – 16%. However, in the future, this system’s toolbox will be strengthened by codon-optimising CAS9 gene for expression in C. krusei (Weninger et al. 2016; Raschmanová et al. 2018), and placing the gRNA under a promoter that is specific for this yeast (Enkler et al. 2016; Morio et al. 2020). Strikingly, in comparison with the wildtype strain, the obtained auxotrophic mutants (ade2Δ/Δ, ura3Δ/Δ) demonstrated a reduced-filamentation phenotype. This may possibly suggest their reduced virulence. However, virulence studies, particularly in an intractable and simple host like C. elegans, are warranted (Madende et al. 2020). Similar studies have reported reduced virulence of mutants of C. albicans with disrupted URA3 and ADE2 (Kirsch and Whitney 1991; Donovan et al. 2001; Staab and Sundstrom 2003). To the best of our knowledge, this is the first development and usage of the CRISPR-Cas9 system for genome engineering in C. krusei. 5.4 Influence of arachidonic acid and fluconazole on the expression and function of Abc1p Although the role of FLC target, Erg11p, in the intrinsic FLC resistance of C. krusei has been established (Venkateswarlu et al. 1997; Orozco et al. 1998; Fukuoka et al. 2003). The role of efflux pump transporters, including Abc1p (the homolog of Cdr1 protein in C. albicans), in this resistance remains obscure. Two earlier studies by Katiyar and Edlind (2001) and Guinea and co-workers (2006) have documented that no ABC transporters are involved in this yeast’s innate FLC resistance. However, a later study noted a possible role of Abc1p in this resistance (Lamping et al. 2009). Despite their roles in antifungal resistance, no class of antifungal targets the efflux pumps. Interestingly, AA (a type of PUFA), has been demonstrated as a potential inhibitor of Abc1p homolog (Cdr1p) in C. albicans (Fourie 2020; Kuloyo 2020). The construction of a Green Fluorescent Fusion (GFP) fusion of Abc1p, using the adapted CRISPR-Cas9 system was attempted in order to gain valuable insights into the influence of AA and FLC on the localisation, expression, and activity of Abc1p in C. krusei. However, this was unsuccessful, possibly due to Page | 200 the failure of the yeast to incorporate the supplied ABC1-GFP fusion donor DNA, after the creation of double-strand breaks (DSBs) by the Cas9 nuclease. Furthermore, with the use of alternative analytical techniques (western blot analysis and efflux pump assay), FLC was found to enhance both the expression and functionality of Abc1p. This may possibly indicate that this transporter plays a role in FLC resistance. Similar studies have reported the mechanism of resistance to itraconazole and voriconazole to be, in part, due to the overexpression of Abc1p and Abc2p (Tavakoli et al. 2010; Ricardo et al. 2014; He et al. 2015). However, AA (at a higher concentration), reduces the expression of Abc1p, and consequently extenuates its functionality, even in the presence of FLC. 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Communications Biology 2:305 https://doi.org/10.1038/s42003-019-0538-8 Page | 208 APPENDIX A: ETHICAL CLEARANCE FORM Page | 209 APPENDIX B: DEPOSITED MUTANTS’ FORMS Page | 210 Page | 211