Department of Biochemistry

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Bioinformatic characterisation of genes associated with coenzyme A biosynthesis in mycoplasmas and expression and isolation of dephospho-coenzyme A kinase from Mycoplasma sp. Ms02
(Stellenbosch : Stellenbosch University, 2018-03) Ras, Tertius Alwyn; Botes, Annelise; Strauss, Erick; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The South African ostrich industry is internationally the leading provider of ostrich products. The increasing popularity of ostrich products has resulted in the adjustment of production strategies, which includes intensifying rearing conditions by using feedlot systems. However, this intensive rearing strategy creates an ideal environment for pathogens, such as mycoplasmas, to spread. There are three Mycoplasma species that infect ostriches, which are associated with respiratory diseases. These mycoplasma infections can result in production losses, which not only have an economic impact on the ostrich industry but also significant socio-economic implications. Hence, there is a need for specific and cost-effective treatment against these ostrich-infecting mycoplasmas. The enzymes involved in the biosynthesis pathway of coenzyme A have long been regarded as potential targets for drug development. These enzymes could, therefore, offer a solution to the control of mycoplasma infections in ostriches. Since the coenzyme A biosynthetic pathway is relatively unexplored in mycoplasmas, the first aim of this study was to determine the presence or absence of enzyme-encoding genes involved in this pathway in Mycoplasma species. This was done using a bioinformatics approach. Of the 62 Mycoplasma species investigated, there were eight species (13%) found to have none of the enzyme-encoding genes, while the remaining species had at least one. Additionally, twelve enzyme-encoding gene homologues were identified and their predicted identities confirmed by evaluating the conserved and functional motifs and domains. The enzyme-encoding gene found to be most common amongst the investigated species was that of dephosphocoenzyme A kinase (DPCK), the final enzyme in the biosynthesis pathway. Furthermore, there was no correlation between the number of identified coenzyme A biosynthetic pathway enzyme-encoding genes in a species and the phylogeny of the respective proteins. There was also no correlation with the 16S rRNA phylogenetic groupings. Given the common presence of the DPCK-encoding gene, the second aim of this study was to recombinantly express the DPCK of the ostrich-infecting Mycoplasma sp. Ms02 (Ms02) and isolate the protein using a His-tag. The Ms02 DPCK-encoding gene was successfully amplified, cloned and mutated by site-directed mutagenesis to allow for expression in a nonmycoplasma host. However, the soluble expression and isolation of the Ms02 DPCK protein proved to be challenging. Using a variation of methods, the protein was eventually solubilised using a sarkosyl treatment method. A pure isolate of the Ms02 DPCK protein could, however, not be attained when using immobilised metal affinity chromatography (IMAC) purification. Subsequent activity testing of the isolated DPCK enzyme, using an HPLC-based method, also showed no activity.
Characterising the gut microbiome of ostrich chicks reared under intensive conditions
(Stellenbosch : Stellenbosch University, 2020-12) Heitmann, Sinjon; Botes, Annelise; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Every year the ostrich industry suffers severe losses from the high mortality rate of intensively farmed ostrich chicks during early post-hatch development. One of the major contributors to the high mortality is enteritis, an enteric disease that stems largely from microbial imbalance. Efforts to reduce and prevent enteric diseases in ostrich chicks requires in part an extensive understanding of the changes in microbial composition within gastrointestinal tract (GIT). This study characterises the successional development of the microbiota present in the GIT of ostrich chicks reared under intensive conditions within the first three months post-hatch. In targeting the microbiota present in the small intestine, caeca, colon and faeces, the changes in bacterial composition and abundance provide insights unique to its development in the gut region and the development of the GIT. To achieve this, samples were taken from three ostrich chicks at five time points (15 chicks). For each time point the samples per gut region were pooled, microbial genomic DNA extracted and used for 16S metagenomic sequencing on the Ion Torrent platform. To improve definition at lower taxonomic levels, seven of the nine hypervariable regions in the 16S rRNA gene were sequenced. The raw sequence data was processed, and the bioinformatic analyses performed using the Ion Reporter software. Analyses of the gut regions over time found a progressive increase in bacterial diversity and stability despite the presence of both colonisation and extinctions events. Initial colonization of the GIT by week 2 coincided with the change in nutritive source from yolk to feed, and with it the introduction of a wide range of taxa including members from the Firmicutes, Bacteroidetes, Proteobacteria and Tenericutes phyla. Yet the changes in bacterial composition and abundance over time were not uniform between gut regions. The small intestine and colon regions were found to have substantial dissimilarities to remaining gut regions from week 0 - 4 and week 6 - 12, respectively. The changeover from small intestine to the colon was marked by the chronological shift of some species such as C. butyricum, C. disporicum and T. sanguinis, and with them the localised proliferation of potentially pathogenic species. The movement of C. butyricum away from the small intestine may remove its protective influence and allow the opportunistic proliferation of pathogenic species. The changeover between the small intestine and colon correlated both with the change in diet, as a part of the intensive rearing system, and the development of the colon into a more efficient fermentation chamber. Furthermore, the developed colon did not present the greater abundance of fibrolytic species from the Ruminococcaceae or Bacteroidaceae families as anticipated. Rather, a greater abundance of fibrolytic species from the Clostridiaceae family were present such as C. butyricum, C. chartatabidum, C. disporicum and C. paraputrificum, which suggest an accumulation of resistant starches and starch components in the colon. Furthermore, differences in bacterial composition were established in the core microbiota of the different gut regions, which shows that faecal samples do not provide a complete representation of GIT microbiota. Ideally the gut regions should be examined individually and together to understand the full impact that changes in diet have on the GIT. An examination of the distribution of relative abundance data may serve as a reference in adapting feeding strategies and strategies to manage GIT infections in intensively reared ostrich chicks.
The development of a DNA vaccine against Mycoplasma nasistruthionis sp. nov. for use in ostriches
(Stellenbosch : Stellenbosch University, 2015-12) Wium, Martha; Botes, Annelise; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Mycoplasma nasistruthionis sp. nov. str. Ms03 (Ms03) is one of three Mycoplasma species that were identified from ostriches. Mycoplasmas infections have been implicated in ostrich chick mortalities, growth retardation and downgrading of ostrich carcasses. Currently there is no vaccine available for the treatment of mycoplasmosis in ostriches. This study investigated the development of DNA vaccines against Ms03 infections in ostriches. To this end, the Ms03 genome was sequenced and annotated. The vaccine candidate gene, oppA, was identified within the genome sequence and characterized before DNA vaccines containing the oppA were developed and tested. The genome of Ms03 was sequenced and the resulting 172 contigs were annotated. This dissertation presents the first Ms03 draft genome and annotation which contributed to the understanding of Ms03 as a miniature genetically independent organism. In Ms03, genome replication, cell division, RNA transcription, protein translation and glycolysis resemble that of the closely related Mycoplasma synoviae 53. Purine and pyrimidine metabolism was incomplete and de novo synthesis thereof was not possible. Amino acid synthesis in Ms03 was mostly absent and only the genes that convert aspartate to asparagine and glycine to serine were found. More importers than exporters were annotated owing to the lack of synthesis pathways in Ms03, which is typical for mycoplasmas that have parasitic life styles. Two oligopeptide permease (opp) operons were annotated within the Ms03 genome. The potential of the oppA as a vaccine candidate gene was evaluated by investigating the need for a substrate-binding domain (OppA) as part of the OppBCDF transporter within Mycoplasma species. An oppA homologue could be identified for each oppBCDF operon in all species and therefore must play an essential role in oligopeptide transport. All mycoplasmas (except for hemoplasma) had one, two or three opp operons that could be divided into three types (Type A, B and C). Each type had unique InterPro and MEME domains and motifs which together with the phylogenetic analysis suggest unique roles in their survival under different conditions. Ms03 had a Type A and a Type B opp operon, the Type A oppA was used as vaccine candidate gene. The Type A oppA was cloned and site-directed mutagenesis was used for codon correction before the mutated gene was sub-cloned into three DNA vaccine vectors. The three DNA vaccines (pCI-neo_oppA, VR1012_oppA and VR1020_oppA) were used to vaccinate ostriches and the OppA-antibody response was analysed by ELISA. The VR1020_oppA and pCI-neo_oppA constructs elicited a primary immune response in ostriches, indicating that the OppA protein was expressed in vivo and was immunogenic. This can therefore be viewed as the first step in the development of a DNA vaccine for the control of mycoplasma infections in ostriches.
Development of an oral vaccine against the ostrich-specific mycoplasma, Mycoplasma struthionis
(Stellenbosch : Stellenbosch University, 2013-03) Van Tonder, Amanda; Bellstedt, D. U.; Botes, Annelise; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The ostrich-specific mycoplasmas Mycoplasma struthionis, Ms02 and Mycoplasma nasistruthionis, are associated with respiratory disease in ostriches, which is threatening the South African ostrich industry. Antibiotics are available to manage Mycoplasma infections, but a need to prevent infections led to an investigation into the development of vaccines against the ostrich-specific mycoplasma. After commercially available poultry mycoplasma vaccines proved to be unsuccessful in protecting ostriches against ostrich mycoplasma infections, the genome of M. struthionis was analysed and the OppA gene identified as a good vaccine candidate. The gene was isolated and used to develop the pCI-neo, VR1012 and VR1020 naked DNA vaccines. M. struthionis infects the respiratory tract of ostriches, therefore a vaccine that results in mucosal immunity is required. The use of a bacterial vector for DNA vaccines has been shown to elicit both a humoral and mucosal immune response. Salmonella enterica serovar typhimurium SL3261 was used to develop mucosal pCI-neo, VR1012 and VR1020 DNA vaccines. The pCI-neo mucosal DNA vaccine was found to be unstable in vivo and the stable mucosal VR1020 and VR1012 DNA vaccines were considered for subsequent vaccine trials. The tissue plasminogen activator (TSA) signal peptide found in the VR1020 plasmid to direct the secretion of the membrane protein, OppA, makes it a good candidate vaccine to compare against the naked DNA vaccine. A preliminary vaccine trial conducted with this vaccine was influenced by various factors including avian influenza and the statistical results proved to be invalid, but enzyme-linked immunosorbent assays (ELISAs) were developed for the successful measurement of the immune response of the ostriches. The dose of the mucosal vaccine administered in the preliminary trial might not have been enough to elicit an effective immune response in the ostriches. Different doses of the mucosal VR1012 and VR1020 DNA vaccines were therefore used in a second trial, but the trial was also influenced by a variety of factors. Even though the results of the vaccine trials were not successful, a few observations were made that could be used to improve future trials and reduce the effect of the factors on the vaccine trials, such as the effect of prior infection, as well as the stress on the ostriches.
Evaluation of DNA vaccines against Mycoplasma nasistruthionis sp. nov. str. Ms03 infections in ostriches and the production of IgA heavy chain proteins
(2017-03) Jonker, Hester Isabella; Botes, Annelise; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Ostrich products have internationally become very popular with South Africa being the world leader in this industry. An increase in the demand for ostrich products has influenced production strategies by intensifying rearing conditions through the use of feedlot systems. Intensive rearing creates ideal conditions for the spread of pathogens such as mycoplasmas, which is associated with a respiratory disease syndrome amongst feedlot ostriches. The three ostrich-infecting mycoplasmas, Ms01, Ms02 and Ms03, together with other secondary pathogens result in reduced production. Since there are no vaccines available against these ostrich-infecting mycoplasmas, three DNA vaccines have been developed in this laboratory. Each vaccine consisted of a eukaryotic expression vector (pCI-neo, VR1020 or VR1012) containing the Ms03 oppA gene as antigen. The first objective of this study was to re-evaluate the anti-OppA immune response elicited by the pCI-neo and VR1020vaccines in ostriches. A vaccination trial was conducted and both vaccines were administered intramuscularly at 100, 600 and 1200 μg/ml doses followed by a booster vaccination. The anti-OppA immune response elicited by these vaccines in the ostriches was measured by means of the ELISA technique. All of the VR1020 vaccine doses as well as the 100 and 600 μg/ml doses of the pCI-neo vaccine were able to elicit a statistically significant anti-OppA immune response after administration of a booster vaccination. Since mycoplasmas target the respiratory system of ostriches a mucosally administered vaccine should also be considered. Opposed to the intramuscular route of vaccination, which results in humoral immunity represented predominantly by IgG, mucosal administration would result in mucosal immunity represented by IgA production. For the measurement of IgA production, the ELISA requires secondary anit-IgA antibodies. Although the whole antibody is typically used for the production of secondary antibodies, it is possible to use only the region representing the heavy chain constant region. This can then be produced as a recombinant protein that will allow an easy reproducible source for the production of secondary antibodies. The second objective of this study was therefore to evaluate the baculovirus-insect expression system for the production of the ostrich IgA heavy chain constant region (IgAH) protein, as this system will allow glycosylation of the protein product. The IgAH gene was inserted into the pAB-6xHis transfer plasmid and together with the ProFold-ER1 baculovirus used to co-transfect Sf9 insect cells for the production of ProFold-ER1_IgAH by means of homologous recombination. The IgAH protein was successfully expressed as confirmed by using HisProbe-HRP as well as previously produced rabbit anti-ostrich IgA antibodies during western blot analysis.
Evaluation of DNA vaccines developed against Mycoplasma struthionis sp. nov. str. Ms01 in ostriches
(Stellenbosch : Stellenbosch University, 2016-03) De Wet, Bertus; Botes, Annelise; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The vast demand for ostrich meat has made South Africa the leader in not only the production of ostrich meat but also ostrich associated products such as feathers and leather. Ostrich specific mycoplasmas (referred to as Ms01, Ms02 and Ms03) cause respiratory tract infections with subsequent reduction in physical growth rate and therefore reduced production. To date no vaccines are available to combat these infections in ostriches. In this laboratory three DNA vaccines (pCI-neo, VR1012 and VR1020) have been developed with each containing the Ms01 oppA gene as antigen. The aim of this study was to evaluate these developed DNA vaccines in a mammalian cell culture based system as well as an ostrich vaccination trial. COS-1 cells were transfected with the three developed DNA vaccines. Transcription of the oppA gene was proven for all the plasmids. Translation into the OppA protein was shown to be limited to the VR1020_oppA plasmid. The protein was visualised by SDS-PAGE and detected by western blot using chemiluminescence. Two of the vaccines, VR1020_oppA and pCI-neo_oppA, were administered in three concentrations (100 μg/ml, 300 μg/ml and 600 μg/ml) to ostriches during a vaccination trial followed by a booster injection. The ability of the vaccines to elicit anti-OppA antibodies was measured using ELISA. The pCI-neo_oppA vaccine failed to induce an immune response against the antigen after both the first and booster vaccinations. The VR1020_oppA vaccine on the other hand was able to elicit an anti-OppA immune response.
The indentification, contiguous sequence annotation, cloning and site-directed mutagenesis of the P100 vaccine candidate gene of the ostrich mycoplasma Ms02
(Stellenbosch : University of Stellenbosch, 2010-12) Steenmans, Shandre; Bellstedt, D. U.; Botes, Annelise; University of Stellenbosch. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The ostrich industry in South Africa is currently threatened by respiratory disease in feedlot ostriches which causes a dramatic loss in production. Ms01, Ms02 and Ms03 were identified as the three ostrichspecific mycoplasmas to be associated with this respiratory disease in ostriches of South Africa. The ostrich-specific mycoplasmas have a major impact on ostrich production and for this reason there is a serious need for treatment for these infections. For this reason, the ostrich industry has undertaken an investigation into the development of vaccines against mycoplasma infections. In this study, an approach to DNA vaccine development will be investigated and applied, specifically for the ostrich mycoplasma Ms02. Firstly, the whole genome of Ms02 was sequenced using GS FLX sequencing technology. The contiguous sequences obtained from the whole-genome sequencing were analysed bioinformatically which included the annotation of the contiguous sequences and the subsequent search for a vaccine candidate gene for the development of a DNA vaccine. The P100 gene of Ms02, which showed a high degree of homology with the P100 gene of the human pathogen M. hominis, was chosen as a vaccine candidate gene for the development of a DNA vaccine. The P100 gene was successfully cloned and subsequently modified by means of site-directed mutagenesis to correct for alternative codon usage, where after the modified P100 gene was subcloned into the mammalian expression vector, pCI-neo for vaccination trials in the near future.
The ostrich mycoplasma Ms01 : the identification, isolation, and modification of the P100 vaccine candidate gene and immunity elicited by poultry mycoplasma vaccines
(Stellenbosch : Stellenbosch University, 2009-03) Pretorius, Benita; Bellstedt, D. U.; Botes, Annelise; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The South African ostrich industry is currently being threatened by respiratory disease in feedlot ostriches with dramatic production losses. Three ostrich-specific mycoplasmas, Ms01, Ms02 and Ms03 were identified to be associated with respiratory disease in ostriches in South Africa. There is currently no registered mycoplasma vaccine available for use in ostriches. In order to prevent mycoplasma infections in South African ostriches, the ostrich industry has launched an investigation into possible strategies for vaccine development. This thesis describes different strategies for the establishment of immunity in ostriches against the ostrich-specific mycoplasmas. Firstly, the effectiveness of existing poultry mycoplasma vaccines to provide protection in ostriches against ostrich mycoplasma infections was tested. To this end, ostriches received primary and secondary vaccinations with poultry mycoplasma vaccines against Mycoplasma synoviae or Mycoplasma gallicepticum, respectively, after which protection against ostrich-specific mycoplasma was evaluated. Even though the specific identity of the ostrich-specific mycoplasmas (Ms01, Ms02, and/or Ms03) responsible for subsequent infection of immunized ostriches was not determined, it was concluded that poultry mycoplasma vaccines do not provide protection against these mycoplasma infections in ostriches. This appears to be the result of low levels of antibody crossreactivity between mycoplasmas, highlighting the necessity for the development of specific vaccines against each of the individual ostrich-specific mycoplasmas. Secondly, the development of a DNA vaccine against Ms01 was investigated. With the aim of developing an Ms01-specific DNA vaccine, the entire Ms01 genome was sequenced using GS20 sequencing technology. Bioinformatic searches were launched for the identification of an appropriate vaccine candidate gene in the Ms01 genome. The P100 gene, showing a high degree of homology with the P100 gene of the human pathogen M. hominis, was subsequently identified. After successful cloning, and modification of ten specific codons within the gene to correct for alternative codon usage, the modified P100 gene of Ms01 is now ready for insertion into a suitable DNA vaccine vector, for subsequent use as a DNA vaccine in ostriches.
The ostrich mycoplasma Ms02 partial genome assembly, bioinformatic analysis and the development of three DNA vaccines
(Stellenbosch : Stellenbosch University, 2013-03) Strydom, Marliz; Botes, Annelise; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The South African ostrich industry is under enormous threats due to diseases contracted by the ostriches. H5N2 virus (avian influenza) outbreaks the past two years have resulted in thousands of ostriches having to be culled. However, the more silent respiratory infectious agents of ostriches are the three ostrich-specific mycoplasmas. Named Ms01, Ms02, and Ms03, these three mycoplasmas are responsible for dramatic production losses each year, due to their intrusive nature and the fact that no vaccines are currently available to prevent mycoplasma infections in ostriches. The use of antibiotics does not eradicate the disease completely, but only alleviates symptoms. The ostrich industry commissioned investigations into the development of three specific vaccines using the relatively novel approach of DNA vaccination. The concept of DNA vaccine development is based on the availability of complete genome sequences of the pathogen against which the vaccine is to be developed. This is necessary in order to identify vaccine candidate genes through comparative genomic studies. The Ms02 genome has previously been sequenced, resulting in 28 large contiguous sequences. This thesis used the technique of Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR) to attempt assembly of these 28 contiguous sequences. The number was reduced to 14 large contiguous sequences, which were then subjected to repetitive sequence analysis and open reading frame analysis. Bioinformatic software was also used to predict the origin of replication. The extent of repeats in the Ms02 genome is illustrated, as well as the problems with genome assembly when dealing with repetitive-rich and A+T-rich genomes as those of mycoplasmas. Previous studies determined the mycoplasma oppA gene to be a good vaccine candidate gene, due to its cytadherent properties. This thesis describes the development of three DNA vaccines containing the Ms02 oppA gene, and a preliminary attempt to prove expression of one of these vaccines in a cell culture-based system. The DNA vaccine vectors pCI-neo, VR1012, and VR1020 were chosen for the vaccine development. The Ms02 oppA gene was also cloned into the prokaryotic expression vector pGEX-4T-1 in order to express the OppA protein for purification. The purified protein may be used in future serological tests in ostrich vaccination trials. In this study the protein was used to elicit anti-OppA rabbit antibodies, which were used to attempt detection of the pCI-neo-driven OppA protein expression in an MDA cell line in a transfection study. However, preliminary findings could not detect expression, but did indicate that the currently used colorimetric western blot technique may not be sensitive enough. It is suggested that different cell lines need to be investigated. Further optimisations are also required to decrease the observed non-specific binding.
Preliminary investigations into the use of DNA vaccines to elicit protective immune responses against the ostrich mycoplasma Ms01
(Stellenbosch : Stellenbosch University, 2012-03) Brandt, Sonja; Bellstedt, D. U.; Botes, Annelise; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Mycoplasmas are evolutionarily advanced prokaryotes which have acquired the elite status of “next generation” bacterial pathogens and necessitate new paradigms in completely understanding their disease potential. The recurrent failure to eradicate mycoplasmal diseases from humans and animals through the use of antibiotics and other techniques have led to the conclusion that the most promising approach would be the development of an effective vaccine with which to control mycoplasma infections. The identification of three species of Mycoplasma which infect South African ostriches, causing huge losses to the South African ostrich industry each year, has thus prompted a search for new vaccination strategies with which to control and eradicate them. This study investigated the use of three potential DNA vaccines, utilizing the adherence-associated Ms01 OppA protein as antigenic determinant to generate antibody responses against the ostrich-infecting Ms01 organism. A vaccine trial in which the antigenic potential of the pCIneo, VR1012 and VR1020 DNA vaccines were evaluated in ostriches, necessitated the development of an enzyme-linked immunosorbent assay (ELISA) for serological analysis. To this end, the Ms01 oppA gene was isolated, cloned into a prokaryotic expression vector and expressed as a recombinant GST-fusion product in Escherichia coli. The successful expression and purification of the recombinant protein enabled its subsequent utilization as antigen in the generation of an ELISA. The ELISA displayed a high signal to background ratio. Using this ELISA, it could be shown that ostriches already possessed antibodies to the Ms01 organism prior to vaccination, a probable result of previous exposure. The expected antibody response pattern could not be detected in ostriches in response to the vaccinations, and therefore no final conclusion as to the immunostimulatory capabilities of the DNA vaccines could be drawn. Further vaccination trials in which ostriches that do not possess immunity to ostrich mycoplasmas, are required in order to obtain conclusive results.

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