Microbial, Biochemical and Food Biotechnology

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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.
Open Access
ADH2 regulation in the yeast Saccharomyces cerevisiae
(University of the Free State, 2004-05) Khoboko, Mojabatho Portia; Albertyn, J.; Du Preez, J. C.
English: The aim of this study was to investigate the ability of ethanol to repress the expression of ADH2 in the genome of Saccharomyces cerevisiae. To achieve this, an expression cassette (ADH2::LacZ) using LacZ as a reporter gene was constructed using the YIp356R shuttle vector. A 1000 bp up and downstream region, flanking the open reading frame of ADH2, was fused to the 5’-end and 3’-end of the LacZ gene in the YIp356R shuttle vector. Numerous attempts were made to transform the expression cassette (ADH2::LacZ) into S. cerevisiae (strain W303) containing a deleted ADH2 (adh2Δ::URA3), to displace the deletion cassette through homologous recombination, thereby placing ADH2::LacZ in the place of the ADH2 in genome of S. cerevisiae. This was unfortunately not successful and it was decided to use an alternative approach. In this case the expression cassette was cloned into the integrative vector YIplac211 and transformed into S. cerevisiae. For initial confirmation, the yeast transformants were grown on selective plates containing X-gal, which allows for the detection of β-galactosidase activity through the production of blue coloured colonies. The detection of the blue colour confirmed that the expression cassette was successfully constructed and integrated into the genome. Two randomly selected transformants were cultivated on 20 g glucose l-1 as sole carbon source, to study glucose repression and on three different ethanol concentrations to study the effect of ethanol on the expression of ADH2. Selection was maintained by growing the yeast in a URA– chemically defined media (pH 5.5) at 30ºC. Samples were taken at appropriate intervals to perform β-galactosidase assay, assess utilization of substrate (ethanol and glucose), ethanol formation and biomass determination. During growth on 20 g glucose l-1 the production of β-galactosidase was apparent only when glucose concentrations were very low (2.3 g l-1), indicating that glucose levels have to decrease to a critical level before ADH2 expression can resume. The highest final biomass was produced during growth on 20 g glucose l-1. During growth on the three different ethanol concentrations the highest β-galactosidase maximum specific activity was obtained during growth on 20 g ethanol l-1 (3 643 U mg-1) and the lowest during growth on 5 g ethanol l-1 (2 533 U mg-1). Although the maximum specific activity obtained during growth on 30 g ethanol l-1 were higher than that obtained during growth on 5 g l-1, the production rate was the lowest (93 U mg-1h-1) during growth on 30 g ethanol l-1, suggesting that 30 g ethanol l-1 concentration has negative effect on the expression of ADH2. However this slow production might have been due to the slow growth during this cultivation and not due to ethanol repression. The possible repression of ADH2 is further disputed by the high β-galactosidase production on 30 g ethanol l-1.
Open Access
Characterization and expression of protein(s) involved in gold nanoparticle formations by Thermus scotoductus SA-01
(University of the Free State, 2010-05) Erasmus, Mariana; Van Heerden, E.; Albertyn, J.
English: Developments in the biosynthesis of nanoparticles have increased significantly during the last few years as a result of the growing interest in the unique properties displayed by nanoparticles. These particles are extremely small in size and have a large surface to volume ratio, giving them unique physical and chemical properties at this scale that differs considerably from when they are used in larger form. These exceptional properties are used in a wide variety of applications, ensuing nanotechnology to become a multidisciplinary field. Research into application structure types are extended daily and as a result, the next few years will be crucial as applications for nanomaterials in the industry are most likely to be increased. Gold nanoparticles is receiving more and more attention because of its wide variety of uses in optical, electronic, magnetic, catalytic, and biomedical applications, but even more due to them being the most stable of all the metal nanoparticles. Several methods are used to produce these metal nanoparticles, but are mostly making use of toxic chemicals in the synthesis protocol, which are harmful to the environment and human health. To overcome this problem, researchers are making use of more “greener” alternatives through the use of biological systems and microorganisms in nanoscience and nanotechnology. These microorganisms have unique potential in producing nanoparticles that are environmentally friendly and display different shapes, sizes and distributions. Among the different microorganisms used, bacteria have received the most attention in the nanoparticle production process, but have not been as successful as chemical synthesis to produce monodisperse noble metal nanoparticles. In this study, successful gold reduction and nanoparticle formation with different shapes, sizes and distribution was obtained; however, these particles were not monodisperse. This was achieved with a thermostable protein of ± 70 kDa that was identified as an ABC transporter, peptide-binding protein and which was purified from Thermus scotoductus SA-01; an extremophile and thermophilic bacterium that was isolated from groundwater samples from Mponeng (a deep South African gold mine in the Witwatersrand Supergroup operated by AngloGold Ashanti) at a depth of 3.2 km with ambient rock temperature of 60ºC. The protein was expressed in Escherichia coli and Thermus thermophilus HB27, a mesophilic and a thermophilic expression host respectively. It was found that the expression host might have an influence on the way the protein is folded and therefore influence nanoparticle formation. Expression studies was also done on the protein that either included or excluded Histidine-Tags and a leader peptide, but it was found that neither the His-Tags nor the leader peptide had any influence on the nanoparticles produced. Gold reduction and nanoparticle formation was obtained through reduction of a surface exposed disulphide bond in the ABC transporter, peptide-binding protein, using sodium dithionite as electron donor and reducing agent. In general it was found that nanoparticle formation was dependant on environmental parameters but control of this process was not complete. Chemical reduction did influence the nanoparticle formation process in some instances, but overall it could be seen that the presence of the protein played a significant role in slowing down the reaction rate, yielding a level of control over the nanoparticles produced and ensuring a more environmentally friendly, biological process for the production of gold nanoparticles.
Open Access
Characterization of the putative haemagglutinin in haemophilus paragallinarum
(University of the Free State, 2001-12) Barnard, Tobias George; Van Heerden, E.; Albertyn, J.; Bragg, R. R.
Haemophilus paragallinarum , the causative agent of infectious coryza (IC), an acute respiratory disease in chickens and fowl, was first isolated in 1931 by De Blieck (1932). The first serious, documented outbreak in South Africa occurred in 1968 (Buys, 1982) on a multi-age layer-farm, soon the bacterium spread to most large production sites and established itself as the most common bacterial infection in layers (Bragg, 1995). The disease has a low mortality rate but leads to a drop in egg production of up to 40 % in layer hens and increased culling in broilers and thus poses significant financial liability to chicken farmers (Arzay, 1987; Bragg, 1995). One of the reasons for the success of survival for this bacterium is that after recovering from infection, birds become carriers of the bacterium, therefore aiding the spread of H. paragallinarum (De Blieck, 1948). Secondly, the bacterial strain belongs to one of nine serovars, which makes combating the spread of the disease through inactivated vaccination ineffective especially due to low cross protection among these serovars. (Rimler et al., 1977; Kume et al., 1980a). Various potential factors have been identified as potential virulence factors, e.g. the haemagglutinin protein. This protein plays a crucial role in adherence of the bacteria to the host's cells and is considered a possible virulence factor (Sawata et al., 1982; Yamaguchi et al., 1989). Sawata and co-workers (1982) reported at least three different haemagglutinins from H. paragallinarum strain 221 with one, HA-L, being serovar specific with the other common types shared by the different serovars in one serogroup.It would therefore be important to understand the working and interaction of the various virulence factors of H. paragallinarum, especially the haemagglutinins, in order to combat this bacterium.
Open Access
Charactt[sic]erization of a plasmid conferring NAD independence in Haemophilus paragallinarum
(University of the Free State, 2003-05) Van Zyl, Anna Elizabeth; Albertyn, J.; Bragg, R. R.; Van Heerden, E.
English: Members of the family Pasteurellaceae are classified in part by whether or not they require NAD+ supplement for growth on laboratory media. It is known that this phenotype is determined by a plasmid whose presence allows NAD+-independent growth of Haemophilus paragallinarum. In this study, this 6-kb plasmid, which was previously shown to be responsible for NAD+ independent growth of H. paragallinarum on defined media, was isolated. Isolated plasmid DNA was shredded by sonification and subcloned into vector PGEM-T easy. The recombinant plasmid was transformed into E.coli the transformants isolated were sequenced. Sequence analysis revealed one open reading frame of 1119bp that is predicted to encode a protein with a molecular mass of 43kD. Compared with the sequence databases, this protein was found to have significant sequence homology to Quinolinate phosphoribosyltransferase of Bacillus anthracis this enzyme is responsible for the production of nicotinic acid mononucleotide (NAMN) from nicotinate and quinolinate. A 3284bp nucleotide fragment of the plasmid revealed four additional open reading frames. Proteins encoded on this fragment of the plasmid all have significant homology to proteins from H. influenzae of which all have functions related to production and immunity of the bacteriocin haemocim. This bacteriocin produced by most type b-encapsulated strains of H. influenzae, is toxic to virtually all non-type b strains of H. influenzae idependent of encapsulation. This bacteriocin is thought to inhibit DNA synthesis, of susceptible strains. Purification of this bacteriocin and testing its toxicity to other pathogens as a possible antimicrobial drug might form the bases of a future study. Previous work has indicated that plasmid bearing strains of H. paragallinarum are less virulent, thus creating the possibility that more virulent wild type strains can be transformed and used as live vaccines. The influence of transformation with this plasmid on other members of the family Pasteurellaceae and the possibility of creating live vaccines should be further investigated Since species of the genus Haemophilus cannot easily be transformed with plasmid, this naturally occurring plasmid could be modified to create a vector, which has specific application in the transformation of Haemophilus species.
Open Access
Cloning and characterization of the capsule transport gene region from Haemophilus paragallinarum
(University of the Free State, 2001-11) De Smidt, Olga; Albertyn, J.; Bragg, R. R.; Van Heerden, E.
English: Haemophilus paragallinarum causes an acute respiratory disease of chickens known as infectious coryza (IC), a disease first recognized as a distinct entity in the late 1920's. Since the disease proved to be infectious and primarily affected nasal passages, the name "infectious coryza" was adopted (Blackall, 1989). Infectious coryza may occur in both growing chickens and layers. The major economic effect of the disease is an increased culling rate in meat chickens and a reduction in egg production (10-40%) in laying and breeding hens. The disease is limited primarily to chickens and has no public health significance (Yamamoto, 1991). The most common clinical signs are a nasal discharge, conjunctivitis, and swelling of the sinuses and face. Various sulfonamides and antibiotics are useful in alleviating the severity and course of infectious coryza; however, none of the therapeutic agents has been found to be bactericidal. Relapse often occurs after treatment is discontinued, and the carrier state is not eliminated (Yamamoto, 1991). All the commercially available bacterins against IC, consist of inactivated broth cultures of a combination of two or three different serotypes. Although vaccines against IC have been used in South Afr ica since 1975, it became apparent in the 1980s that the vaccines were becoming less effective in controlling the disease (Bragg et al., 1996). This could have been due to the emergence of a previously unknown serovar, or even serogroup and the possibility of changes in the population dynamics. Vaccine efficiency is therefore a problem and an alternative to available vaccines is needed. Capsules have long been associated with virulence properties of bacteria. The role that the capsule play in the virulence of bacterial species related to H. paragallinarum has been investigated by several workers (Kroll et al., 1988; Inzana et al., 1993; Boyce and Adler, 2000). Mutation, deletion or allelic exchange of gene/s involved in the transport of capsule polysaccharides in related species like Haemophilus influenza, Actinobacillus pleuropneumoniae and Pasteurella multocida, resulted in organisms with reduced virulence. The noncapsulated mutants of Actinobacillus pleuropneumoniae reported by Inzana et al. (1993) showed extreme stability and induceda protective immune response without any symptoms of disease. This not only proves the capsule�s involvement in virulence of bacteria but also offers the opportunity to investigate the possibility of producing live vaccines. The aim of this study was an attempt to understand the genetic organization of the capsular genes of H. paragallinarum in comparison to related HAP organisms and the possibility of producing a mutant lacking the capsule. The goals were: 1. Isolation and cloning of the capsule transport gene locus. 2. Sequencing and characterization of the locus 3. Transplacement of a gene/s to produce a noncapsulated mutant of H. paragallinarum.
Open Access
Cloning of the XynA gene from Thermomyces lanuginosus and expression in Saccharomyces cerevisiae
(University of the Free State, 2001-11) Nel, Sanet; Albertyn, J.; Van Heerden, E.; Bragg, R. R.
English: The xylanase from Thermomyces lanuginosus (XynA) was cloned into two shuttle vectors, pRS416 (single copy vector) and pRS426 (multi-copy vector) adjacent to a PDC1 promoter (designated pRS416:XynA and pRS426:XynA). An expression cassette for this xylanase was constructed by cloning of the XynA gene into a modified a-agglutinin (Aga1) gene from Saccharomyces cerevisiae. This modification entailed the deletion of the binding domain coding region of the Aga1, and the cloning of the XynA gene into this deleted binding domain region, which is flanked by a stalk-like protein coding region. This fusion protein was cloned into two shuttle vectors (pRS416 and pRS426), flanking the PDC1 promoter (designated pRS416:Aga1::XynA and pRS426:Aga1::XynA). The aim of the cassette was to immobilize the expressed enzyme on the cell surface of the yeast cell with the expression of the xylanase on the stalk of the Aga1, however, extracellular secretion of the enzyme was obtained upon expression. Enzyme assays performed on pRS416:XynA and pRS426:XynA yielded very low activity [0.1505 U/ml (2.5088 nKat/ml) and 0.0909 U/ml (1.5153 nKat/ml) respectively], whereas pRS416:Aga1::XynA and pRS426:Aga1::XynA yielded activities of 1.7035 U/ml (28.3973 nKat/ml) and 1.7319 U/ml (28.8707 nKat/ml) respectively. The partial characterization of this extracellular secreted recombinant xylanase (pRS416:Aga1::XynA and pRS426:Aga1::XynA) yielded an optimum temperature of 70 °C and an optimum pH of 6.0-7.0. Thermal stability for the recombinant xylanase was determined for temperatures 50 °C, 60 °C and 70 °C, and the activation energy for pRS416:Aga1::XynA and pRS426:Aga1::XynA were calculated as 34.86 kJ/mol and 53.59 kJ/mol respectively.
Open Access
Cloning, expression and characterization of tannase from Aspergillus species
(University of the Free State, 2002-01) Albertse, Ewald Hendrik; Van Heerden, E.; Albertyn, J.; Litthauer, D.
Tannin Acyl Hydrolase (E.C. 3.1.1.20) is commonly referred to as tannase. Teighem accidentally discovered this unique enzyme in 1867 (Teighem, 1867). He reported the formation of gallic acid when two fungal species were exposed to an aqueous solution of tannins. The fungal species were later identified as Penicillium glaucum and Aspergillus niger (Lekha & Lonsane, 1997). Tannase is responsible for the hydrolysis of ester and depside linkages in tannins to liberate gallic acid and glucose. This was a very interesting observation due to the usual complexation of proteins with tannic acid and naturally occurring tannins to form water insoluble complexes that inactivates enzymes (Haworth et al., 1985). Tannins have since been shown to be the natural substrate for the tannase enzyme. The enzyme also attacks gallic acid methyl esters, but it possesses high specificity towards the acyl moiety of the substrate. It has been known that certain moulds and fungi belonging to the species Aspergillus and Penicillium produce the enzyme (Rajakumar & Nandy, 1983). According to the work done by Yamada et al., (1968) the enzyme was mainly found intracellularly although the culture broth also contained the enzyme. Aspergillus niger, A. flavus and A. oryzae were found to be the best tannase producers on tannic acid as a sole source of carbon. From these growth studies it became evident that the tannase enzyme was an inducible enzyme (Gupta et al., 1997, Jean et al., 1981 and Mattiason & Kaul, 1994).
Open Access
Construction of self-sufficient CYP153 chimeras
(University of the Free State, 2010) Randall, Charlene; Smit, M. S.; Albertyn, J.
Cytochrome P450 monooxygenases are a superfamily of heme-containing enzymes that are found in all domains of life. P450s catalyse diverse reactions, many of which are difficult reactions to accomplish, even with the use of chemical catalysts. One such reaction is the terminal hydroxylation of alkanes, the first step in alkane degradation. The CYP153 family, found in alkane-utilising bacteria, is one of only two P450 families that can catalyse this reaction. One of the long-term goals of our group’s research is the directed evolution of terminal alkane hydroxylases, using preferably a self-sufficient terminal alkane hydroxylase as the starting point. There are, however, no naturally self-sufficient CYP153s. Therefore, the first aim of this study was to create a self-sufficient CYP153 by fusing a CYP153 heme domain to the reductase (PFOR) domain of a self-sufficient P450. The gene encoding the heme domain of CYP153A6 from Mycobacterium sp. HXN-1500 was ligated to the DNA encoding the PFOR reductase domain of CYP116B3 from Rhodococcus ruber DSM 44319. The fusion gene was expressed in E. coli using a pET28a plasmid. The resulting protein was misfolded and expressed mainly in the insoluble fraction in the form of inclusion bodies. Factors possibly responsible for this were investigated including the expression conditions, the effect of an N-terminal His-tag on protein folding, the effect of the linker region sequence on protein folding, and the possibility of rapid expression resulting in protein misfolding, but with all of these experiments only low levels of P420s were observed in the soluble fraction and no P450 forms were detected. A whole-cell octane bioconversion experiment conducted using the expressed fusion revealed the presence of P450 forms of the protein, but no 1-octanol was produced, indicating that octane possibly facilitated the correct folding of the CYP153A6 heme domain but that the heme domain and the PFOR reductase domain were unable to form a functional complex. This theory does, however, require further research. In this study, CYP153A6 and its redox partners, ferredoxin reductase and ferredoxin were expressed in E. coli using the pET28b plasmid. Expression of CYP153A6 in E. coli using this plasmid has not previously been reported in literature. Whole-cell octane bioconversions conducted using the expressed CYP153A6 resulted in the production of 42 mM of 1-octanol after 24 hours, with the P450 concentration increasing during this time, a trend which was also observed with the fusion. The second aim of this study was to apply cassette PCR to the fusion to generate diverse selfsufficient terminal alkane hydroxylases, which would provide the genetic diversity required for directed evolution. Degenerate primers designed according to conserved N- and C-terminal regions of CYP153A amino acid sequences were used to amplify internal CYP153A gene fragments from environmental DNA extracted from enrichments of soil sampled at a dieselcontaminated site in the Eastern Cape. Three different sequences were identified, one of them being CYP153A6, which was excluded from the rest of the study. The two remaining sequences and two sequences originating from another project using environmental DNA from samples from the Beatrix Goldmine in the Free State were linked to the 5’- and 3’-ends of the CYP153A6 gene, generating full-length chimeric CYP153A genes. Because of the fact that the expression of the fusion was unsuccessful, the functionality of these chimeric genes was tested using the above-mentioned functional CYP153A6 operon. Expression was observed in the insoluble fraction in the form of inclusion bodies, with the proteins being misfolded. A whole-cell octane bioconversion did not result in P450 forms of the proteins and no 1-octanol was produced, indicating that these chimeras were non-functional.
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.
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.
Open Access
The development of a CRISPR-Cas9 gene editing system for Cryptococcus deneoformans
(University of the Free State, 2019-01) Du Plooy, Lukas Marthinus; Albertyn, J.; Sebolai, O. M.; Pohl, C. H.
The pathogenic yeasts, Cryptococcus neoformans and C. deneoformans, are responsible for potentially fatal meningoencephalitis in immunocompromised individuals, most notably in patients who have AIDS. Only three drugs are commonly administered to treat cryptococcal infections and most are not readily available in developing countries most affected by the AIDS pandemic. Cheaper and more widely available drugs are therefore needed. Developing molecular methods to disable genes encoding virulence factors could help to elucidate the mechanism of action of potential drugs against these fungal pathogens. Previously, researchers mostly relied on biolistic transformation to deliver DNA into cells for homologous integration into the targeted site. Recent developments with CRISPR-Cas9-based systems for gene targeting made it possible to utilise another transformation technique, electroporation, to knock genes out. In this study, a one-step CRISPR- Cas9 system was developed to be delivered into cells with electroporation. This system consists out of two plasmids, carrying a nourseothricin and G418 resistance marker respectively, as well as a CAS9 gene. A third plasmid was used to construct guide DNA, which was then amplified and cloned into the two CRISPR-Cas9 plasmids carrying the CAS9 gene. The plasmids carrying the CRISPR-Cas9 components were maintained transiently for expression of the CRISPR-Cas9 genes before these constructs were degraded by the cells. Donor DNA was also constructed to remove parts of the biosynthetic genes ADE2 and HIS3 to obtain adenine and histidine auxotrophic mutants with visually discernible phenotypes. A second round of transformation can then introduce new donor DNA to repair these auxotrophic genes whilst disrupting virulence genes for virulence studies. Electroporation proved to be very inefficient in this study and gene targeting was unsuccessful. Using large amounts of plasmid and donor DNA yielded the best results, although no more than 8 colonies were seen on a few selective media agar plates. Inefficient transformation could be due to old and faulty electroporation equipment or ineffective delivery of the electrical current to cells. In the future, other transformation methods will be employed to deliver the plasmids constructed in this study into C. deneoformans cells. This system can then be used to remove virulence genes to study their role in infection, which could help to elucidate the mechanism of action behind potential drugs. For instance, a capsule-less mutant could reveal what effect a drug targeting capsule synthesis will have on the ability of these yeasts to cause disease.
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.
Open Access
The development of a wide range CRISPR-Cas9 gene editing system
(University of the Free State, 2019-01) Bisschoff, Eduvan; Albertyn, J.; Pohl-Albertyn, C. H.
CRISPR is a revolutionary method to effectively and efficiently alter the genomic make-up of an organism. Unlike any other genetic engineering tool or technique, CRISPR is remarkably cheaper, simpler and faster to perform. In biotechnology, the best eukaryotic organism for research is yeast, due to their fast growth rate and ease of manipulation compared to multicellular organisms. Hence the aim of the study was the development of a wide range CRISPR-Cas9 system for a wide variety of different yeasts for easy and fast gene editing. The system were validated in Saccharomyces cerevisiae and six other non-conventional yeast. System construction began with the incorporation (separately) of three different optimized CAS9 (optimized for expression in Pichia pastoris, Candida albicans and Homo sapiens) genes into the wide range pKM180 vector. The three different CAS9 construct were then tested for correct expression of the Cas9 protein and the effects thereof in all the yeasts. Through western blot analysis it was observed that all three of the different Cas9 proteins were expressed successfully in the different yeasts. However, all of the Cas9 proteins had a negative effect on the growth of the yeast. For the completion of the CRISPR-Cas9 system, a Ribozyme‐gRNA‐Ribozyme cassette was incorporated into the wide range CAS9 vector, containing the C. albicans optimized CAS9. The system was then validated with successful disruption of the ADE2 gene in all of the yeasts. This proved that the wide range CRISPR-Cas9 system was applicable in a wide variety of different yeasts, thus allowing for rapid, cost-effective genetic manipulation of biotechnologically relevant yeast strains.
Open Access
Effect of cultivation conditions on the dimorphism of and heterologous protein production by Arxula adeninivorans
(University of the Free State, 2007-11) Jansen, Arina Corli; Du Preez, J. C.; Albertyn, J.; Killian, S. G.
Arxula adeninivorans strain LS3, a dimorphic yeast with potential for biotechnological applications, has in recent years has been studied for the production of heterologous proteins. The growth kinetics of this species and the effect of environmental conditions on its morphology under controlled cultivation conditions have not been well documented. Because genetic transformation might affect the yeast physiology, a comparative investigation of the growth characteristics of strains LS3, G1211 (LEU2+) (the auxotroph derived from strain LS3) and LS3/pXynA, derived from strain LS3 by transformation with the Thermomyces lanuginosus xylanase gene under the control of the TEF1 promoter, was conducted, focusing on the effect of temperature and dissolved oxygen tension (DOT) on the morphology and growth parameters of the strains. This xylanase gene (XynA) was integrated in the 25S rDNA locus and expressed under control of the strong constitutive Arxula-derived TEF1 promoter. The plasmid copy number integrated into the rDNA locus was unknown. Little to no activity was found with the A. adeninivorans LS3 transformants, namely only 5.86 nkat ml-1-1 (0.35 U ml) compared to the 4 418 nkat ml-1-1 (265 U ml) obtained with the T. lanuginosus strain SSBP positive control. The protein itself might have been defective and since it accumulated intracellularly, this could also have resulted in the diminished activity observed. Cultivation in a temperature gradient incubator over a wide temperature range revealed significant differences in the maximum specific growth rates of the strains LS3 and G1211(LEU2+), namely 0.48 and 0.52 h-1, respectively. The minimum and maximum temperatures were 18 and 46°C, respectively, with the optimum temperature in the range of 37 to 39°C. In accordance with the literature, in shake flask cultures the morphology of LS3 changed with an increase in temperature from predominantly budding cells at 30°C to pseudohyphae at 42°C and resembled a mycelial culture at 45°C. By contrast, the morphology of strain G1211 (LEU2+) remained pseudohyphal at 45°C. During batch cultivations no true hyphae were observed, but a change in morphology, from budding cells to pseudohyphae, was observed during cultivation at different dissolved oxygen concentrations at different temperatures for LS3 and LS3/pXynA. G1211 (LEU2+) remained a pseudohyphal culture. The critical dissolved oxygen concentration (Ccrit) value, determined from the intersection of tangent lines of a plot of specific rate of oxygen uptake as function of dissolved oxygen concentration, of strains LS3 and G1211 (LEU2+) was 0.016 mmol l-1-1 (9% of saturation) and 0.013 mmol l (7% of saturation), respectively. This revealed that A. adeninivorans strain G1211 (LEU2+) had a slight but significantly lower Ccrit than strain LS3. This study showed that insertion of plasmid pAL-ALEU2m has a significant effect on the specific growth rate and on the morphology of Arxula adeninivorans LS3. Insertion of plasmid pXynA had only a slight effect on the morphology but no effect on the specific growth rate.
Open Access
An epidemiological survey of Newcastle disease virus in South Africa
(University of the Free State, 2001-05) Mashope, Barbara Keitumetse; Albertyn, J.; Bragg, R. R.; Van Heerden, E.
English:The primary objective in this study was the investigation of the molecular epidemiology of NDV strains isolated during epizooties in South Africa in 1998. Isolates purported to be NDV were collected from the Onderstepoort Veterinary Institute, the University of Pretoria, and EarlyBird Farms, Standerton. Four of the twenty-two isolates collected were identified as NDV, and successfully grouped into genotype VIIb, previously described by Herezeg et al., (1999). The isolates were propagated in 9-10 day-old embryonated SPF eggs. Embryo mortality was observed, allantoic fluid harvested, and genomic RNA extracted using TRIZOL ® reagent, containing guanidine thiocyanate. An RT-PCR based detection method was used to screen the isolates received (Ballagi- Pordány et al., 1996). The optimised method entailed initial reverse transcription in a reaction mixture containing IIlM random hexamers p(dN)6, 200 U Moloney Murine Leukemia (M-ML V) RT and 25 U RNasin. Aliquots of 5 ul, of the cDNA mix were used as template in subsequent PCR amplification reactions. A 1349bp region of the fusion protein gene was amplified using primers ONDVlaa and ONDV 4aa. Fusion protein amplicons were obtained from field isolates, M89/98 (Ost Pool I), M89/98 (Av Pool 2), M57/98, and M308/98 obtained from OP. Restriction enzyme profiles of the fusion protein amplicons using restriction enzymes HinfI, BstO I, and Rsa I displayed fragment patterns that allowed their grouping into genotype VIIb, described by Herezeg et al., (1999). Non-group specific bands observed upon digestion of the amplicon of strain M308/98 with Hinf I released fragments not consistent with those observed in genotype VIIb isolates. These fragments suggest the presence of mutations in this area of the genome. Amplification of a region comprising 88% of the matrix protein gene was facilitated using primers Ml and M2. These amplicons were subjected to RE analysis using restriction enzymes Mbt) I and Hinf I. Analysis of the restriction profiles produced revealed that these four strains were not re-isolated forms of the commonly used live vaccine LaSota strain. A region of the fusion protein gene encoding the fusion protein cleavage activation site was amplified by means of a nested peR, and sequenced. Initial peR amplified a region spanning from the M gene nt 778 to F gene nt 545 using primers Kl and K2. These amplicons were purified and used as template in a nested peR using primers MV1 (M gene nt 1163) and B2 (F gene nt 470). Sequence analysis revealed that the amino acid sequence at the fusion protein cleavage site of strains M89/98 (Ost Pool I), M57/98,and M308/98 was RRQKR -l-F, indicative of velogenie strains. Strain M89/98 (Av Pool 2) displayed a sequence at the fusion protein cleavage site that is characteristic of lentogenie strains GRQGR-l-L. This finding suggests that genotype VIIb isolates are heterogeneous, composed of strains of varying pathogenicity . Phylogenetic analysis based on a 378nt long region of the nested peR amplicon allowed the grouping of all these isolates into genotype VIIb. A 1.5-1% divergence was observed between group VIIb isolates collected in 1993, and 1995, and those used in this study, collected in 1998. This genetic distance is consistent with a 0.5-1% divergence in strains per year. The remaining field strains not detected by RT-peR, but that displayed embryo mortality indicative of pathogenic agents were subjected to HAlHI tests. Strains M193/98 (Ost Pool 6), M183/98 (Ost Liv B), and M193/98 (Ost Pool 5) agglutinated chicken erythrocytes. Haemagglutination was not inhibited by anti-NDV serum. These results led to the suspicion of the presence of an unidentified pathogen, most likely avian influenza, as all three samples were collected from ostrich hosts, from which this virus has previously been isolated in South Africa.
Open Access
The epidemiology and antifungal sensitivity of clinical Cryptococcus neoformans and Cryptococcus gatti isolates from Bloemfontein, South Africa
(University of the Free State, 2013-06) Ogundeji, Adepemi Olawunmi; Sebolai, O. M.; Pohl, C. H.; Kock, J. L. F.; Albertyn, J.
English: In this dissertation, an attempt was made to study the epidemiology of cryptococcosis by first estimating the incidence rates over a two-year period, 2011 and 2012. The major findings from this part of the study included establishing that: 1) cases were more prevalent among Blacks (Africans, Coloureds and Indians), and this is in line with the assertion by WHO that diseases such as cryptococcosis are more poverty-related, 2) the distribution pattern of cryptococcosis across different age groups mirrored that of HIV-infected persons, and 3) the number of cryptococcosis cases were quite low for the study period (representing less than 0.1 % of the Bloemfontein population), this was surprising and unexpected given the huge HIV positive population in South Africa, and by extension in Bloemfontein, that is at risk of acquiring this AIDS-defining illness. It is documented in literature that the currently employed methods for the routine diagnosis of cryptococcosis often yield inconsistent test results, thereby; influencing the number of reported cases, which are important for health officials. But more importantly, these inconsistencies have far reaching consequences as they may negatively influence patient outcomes. Therefore, we sought to investigate the usefulness of molecular methods in identifying the etiological agents of cryptococcosis viz. Cr. neoformans and Cr. gattii. Here, the ITS, including the 5.8 gene, intra-specific variation between the tested strains allowed for their delineation into three traditional varieties of Cr. neoformans. To be specific, we identified: 1) 51 strains of Cr. neoformans var. grubii, 2) 13 strains of Cr. neoformans var. neoformans, and 3) 6 strains of Cr. neoformans var. gattii. Given the geographical distribution of Cr. gattii, thought to be limited to the tropics, we sought to confirm the six positive cases obtained from the molecular identification study by cultivating all 70 strains on CGB media. Here, only the six strains of Cr. gattii (constituting Cr. neoformans var. gattii) were able to turn the media blue via hydrolyzing glycine whereas all 64 Cr. neoformans (constituted by Cr. neoformans var. neoformans and Cr. neoformans var. grubii) strains we unable to do so. Thus confirming the molecular test results. Perhaps, the important finding from the molecular study, is the uncovering of a restriction site for the enzyme SspI, which is present only in the distinct species, Cr. neoformans but absent in the distinct species, Cr. gattii. This is important as this eliminates sequencing from the identification process, thus shortening the time required to obtain test results and simultaneously cuts down the operational costs. In addition, it also makes it easier to optimise the protocol, as laboratory technicians will require no specialised training. Although a patient’s outcome is dependent on the timely release of an accurate diagnosis, treatment is also crucial. Today, the widespread usage of antifungals has led to increased resistance. Therefore, there is a constant need to find alternative drugs in order to improve patient outcomes. In this part of the study, we considered antimitochondrial drugs i.e. aspirin and oligomycin, as possible candidate drugs for controlling the growth of Cr. neoformans and Cr. gattii. In vitro susceptibility results, based on a direct comparative experiment, revealed that aspirin was more inhibitory than oligomycin, with 1 mM aspirin yielding at least 70 % growth reduction. Meanwhile, the checkerboard assay revealed that aspirin was not synergistic with fluconazole, however; it was indifferent, which is a frequent outcome in combined therapy. In future, it will be prudent to directly compare aspirin with fluconazole. Nonetheless, in this study, aspirin was proven to be useful as an antifungal agent with the highest concentration tested, 1 mM aspirin, being well within the recommended doses in the blood.
Open Access
Establishment of serological and molecular techiques to investigate diversity of psittacine beak and feather disease virus in different psittacine birds in South Africa
(University of the Free State, 2004) Kondiah, Kulsum; Bragg, R. R.; Albertyn, J.
Psittacine beak and feather disease (PBFD) is a readily recognizable disease of wild and captive psittacines in Australia but it is also a problem worldwide wherever captive species are bred. The disease caused by beak and feather disease virus (BFDV) is characterized by the progressive development of feather dystrophy and loss. Although the occurrence of PBFD in South Africa has been reported only recently, it is already rampant and threatens the extinction of the endangered Cape parrot and black-cheeked lovebird. Currently no vaccine for PBFD is commercially available but the loss of approximately 10-20% of breeding stocks of psittacines annually in South Africa alone is enough to realise the importance of one. Genetic and antigenic differences in BFDV are significant aspects for production of a vaccine but the lack of a culture system for BFDV has limited studies into its genetics, antigenicity and pathogenicity. The objective of the study thus became to establish techniques that could be used to investigate genetic and antigenic differences that may be present in BFDV in South African psittacines. Molecular investigations involved the testing dried blood samples for BFDV nucleic acid using polymerase chain reaction (PCR). A region within the Rep gene was amplified and digested with HaeIII to yield restriction length fragment polymorphisms (RFLPs). Six RFLPs were identified, cloned, sequenced (UFS 1- 6) and phylogenetically analysed. BFDV was purified from body organs of PBFD- affected birds by cesium chloride density gradient centrifugation and fractions tested by PCR and haemagglutination (HA) assays. BFDV-specific antibodies were raised in two rabbits by inoculation with purified BFDV in Freund’s incomplete adjuvant and tested by haemagglutination inhibition (HI) assays and an enzyme-linked immunosorbent assay (ELISA). HA and HI assays were attempted using erythrocytes from African grey parrots and Brown-headed parrots. HI assays were also used to test parrot sera for the presence of antibodies to BFDV. UFS 1-6 were closely related to known BFDV isolates and UFS 1 may represent a unique genotype in South Africa as it was separated from its closely related isolates by a 90% bootstrap value. UFS 3, 4 and 5 isolated from budgerigars belong to the budgerigar lineage as they clustered with isolate BG3-NZ. Together with three previously identified genotypes, UFS 1 indicates the introduction of BFDV into southern Africa on four separate occasions. Purification of BFDV from organs was successful but yielded low titres possibly because low quantities of virus were present in the organs or because little virus was circulating in the bird upon its death. PCR and HA assays confirmed the presence of BFDV in fractions; the two results correlated well. HA and HI assays were successfully established using erythrocytes from African grey parrots and Brown-headed parrots. Antibodies to BFDV were successfully detected in sera of three parrots by the HI assay. However, the assay could not detect non-psittacine raised antibodies (rabbit-raised) due to non-specific reactions. BFDV-specific antibodies were successfully raised in rabbits and were verified by the use of an ELISA. The high level of genetic diversity observed in the study compels further investigation into the genetics of BFDV as such levels of diversity may become a limiting factor in the applicability of PCR as a diagnostic test. The entire diversity of BFDV has not been studied and future work may lead to the identification of more BFDV strains, an important factor in vaccine development. The rabbit- raised antibodies together with the HA and HI assays and ELISA can be used to study the antigenic differences that may be present in known BFDV isolates that may also lead to the identification of more strains of the virus.
Open Access
Expression and localization of four putative fatty aldehyde dehydrogenases in Yarrowia lipolytica
(University of the Free State, 2006-01) Müller, Walter Joseph; Albertyn, J.; Smit, M. S.
English: The dimorphic fungus Yarrowia lipolytica is an n-alkane assimilating yeast. During nalkane oxidation toxic fatty aldehydes are formed that are further oxidized by fatty aldehyde dehydrogenases (FALDH) to carboxylic acids that then enter the β-oxidation pathway. Very little research emphasis has been placed on FALDHs in yeasts and their precise role in n-alkane metabolism. This study aimed at contributing to the limited knowledge of yeast FALDHs and in particular the four putative Y. lipolytica FALDHs (YlFALDH1 - 4) that were recently identified in the fully sequenced genome of Y. lipolytica E150. The contribution made from this study to YlFALDHs was with reference to their promoter expression and subcellular localization. The promoter and terminator of each YlFALDH was initially used to construct reporter cassettes in conjunction with β-galactosidase (lacZ) by utilizing the Sticky-end PCR (SEP) and Enzyme-free cloning methods. These two methods proved to be unsuitable for the expression study. The promoter region of each YlFALDH was then cloned into the pINA781 expression vector containing lacZ to study the expression further. With the aid the pINA781 integrative vector and qualitative plate assays, with 5-bromo-4-chloro-3- indolyl-β-D-galactosidase (X-gal), it was observed that the promoters of YlFALDH1 and 2 were inducible by dodecane and hexadecane but not by oleic acid, glucose or glycerol. The promoter of YlFALDH2 also seemed to display the same level of transcriptional strength as the inducible POX2 promoter. Induction of the YlFALDH3 and 4 promoters was not observed. Localization of the YlFALDH proteins was studied with the aid of green fluorescent protein (GFP) from Aequorea victoria and putative localization sequences (LS) from each YlFALDH isozyme. The putative Y. lipolytica LSs comprised of the last 150 bp of the COOH-terminal of the YlFALDH proteins. These LSs were modeled from Rattus norvegicus FALDH that possesses a 35 amino acid hydrophobic protein anchor at its COOH-terminal. For localization studies, chimerical JMP5 molecules were created with an inducible ICL1 promoter, GFP and putative Y. lipolytica LS from each isozyme. Chimerical molecules were also constructed with a pKOV136 vector that contained a constitutive TEF promoter, a GFP-LS fragment (from a JMP5-chimera) and LIP2 terminator. No fluorescence was observed with epifluorescence or confocal laser microscopy when either of the JMP5- or pKOV136-chimeras were transformed into Y. lipolytica E150 and Po1g respectively. Consequently the subcellular localization could not be identified.
Open Access
Fatty alchohol and fatty aldehyde dehydrogenases of Yarrowia lipolytica
(University of the Free State, 2005-05) Matatiele, Puleng Rose; Smit, M. S.; Albertyn, J.
The cytochrome P-450 monooxygenase and b-oxidation systems of alkane-utilizing yeasts have been studied extensively, whereas very little is known about the fatty (long chain) alcohol and fatty aldehyde oxidizing enzymes. With the recent completion of sequencing of the genome of Yarrowia lipolytica, an alkane-degrading yeast, several putative aldehyde dehydrogenases (ALDHs) have been identified. Four of these were identified as fatty ALDHs (FALDHs). Northern blot analysis and RT-PCR showed that one of the FALDH genes, labelled FALDH4, is induced during growth of Y. lipolytica on alkanes, whereas another aldehyde dehydrogenase gene, labelled ALDH1, was constitutively expressed. Functional analysis of the four FALDH isogenes was initiated by single gene deletion of the four fatty aldehyde dehydrogenase isogenes in all possible combinations. The Cre-loxP recyclable tools system was used for gene disruption. Growth properties of the triple and quadruple deletion strains on alkanes were investigated. A slightly arrested growth in hexadecane was observed in two strains, the triple deletion mutant with intact FALDH2 isogene and the quadruple deletion mutant with all four FALDH isogenes deleted. Very strong hydrophobicity during growth of these mutants in hexadecane was also observed. At this stage one can only say that disruption of FALDH isogenes had a slight negative effect on growth of this yeast on alkanes.; However, it is not yet clear which individual isogenes are the most important for alkane metabolism in this organism. Although fatty aldehyde dehydrogenase (FALDH) activity has been detected in fungi no FALDH genes have yet been cloned, sequenced and expressed. Through BLAST searches using the human FALDH sequence as query we have identified 28 FALDH/FALDH-like gene sequences of which nine are from molds and 19 from yeast species. A comparative study of these sequences showed that fungal FALDH sequences may fall into several different subclasses of the ALDH3 family. Unique features of these proteins included presence of several transmembrane domains and in particular relatively long C- and N-termini. Searches of the sequenced Y. lipolytica genome for fatty alcohol oxidase (FAOD) and fatty alcohol dehydrogenase (FADH) encoding genes, which could be involved in the oxidation of fatty alcohols to aldehydes, yielded only one putative FADH encoding gene. However, FADH activity during growth on n-alkanes was very low and Northern-blot analyses showed that this gene was only eakly expressed during growth on hydrocarbon and non-hydrocarbon substrates.