Browsing by Author "Bothma, Karli"

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    Antibody production against Staphylococcus aureus CoA biosynthesis enzymes and their application in protein level quantification
    (Stellenbosch : Stellenbosch University, 2021-04) Bothma, Karli; De Villiers, Marianne; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Antimicrobial resistance has become an increased burden worldwide as more and more human pathogens are becoming resistant to current antimicrobials. Therefore, the identification of novel drug targets and development of new antimicrobial drugs are currently of high priority. A drug target that has gained increased attention is the coenzyme A (CoA) biosynthesis pathway. CoA is an essential cofactor that is necessary for life in all organisms, including human pathogens, making it an attractive target for the development of new antimicrobial drugs. The CoA biosynthesis pathway of Staphylococcus aureus, which is the leading cause of hospital-associated infections, was the focus of this study. Although various studies have investigated this pathway in S. aureus as a possible drug target, there is still a lot that needs to be elucidated in this regard. One gap in our knowledge is that the levels of the CoA biosynthesis enzymes (PanK, CoaBC, PPAT and DPCK) under physiological conditions are currently unknown. This study therefore aimed to develop immunological techniques which could be implemented as tools to quantify the levels of these enzymes at different growth phases of S. aureus cultivated under physiological growth conditions. To achieve this aim, the four CoA biosynthesis enzymes of S. aureus were recombinantly expressed and purified using established methods. Polyclonal antibodies were raised in rabbits by immunising the animals with the respective enzymes adsorbed to acid-treated, naked Salmonella minnesota R595. This method has been used to successfully produce antibodies to a wide variety of antigens, especially in cases where only small amounts of the antigen were available. With these antibodies, indirect competition enzyme-linked immunosorbent assays (ELISAs) with excellent standard curves were obtained for the quantification of each of the respective enzymes. Furthermore, cross-reactivity studies performed with ELISA and western blot revealed that the anti-SaPanK, anti-SaPPAT and anti-SaDPCK antibodies showed limited cross-reactivity. In an attempt to quantify the amount of cross-reactivity of each antibody-antigen pair, however, it was found that only the cross-reactivity of the anti-SaPPAT antibodies had an effect on the final optimised assay. In this study an original, highly sensitive ELISA method for the detection and quantification of each of the enzymes of the CoA biosynthesis pathway of S. aureus was developed. These assays provide a cost-effective method for enzyme level quantification that have the potential to provide better insight on the levels of the different enzymes under physiological conditions and ultimately aid in the development of new antimicrobial drugs.