Doctoral Degrees (Microbial, Biochemical and Food Biotechnology)
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Browsing Doctoral Degrees (Microbial, Biochemical and Food Biotechnology) by Author "Albertyn, J."
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Item Open Access Activation of the SARS-CoV-2 spike protein by 𝘤𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 secreted proteases(University of the Free State, 2023) Mjokane, Nozethu; Sebolai, O. M.; Pohl, C. H.; Albertyn, J.; Gcilitshana, O. M. N.The thesis is not structured in a classical way. As such, it is composed of a literature review section (Chapter 1) and two research chapters (Chapters 2 and 3). A general discussion section (Chapter 4) and addendums are also included. As some chapters are in a publication format, repetition of essential information could not be avoided. Chapter 1 reviews the emergence of SARS-CoV-2 and its impact. In particular, it considers the co-infection of this virus with respiratory fungal pathogens, which are major independent risk factors that complicate COVID-19 by causing a more severe infection resulting in higher mortality than that of either infection on its own. These fungal pathogens secreted furin-like proteases to further their virulence during host invasion. In this context, the thesis argues that it is foreseeable that the virus could also access these fungal furin-like proteases and pervert them in order to activate its latent spike protein. Therefore, this set up a number of questions, which are addressed in the thesis concerning the possible activation of the viral latent spike protein by fungal furin-like proteases. In Chapter 2, it was sought to characterise 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 (𝘊.) neoformans proteases and assess if they could theoretically bind to the SARS-CoV-2 spike protein. To be specific, previous papers reporting on cryptococcal serine proteases were perused, and this made it possible to select a number of proteases, namely cryptococcal serine carboxypeptidase (CNBF4600), cryptococcal cerevisin (CNBJ2870) and cryptococcal peptidase (CNBA1340), cryptococcal peptidase (CNAG_00150) and cryptococcal cerevisin (CNAG_04625). By designing specific primers, it was possible to show that these serine proteases were expressed in 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 H99, the prototypical cryptococcal strain used in this thesis. Therefore, the expressed gene products were expected to be secreted into the culture media. This was important for the work that follows in Chapter 3. Through using the computational programme, High Ambiguity Driven protein-protein DOCKing (HADDOCK), it was possible to show that some of the selected cryptococcal serine proteases could interact with the coronavirus spike protein and yield a binding affinity greater than and comparable to furin. However, as HADDOCK is a computational programme, the predicted binding affinities might not correlate with the experimental binding affinities in solution, more so since the used 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 proteases structures were predicted and not solved. To account for this, Chapter 3 sought to provide enzymatic evidence using the collected culture media – in the form of supernatant. To do this, a mimetic fluorogenic peptide of the SARS-CoV-2 spike protein was designed and modified to have intra-molecular fluorescence quenching capability using 7-methoxycoumarin-4-yl acetyl (MCA) at the N-terminus and N-2,4-dinitrophenyl (DNP) at the C-terminus. The assay was performed using the cryptococcal supernatant. For reference, recombinant furin was included as this is the serine protease present in humans that catalyses the activation of the spike protein. Here, it was determined that cryptococcal serine proteases present in the supernatant could cleave the mimetic spike protein at S1/S2 site with biochemical efficiency comparable to furin. To test the veracity of these data, SARS-CoV-2 pseudovirion containing a full-length spike protein was used. It was possible to show that the pseudovirion could be transduced into HEK-293T cells in the presence of the cryptococcal supernatant. Chapter 4 takes into account the obtained results and provides a summary of the major observations. Of note, the thesis theorises that yeast kexin proteases are responsible for the observed activity. This is because there is a functional homology between yeast kexin proteases and furin (both are convertases); thus, it is reasonable that the supernatant (which contains yeast kexin proteases) could activate the latent SARS-CoV-2 spike protein. The thesis further proves that other respiratory fungal pathogens have yeast kexin proteases that activate the spike protein. This evidence is documented in Addendum no. 1. All things considered, the findings point to the regulation of protease activity as a viable approach to control the activation of the spike protein by either mammalian protease or fungal proteases. To this end, protease inhibitors could be used to control unwanted proteolysis. Addendum no. 2 attempted to show this. Here, it was possible to show that the South African-based medicinal plant Artemisia tea infusion extract and its active compound artemisinin could control the activation of the mimetic SARS-CoV-2 spike protein by furin but not the supernatant. The latter highlights the need to purify the supernatant and isolate yeast kexin proteases. The idea of exploring the control of unwanted proteolysis is also an interventional measure considered by Pfizer, the pharmaceutical company. This American multinational pharmaceutical and biotechnology corporation successfully piloted Paxlovid to control SARS-CoV-2. This drug contains an anti-protease (PF-07321332) that inhibits the protease (SARS-CoV-2 3CLp) responsible for viral replication.Item Open Access Control of Listeria monocytogenes in an avocado processing facility(University of the Free State, 2015-11) Strydom, Amy; Witthuhn, R. C.; Gouws, P. A.; Albertyn, J.Listeria monocytogenes contamination of food is a growing concern for the food industry since it is the causative agent of human listeriosis. Despite increased awareness and strict microbiological standards for this pathogen, countries such as France, Austria and Germany have reported increases in listeriosis outbreaks. The research in this thesis shows how Listeria contamination in a South African avocado processing was almost eradicated. The first aim of this project was to isolate and genetically type the L. monocytogenes strains isolated from the facility. Pulsed field gel electrophoresis (PFGE) was used to group a subset of strains (n=80) according to the digestion of their genomes with the restriction enzyme AluI. These results were compared to polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) of the inlA gene (n=140). The results of the two methods compared well with each other but both indicated a lower genetic diversity among the isolates than expected. These strains were isolated over a period of four years and only two major groups were identified with the PFGE and the PCR-RFLP resulted in only three banding patterns. Also, the origin of the bacterial contamination could not be identified, but results indicated that cross contamination played a role in the persistence of these bacteria in this food processing facility. Secondly, the commercial product ListexTM P100 was assessed for possible use as a biocontrol agent in the facility. The host range of the phage product was determined based on 239 L. monocytogenes strains isolated from this facility, but only 26.7% of these strains were susceptible to the bacteriophage. The strains were also analysed for serotype and no correlation was observed between typing methods or isolation source and date of isolation of the strains, as well as the susceptibility to the phage product. This could indicate that cross contamination played a role in the transfer of bacterial cells since these strains were distributed randomly in the facility. Inoculation of the phage product with L. monocytogenes T162 in brain heart infusion (BHI) broth resulted in significant reductions in the bacterial concentration. However, activity of the phage in avocado pulp and guacamole was very low and no significant reduction in the L. monocytogenes concentration was measured. Lastly, the population of the Listeria strains in the facility were continuously monitored over five years. The final products and processing environment, including floors, equipment, work areas and personnel were tested on-site for Listeria with the ISO 11290-1 method. Based on the prevalence of Listeria, the facility introduced new strategies in processing to counter cross contamination. Results from the 2014 guacamole production season showed almost complete eradication of Listeria spp. in final products (0.17%, n=1170) and the processing facility (0.79%, n=1520). These results indicate that successful management of Listeria spp. in an avocado processing facility can be accomplished with in-house monitoring of the bacterial population and subsequent adjustments to the processing system. The results from this project indicated that the cause of contamination by L. monocytogenes in the facility was due to cross contamination, although a strict quality control system was followed. Despite low genetic variability between the L. monocytogenes strains, the commercial phage product was only effective against 26.7% of strains tested. This is surprising since literature reported very high percentages of susceptible strains to this specific product. Although bacteriophage biocontrol with ListexTM P100 was not effective in this facility, it cannot be concluded that this will be the case for other facilities. Also, bacteriophage product with a broader host range such as a cocktail of different phages, may work well in the processing environment to minimise transfer of bacterial cells to the final product. Control of L. monocytogenes will, however, only be effective if the processing conditions counter cross contamination.Item Open Access Cytochrome P450 monooxygenases from extremophiles(University of the Free State, 2012-01) Müller, Walter Joseph; Smit, M. S.; Van Heerden, E.; Albertyn, J.; Piater, L. A.English: Information indicate that CYP450s are prevalent in members of the bacterial phylum Deinococcus-Thermus as well as the archaeal family Halobacteriaceae that belong to the phyulm Euryarchaeota. A property shared by these phylogenetically distant extremophiles is the production of carotenoid pigments. It became the purpose of this study to use genome sequence information to clone and study new CYP450s from the genera Thermus and Halobacterium and to explore the role of these CYP450s in pigment production. The non-pigmented thermophilic bacterium Thermus scotoductus SA-01 was screened by PCR for the presence of a cytochrome P450 monooxygenase (CYP450). No CYP450 could be found and subsequent genome sequencing confirmed this finding. However, a CYP450 gene (CYP175A) was isolated from the closely related yellow pigmented strain Thermus sp. NMX2.A1 using oligonucleotides based on the DNA sequence of the β-carotene gene cluster from three Thermus strains. The genome sequence of T. scotoductus SA-01, revealed a ferredoxin (Fdx) and ferredoxin reductase (FNR) that were almost identical to those of Thermus thermophilus HB27. In T. thermophilus HB27 the Fdx and FNR are the native redox partners for CYP175A1, a β- carotene hydroxylase. After heterologous expression in Escherichia coli, we attempted to hydroxylate β-carotene with the CYP450 from Thermus sp. NMX2.A1 and the redox partners of T. scotoductus SA-01 using cell free extracts, but no products were detected. Thirty two CYP450s have been identified in the sequenced genomes of thirteen extremely halophilic archaea. Initial attempts to clone and heterologously express a CYP174A2- homologue from a Haloarcula LK-1 strain in E. coli and Pseudomonas fluorescens were unsuccessful. In order to study the physiological role of CYP450s in halophilic archaea and to create a strain that can be used for heterologous expression of CYP450s from halophiles CYP174A1 was deleted from H. salinarum R1. CYP174A1 is the only CYP450 in H. salinarum R1 and H. salinarum R1 is a genetically tractable strain. Upon culturing the wildtype and deletion strains, a difference in red pigmentation of stationary phase cultures was observed; implying that CYP174A1 might play a role in carotenoid synthesis. Microarray analyses revealed that the bop gene, which codes for bacterioopsin (BO) was severely repressed in stationary phase cultures of the deletion strain and sucrose gradient experiments showed a consequent loss of purple membrane (PM) in the deletion strain. The classical causes of bop repression e.g. insertion elements in the bop open reading frame as well as in the brz gene was ruled out by PCR screening. In addition to bop repression, the neighboring vng1459 and vng1468 genes (both part of the bopregulon) were also down regulated, but the genes normally involved in regulation of the bop gene were not affected. Currently the functions of vng1459 and vng1468 are unknown. Retinal, together with BO, is a key component of bacteriorhodopsin (BR) and essential for PM synthesis. Retinal is formed by the central cleavage of β-carotene which can be achieved by monooxygenases or dioxygenases.The Blh and Brp proteins in H. bacterium salinarum are very closely related to a confirmed bacterial 15,15′-β-carotene dioxygenase and studies have shown that deletion of both brp and blh results in complete abolishment of retinal and BR. It is therefore unlikely that CYP174A1 plays a role in retinal biosynthesis. Another possible function for CYP174A1 might be the hydroxylation of β-carotene, since it is known that H. salinarum strains produce hydroxylated carotenoids such as transastaxanthin, but no genes encoding typical β-carotene hydroxylases or ketolases have been identified in the genomes of H. salinarum strains. This will imply that hydroxylated carotenoids play a role in the regulation of bop.Item Open Access Development of a DNA vaccine for the prevention of Psittacine beak and feather disease(University of the Free State, 2008-05) Kondiah, Kulsum; Bragg, R. R.; Albertyn, J.Psittacine beak and feather disease (PBFD) is a readily recognisable dermatologic condition in wild and captive psittacines worldwide. It is caused by Beak and feather disease virus (BFDV) which is classified in the family Circoviridae and the genus Circovirus. BFDV has a circular ss-DNA genome consisting of seven open reading frames (ORFs), three being conserved in all BFDV isolates, ORF 1 which encodes the Rep protein, ORF 2 which encodes the coat or capsid protein (CP) and ORF 5 which encodes a protein whose function is as yet unknown. General symptoms of the disease include the symmetrical loss of feathers, feather abnormalities, beak and claw deformities, weight loss, anorexia and immunosuppression. The inability to grow BFDV in tissue culture or in embryonated eggs has hindered the routine diagnosis of PBFD affected birds and the development of reliable diagnostic tests and an effective vaccination program. PBFD is widespread in South Africa, leading to a loss of at least 10% of psittacine breeding stocks annually. The disease is also a major threat to the already endangered Cape Parrot (Poicephalus robustus) and the black-cheeked lovebird (Agapornis nigrigenis) and it is only a matter of time before we may see the extinction of these and other parrot species due to the lack of a preventative vaccine. The economical and natural implications of the attack by PBFD led to the aims of the present study which were to develop a potential DNA vaccine candidate, develop an expression system for production of recombinant CP as antigenic protein and establish an enzyme linked immunosorbent assay for the detection of BFDV-specific antibodies in parrots. The entire CP gene which has been suggested to encode for the epitopic protein of the virus was amplified by polymerase chain reaction (PCR) and ligated into a bacterial vector, pBAD/His B or a yeast vector, pKOV136 for expression of recombinant CP in Escherichia coli or Yarrowia lipolytica, respectively. Alternatively, CP gene PCR products were ligated into the mammalian expression vector pcDNA™3.1D/V5-His-TOPO® which was the vector of choice for DNA vaccine design and used to transiently transfect Chinese hamster ovary cells. Subsequently, the candidate DNA vaccine was used in a basic vaccine trial where budgerigars (Melopsittacus undulatus) were vaccinated either with the DNA vaccine candidate or a sub-unit vaccine consisting of purified recombinant CP. Expression of recombinant CP was monitored using polyacrylamide gel electrophoresis (PAGE), chemiluminescent and colorimetric detection on Western blots and ELISAs. While expression of the recombinant CP was unsuccessful in the yeast system using pKOV136, expression of recombinant CP was achieved in E. coli cells using the pBAD vector. Recombinant CP was partially purified and applied in both indirect and indirect competitive ELISAs as coating antigen for the detection of BFDV specific antibodies. Using the established ELISAs, BFDV specific antibodies could be detected in naturally infected parrots as well as in budgerigars vaccinated with the DNA vaccine and sub-unit vaccine. Comparable results were obtained when nonpurified recombinant CP was applied in the ELISAs in lieu of partially purified recombinant CP. Vaccinated budgerigars formed BFDV specific antibodies in response to the DNA vaccine and sub-unit vaccine that were detected using the indirect competitive ELISA established in the study. The antibody responses to the sub-unit vaccine were higher than those in response to vaccination with the DNA vaccine candidate. Although the indirect competitive ELISA could not provide an indication of whether these antibody responses are protective, the results obtained during the trial are a preliminary indication that both the DNA vaccine and sub-unit vaccine may be functional in parrots and safe to use as no adverse reactions were observed.Item Open Access Heterologous expression of cytochrome P450 monooxygenases in different ascomycetous yeasts(University of the Free State, 2012-07) Theron, Chrispian William; Smit, M. S.; Labuschagne, M.; Albertyn, J.Cytochrome P450 monooxygenases (P450) are a diverse, ubiquitous family of important enzymes which catalyze a variety of activities. Their outstanding abilities to hydroxylate non-activated hydrocarbons make them attractive enzymes for applications in the fields of chemical synthesis and bioremediation. Large scale applications of P450s are however largely limited by: (i) their requirement for expensive cofactors; (ii) dependence on co-proteins; and (iii) limited enzyme stability. These problems are largely circumvented by using whole-cell biocatalysis. Therefore the identification of appropriate hosts for heterologous expression is important. E. coli has several limitations in its ability to express eukaryotic P450s, therefore yeasts are attractive alternatives, but have scarcely been used for whole-cell applications (Zollner et al, 2010). The aim of this study was therefore to investigate several ascomycetous yeasts for their potential as P450-expressing whole-cell biocatalysts. To perform parallel, unbiased comparisons, the vector, cultivation conditions and assay conditions needed to be consistent between the different yeasts. This was facilitated by a broad-range vector system designed in our group, which allowed genomic chromosomal integration of foreign DNA at the 18S rDNA regions, and expression using a constitutive TEF promoter. Cultures of different yeasts were grown for the same duration at the same temperature in a medium common among strains, prior to activity assays. Using CYP53B1 as a reporter enzyme we demonstrated whole-cell activity for all of the yeasts tested, except for H. polymorpha. The native reductase systems allowed detectable activities of CYP53B1 in all of the other yeasts, with the highest activity (2.3 μmol.h-1 .gDCW -1) found in A. adeninivorans. Coexpression of cytochrome P450 reductases (CPR) from Yarrowia lipolytica (YlCPR), Rhodotorula minuta (RmCPR) and Ustilago maydis (UmCPR) all led to improvements of the CYP53B1 activities, with the highest activity (11.5 μmol.h-1.gDCW -1) obtained with CYP53B1 and UmCPR coexpressed in A. adeninivorans. The effects of the cloned UmCPR also differed between the hosts, with the biggest improvements observed in A. adeninivorans and Y. lipolytica. RmCPR and UmCPR improved the CYP53B1 activity more dramatically than YlCPR, possibly because CYP53B1, RmCPR and UmCPR are all of basidiomycetous origin. The CYP53B1 activity achieved in this study was considerably better than results obtained previously in our group. In the previous study, a Y. lipolytica strain with multiple copies of CYP53B1 and an additional copy of YlCPR both under the regulation of strong inducible promoters was used, under optimised and constant induction conditions. (Shiningavamwe et al, 2006). The activity obtained in this study on the other hand was achieved using an A. adeninivorans strain carrying presumably only one or two copies of CYP53B1 and UmCPR, expressed under the control of a constitutive promoter, and under non-optimised conditions. Activity of the self-sufficient P450s CYP102A1 and CYP505A1 was assayed using hexylbenzoic acid (HBA) as a non-natural substrate, since wild-type β-oxidation pathways would not permit the use of fatty acids as substrates. HBA is hydroxylated at the ω-1 and ω-2 positions of the alkyl chain by both CYP102A1 and CYP505A1 during this study. Better expression of the bacterial CYP102A1 was obtained using K. marxianus and S. cerevisiae, than in A. adeninivorans; whereas expression of the eukaryotic CYP505A1 was better in Y. lipolytica and especially A. adeninivorans. The best activity observed with a self-sufficient P450 was obtained once more with A. adeninivorans expressing CYP505A1 (33 μmol.h-1 gDCW -1). Overall, the vector system allowed successful expression of P450s and CPRs from bacterial, ascomycetous and basidiomycetous fungal origin, in multiple ascomycetous yeast hosts. The differential effects of different CPRs on a class II P450 were demonstrated, and the UmCPR was in this case found to be an excellent P450 reductase. We report heterologous P450 expression in A. adeninivorans for the first time, and it proved to be, of the yeasts tested, the host with the highest potential for efficient P450 expression and whole cell biocatalysis. The findings of this study provide insight for the improvement of the field of eukaryotic P450 research and particularly whole-cell biocatalysis, and could potentially assist in enhancing the applications of these promising enzymes.Item Open Access An investigation into the diversity of and interactions with platinum of a microbial population from a platinum mine(University of the Free State, 2011) Jugdave, Abhita Gyanendra; Van Heerden, E.; Litthauer, D.; Albertyn, J.The mining industry has provided researchers access to the deep subsurface. The deep subsurface is known to harbor a treasure of novel genes and proteins that can be exploited in the biotechnology industry. It has been established that microorganisms in the deep subsurface are potentially novel and are able to endure high temperatures and extreme pH with limited nutrients for survival. Unfortunately most of these organisms are unculturable. Due to the lack of nutrients these microorganisms utilize reduced metals and minerals from the environment as a source of survival in a process known as biogeochemical cycling. Two fissure water samples were collected from two borehole sites at the Northam platinum mine and analyzed through molecular approaches. Microbial biodiversity was determined for borehole NO24FW030908 fissure water sample. The microbial biodiversity was based on the 16S rRNA and 18S rRNA gene clone libraries determined through phylogenetic clustering analyses using ARB software and a comparative analysis was done using DGGE profiling. The prokaryote and eukaryote diversity revealed low diversity at the species level but a high intraspecies diversity probably associated with the novelty of the biome. Unique isolates were cultured from borehole NO212FW050508 fissure water sample. Five isolates showed novelty at the species level and one isolate showed novelty at the genus level. Two isolates, Geobacillus sp. A8 and Geobacillus sp. A12 were sent for characterization at the DSMZ, Germany. Both isolates exhibited similarities at the genus level but significant differences at the species level based on the type strain to warrant different taxonomical positions. These isolates along with Thermus scotoductus SA-01 were analyzed for platinum reduction and the possible formation of metallic platinum. All isolates showed the ability to reduce platinum (IV) to platinum (0). Geobacillus sp. A8 was selected for further characterizations of platinum nanoparticle formation. Platinum nanoparticles were characterized with various tools to show the size and shape using TEM and SEM, to show the composition using XRD and EDS, and to show the particle size distribution using the NanoTrac and NiComp 380 ZLS systems. It has been proposed that a classical hydrogenase activated by a cytochrome c3 is responsible for the two-step reduction of platinum. Therefore, hydrogenase inhibition tests and the TTC test for the presence of an active hydrogenase were done to confirm the presence of a hydrogenase in Geobacillus sp. A8. It was then selected for the construction of the metabolic pathway genome database to study the metabolism of the microorganism in order to identify alternate or novel pathway(s) associated with the genome. Platinum reduction activity tests based on subcellular fractionation revealed platinum reduction and deposition that occurred in the periplasm; therefore, the putative protein involved in platinum reduction was probably periplasmic. The periplasmic fraction was separated into three fraction sizes, greater than 30 kDa, between 10 and 30 kDa and less that 10 kDa. The 10 – 30 kDa fraction revealed positive platinum reduction. The active fraction was analyzed on a SDS-PAGE and revealed three bands that were digested by trypsin and the peptides were analyzed by protein mass spectrometry. Two proteins were identified, an oxidoreductase commonly known as the old yellow enzyme previously shown to reduce chromate (VI), and a hypothetical YajQ protein. Both proteins were expressed and purified and both proteins showed the ability to reduce platinum. Further work would be to elucidate the in situ mechanism involved in the reduction of platinum with hydrogen as the electron donor.Item Open Access Molecular and physiological aspects of alcohol dehydrogenases in the ethanol metabolism Saccharomyces cerevisiae(University of the Free State, 2007-05) De Smidt, Olga; Albertyn, J.; Du Preez, J. C.English: When Saccharomyces cerevisiae is grown on a fermentable carbon source such as glucose, the fermentative alcohol dehydrogenase, ADH I , catalyses the regeneration of NAD+ from NADH and produces ethanol from acetaldehyde. When the fermentable carbon source is depleted, a variety of other enzymes are derepressed in order to utilise the previously excreted ethanol via oxidative respiration and gluconeogenesis . To provide both the carbon source and energy for this system, the yeast cell requires an efficient method for oxidising this previously excreted ethanol. ADH II is a catabolite repressible isoenzyme which primarily functions in the cell to oxidise ethanol to acetaldehyde, which can be metabolised via the tricarboxylic acid cycle or act as intermediate product in gluconeogenesis. ADH III is a mitochondrial isoenzyme participating in the respiratory metabolism by forming part of the ethanol-acetaldehyde shuttle that is important for shuttling mitochondrial reducing equivalents to the cytosol under anaerobic conditions. The physiological roles and regulation of ADH1, ADH2, ADH3, ADH4 and ADH5 were investigated by monitoring transcription levels in chemostat and batch cultivations with Northern blotting and real-time RT-PCR. ADH I was shown to be the key enzyme in the reduction of acetaldehyde to ethanol and also demonstrated ample ability to oxidise ethanol. ADH2 transcription was inhibited by glucose and ethanol in chemostat cultures pulsed with both these carbon sources, but only glucose repression was evident in batch cultures. Northern blot analysis showed that the ADH3 gene was induced during the ethanol phase of the pulses suggested that the mitochondrial ADH III enzyme could also be involved in the first step in ethanol utilisation. The growth kinetics of a strain expressing only ADH III demonstrated that the ADH3 gene product could fulfil the same function as ADH II. ADH4 transcription was detected for the first time in batch cultures and was shown not to be involved in the production or assimilation of ethanol. ADH5 transcription was also demonstrated for the first time and data suggest that ADH V is not involved in ethanol production in a adh1-adh4 deletion mutant.Item Open Access Phenotypic characterisation of Candida albicans mutants with deletions of arachidonic acid responsive genes(University of the Free State, 2015-04) Motaung, Thabiso Eric; Pohl, C.; Albertyn, J.; Kock, J. L. F.; Ells, R.English: Candida albicans is a permanent resident of the healthy microbiome of humans. It co-inhabits the host micro-environment with trillions of species which make up the complete microbiota. Host parameters such as nutrient availability, CO2, and temperature as well as pH fluctuations are some of the key contributory elements to C. albicans phenotypic attributes and virulence. In this study, our analysis was based on arachidonic acid (AA), a polyunsaturated fatty acid, as an inducer of a genome wide transcriptional response. Arachidonic acid is a precursor of prostaglandins (PGs) such as PGE2 which can act on prostanoid receptors and potentially alter important biological functions. Since AA is available in the host, it is of great importance to understand the response of pathogenic yeasts to AA and how this interaction contributes to clinical infections. We analyzed genes up-regulated during AA exposure of C. albicans biofilms. Three mutants, two disrupted for membrane-bound proteins (ABC pheromone exporter transport, Hst6p, and amino acid sensor, Ssy1p) and ORF19.4612 (uncharacterized) were analyzed using phenotypic microarrays (PMs). Additional roles of Hst6p were uncovered in this study, one of which is linked to its up-regulation in the presence of AA. The PM data suggests that Hst6p is required for stress protection and conventional phenotypic screens confirmed this. ΔΔhst6 was found to be sensitive to osmotic, oxidative, cell wall and heavy metal stress. Because AA can induce sensitivity to stress, our phenotypic data suggest that HST6 was up-regulated during AA exposure, to respond to stress. In addition to HST6, other ABC transport genes, also important in stress, were up-regulated. Other groups have recently discovered the role of these proteins in stress in higher organisms such as humans, suggesting that the ABC-stress response system is widely conserved. Another function of Hst6p was suppression of hyphal formation in conditions replete with nitrogen sources. ΔΔhst6 was filamentous under these conditions. In addition, ΔΔhst6 formed highly wrinkled colonies with filamentous borders, whose cells were filamentous, under nitrosative stress. Therefore, our data supports the fact that Hst6p is expressed in all the cell types in C. albicans and has a more generic role as opposed to pheromone export alone. Analysis of phenotypes also revealed additional functions of the amino acid sensor, Ssy1p. According to literature, Ssy1p activates cleavage of Stp1p and Stp2p in response to lower pH and poor nutrient availability. In turn, these proteins activate amino acid permeases, Mep1p and Mep2p, required for pH neutralization and yeast-hyphal transition respectively. We found that under stressful conditions, Ssy1p results in differential stress response. For instance, while the wild type strain cells were inflated, cells of ΔΔssy1 grew normally on high ammonium concentrations and in the presence of nitrosative stress. In addition, ΔΔssy1 was resistant to osmotic and cell wall stress. Furthermore, ΔΔssy1 was sensitive to heat stress at 37 °C and 42 °C. Since AA conversion to PGE2 was conducted at 37 °C, and this fatty acid results in predisposition to stress which may affect membrane fluidity, we argue that SSY1 was up-regulated in response to heat stress. Although the microarray data failed to reveal genes directly involved in PGE2 production, the analysis of the white-gray-opaque-like (WGOL) switching, suggest that up-regulation of ORF19.4612 was necessary for rough pink (RP) cell formation and conversion of AA into PGE2. At 37 °C in RPMI-1640 medium, RP cells of the wild type strain produce significantly more PGE2 from AA. Δorf19.4612 is defective in RP cell formation after 3 days on conditions similar to those of RPMI-1640 medium. Since ORF19.4612 was expressed after 48 h at 37 °C, is therefore not only implicated in WGOL, but also in PGE2 production.Item Open Access Prostaglandin E2 production by Candida albicans and Candida dubliniensis(University of the Free State, 2011-11) Ells, Ruan; Pohl-Albertyn, C. H.; Kock, J. L. F.; Albertyn, J.English: Arachidonic acid (AA) [20:4(n-6)] is released from infected host cells during Candida albicans infection and may serve as carbon source for yeast growth and as precursor for the production of biologically active eicosanoids, such as prostaglandin E2 (PGE2) by C. albicans. Prostaglandin E2 is an important virulence factor in C. albicans and involved in biofilm formation. Biofilms increase damage in host cells and are more resistant to antifungal drugs than planktonic yeast cells. This study evaluated the production of prostaglandins, PGE2 and PGF2α, by biofilms of C. albicans and the closely related C. dubliniensis. Since, the mechanism involved in this production is still unclear, it was of interest to investigate the effect of different AA metabolism inhibitors on PGE2 production by biofilms of these Candida species. The experiments were done by growing Candida biofilms in the presence of AA as well as cytochrome P450 (CYP450), multicopper oxidase, cyclooxygenase (COX) and lipoxygenase (LOX) inhibitors. The PGE2 and PGF2α concentrations were determined by a monoclonal enzyme-linked immunosorbent assay (ELISA) and verified with LCMS/MS. The results obtained indicate the ability of C. albicans and C. dubliniensis biofilms to produce PGE2 and PGF2α, from exogenous AA. The use of different inhibitors suggested that CYP450s and multicopper oxidases are involved in PGE2 production by these Candida biofilms.Item Open Access Role of polyunsaturated fatty acids during infection of Caenorhabditis elegans(University of the Free State, 2023) Mokoena, Nthabiseng Zelda; Pohl-Albertyn, C. H.; Albertyn, J.During polymicrobial infection, interactions between different microbial species can alter host responses and/or microbial virulence and pathogenesis, often complicating patient treatment and resolution of infection. Polyunsaturated fatty acids (PUFAs) are not only crucial for normal function in mammalian systems, but are also proposed to act as endogenous antimicrobial molecules. Thus, we adopted Caenorhabditis elegans to mimic the Pseudomonas aeruginosa and Candida albicans polymicrobial infections found in humans. We determined the influence of arachidonic acid (AA) and eicosopentaenoic acid (EPA) supplementation on mono- and polymicrobial infection, fatty acid (FA) composition and egg retention of the nematodes, as well as on expression of FA metabolic genes. Supplementation with either AA or EPA in mono- and polymicrobial infections resulted in changes in FA profiles and PUFA biosynthesis pathway. We discovered that the degree of egg retention elicited by C. albicans and P. aeruginosa varied, with C. elegans exposed to both C. albicans and P. aeruginosa showing the highest level of egg retention compared to nematodes infected by either C. albicans or P. aeruginosa alone. Interestingly, the AA supplemented infected nematodes showed an increased level of egg retention, while EPA supplemented infected nematodes showed a significant decrease. Using the C. elegans model, we determined the effects of AA and EPA supplementation on the survival of nematodes with mono- and polymicrobial infection. We showed that the survival of the infected nematodes was influenced by PUFA-supplementation. EPA supplementation effectively reduced C. albicans virulence and inhibited hyphal formation, thus leading to a partial rescue of pathogen susceptibility. However, this was not the case for P. aeruginosa infections. In fact, EPA supplemented nematodes infected with P. aeruginosa were more susceptible. Notably hyphal formation is an important component of Candida pathogenesis in mammals. Furthermore, polymicrobial infection resulted in synergistic virulence. However, C. albicans did not produce any hyphae in the co-infection of either AA, EPA supplemented or unsupplemented nematodes, this suggests that the increase in pathogenesis may be associated with increased P. aeruginosa pathogenesis. To further test the role of EPA in hyphal formation of C. albicans, we hypothesised that cytochrome P450 (CYP450) metabolises EPA to 17,18-epoxyeicosatetraenoic acid (17,18-EpETE), inhibiting C. albicans hyphal formation. We showed that 17,18-EpETE inhibits C. albicans hyphal formation in vitro and in vivo in C. elegans and that inhibitors of mammalian EPA-metabolising CYP450 enzymes, 17-octadecynoic acid (17-ODYA) and 6-(2-propargyloxyphenyl) hexanoic acid (PPOH) restored C. albicans hyphal formation in vivo. Lastly, the role of EPA on the physiology of C. elegans as well as C. albicans in vivo was investigated using gene expression analyses. Among the up-regulated genes, we observed several genes with potential roles in lipid metabolism, hyphal formation, detoxification, stress response and immune response. For instance, we observed an up-regulation of several involved in the synthesis of FAs, including fat-3, fat-4, fat-6, cyp-29A2 and cyp-37A1. Other up-regulated genes were those involved in immune response, such as cyp-37B1, daf-16, fipr-22, ilys-2, lys-5, lys-6, spp-12 and fat-3. Interestingly common genes involved in hyphal formation, such as CAS5, CRZ1, CTA4, ERG11, FCR1, SNQ2, TAC1, TEC1, YOR1 and ZCF3 were also up-regulated. Overall, the benefits of EPA supplementation may be two-fold, by inhibiting virulence factors of C. albicans and stimulating the immune response of the host. Thus, PUFA supplementation might be useful in the treatment of infections in patients caused by C. albicans and P. aeruginosa.Item Open Access Towards unravelling the genome of Avibacterium paragallinarum(University of the Free State, 2009) Roodt, Yolandi; Albertyn, J.; Bragg, R. R.Avibacterium paragallinarum is an avian pathogen and has the ability to cause vast economical losses. This bacterium forms part of the Pasteurellaceae family and factors contributing to pathogenicity, immunogenicity and serotyping are not clearly understood. One of the main questions that were addressed in this study was the identification of genetic tools are that is responsible for the NAD+- independence ability of this organism. NAD+ recycling genes were implicated for this bacterium and Av. paragallinarum seem to follow the recycling pathway as set out for the Pasteurellaceae family. Still the question regarding NAD+- independence remains unanswered as no complete pathway could be implicated for this trait. Furthermore, no plasmid(s) could be isolated that conferred this trait and no complete NAD+ synthesis pathway could be implicated. Only two genes were identified to form part of a NAD+-independent pathway which indicated that Av. paragallinarum may use genetic tools for NAD+-independence as set out for bacteria in general and not as the rest of the Pasteurellaceae family members. Plasmid isolation studies revealed only the plasmid p250 identical to the established plasmid p250 for Av. paragallinarum. Literature reports on two other native plasmids namely pYMH5 and pA14, however, these two plasmids could not be detected in any of the strains used in this study during plasmid screens. This study confirms and illustrates the integration of plasmid p250 within the genome of Av. paragallinarum by different serovars. In order to study this bacterium on a genomic level a whole genome sequencing project was launched. This project experienced vast amount of difficulties regarding the assembly of a complete genome for Av. paragallinarum. This bacterium contains numerous repeated regions within its genome which, with current sequencing technology, prevent the genome assembly into a single chromosome. A complete assembled genome can only be achieved when longer read lengths are available in high-throughput sequencing technologies and will thus enable clarification regarding the repeated regions. A pseudo-assembled molecule was sent to the JCVI for genome annotation. Annotated data revealed that Av. paragallinarum contains a high degree of complexity regarding its cell envelope which may shed light on pathogenicity and immunogenicity problems endured. This bacterium contains numerous mobile and extrachromosomal elements which contribute to the inability to close the genome of Av. paragallinarum. Nine site-specific integrases and 10 transposases were identified. These tools could allow for a high degree of genetic diversity within the Av. paragallinarum specie. Alongside these tools two putative prophages was identified for Av. paragallinarum. One prophage resembles the Mu-like prophages and was termed ФAvpmuC-2M, the other resembles the HP2-like prophages and was termed ФAvpC-2M-HP2.Item Open Access Yeasts from Lesotho: their classification and possible applications(University of the Free State, 2004-11) Tarr, Shahida; Kock, J. L. F.; Albertyn, J.; Smit, M. S.In view of the decline of natural habitats due to urban and industrial development, the need to search for new yeasts is pressing since few natural habitats have been thoroughly investigated for yeast species. Yeasts have not been isolated from Lesotho before despite being an ideal environment for yeasts. In addition, isolation of yeasts able to utilize complex substrates, similar to intermediates in the degradation of chlorophenols would be important in their detoxification. Yeasts with these abilities are usually isolated from polluted environments and their presence from the pristine environment in Lesotho would be unexpected. The species Lipomyces starkeyi and L. tetrasporus were found distributed throughout the various habitats while Debaryomyces hansenii, D. hansenii var. fabryi and D. occidentalis were found in 60% - 70% of the different regions. Debaryomyces polymorphus, Dipodascus spicifer, Galactomyces geotrichum, G. reessii, Kluyveromyces lactis , L. kononenkoae, L. mesembrius, L. spencermartinsiae, Pichia anomala, P. fabianii, P. guilliermondii and Yarrowia lipolytica were site specific. The fatty acid profiles of the isolated lipomycetous yeasts are similar to those reported in literature. This corroborates the value of this phenotypic characteristic in the taxonomy of these yeasts. The PCR products of the ITS region of some of the type strains of the family Lipomycetaceae showed high length variation enabling rapid identification. The type strains, the sub-species and the varieties of the family Lipomycetaceae could be differentiated from each other using RFLP profiles obtained with the restriction enzymes used in combination i.e. Cfo I, HaeIII and MboI. The RFLP profiles for the five Lesotho isolates with atypical carbohydrate patterns could be separated into three groups. The first group, comprising of three isolates (52b, 73, 93), gave similar restriction patterns suggesting that they are the same species. Isolate 58b yielded a distinct profile while isolate 97 had similar sized ITS-PCR (1000bp) to that of L. lipofer and L. tetrasporus . It is important to assess the variation in RFLP profiles between various strains from different habitats of a particular species in order to determine the conserved status of this genotypic character. More strains of a species representing the Lipomycetaceae should be subjected to similar RFLP analysis to further determine its conserved status. The D1/D2 sequence data enabled separation of the five isolates into three groups. The first group, comprising of three isolates, showed 1% nucleotide substitutions to L. starkeyi suggesting that these isolates are probably known Lipomyces spp. The other two isolates yielded sequences that were 99% identical to L. kononenkoae subsp. kononenkoae (isolate 58b) and 100% identical to that of L. tetrasporus (isolate 97) further suggesting that these isolates are probably known Lipomyces species. Isolation of basidiomycetous yeasts from unusual carbon sources has been reported, however, this is the first report of isolation of ascomycetous yeasts able to grow on unusual substrates. The substrate that yielded the highest number of isolates was 1,4 cyclohexanedimethanol. Pichia anomala and Y. lipolytica strains (identity confirmed with D1/D2 sequencing) isolated from 2-chlorobutyric acid could not degrade the 2-chlorobutyric acid probably due to toxicity of low molecular weight organic acids at low pH. The ability of P. anomala isolated from 1,4 cyclohexanedimethanol to grow on dodecane and pristane is unusual and this strain should also be subjected to sequencing to confirm identification. Debaryomyces hansenii, P. anomala, P. fabianii, P. guilliermondii and Y. lipolytica could grow on the cyclohexane derivatives although they preferred a shorter straight chain hydrocarbon as confirmed by higher growth rates in the presence of dodecane. The isolates could not grow in the presence of monoterpenes, although they had been isolated from substrates that were supposed to mimic the structures of monoterpenes and which previously had yielded limonene utilizing Rhodotorula species. The inability of these strains to grow on monoterpenes might be due to toxicity of the monoterpenes, because of their hydrophobicity and lipophilicity resulting in partitioning into the lipid bilayer of cell membranes.