Antibody production against Staphylococcus aureus CoA biosynthesis enzymes and their application in protein level quantification

Bothma, Karli (2021-04)

Thesis (MSc)--Stellenbosch University, 2021.

Thesis

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.

AFRIKAANSE OPSOMMING: Antimikrobiese weerstand het ‘n toenemende las geword wêreldwyd soos meer en meer menslike patogene weerstand opgebou het teen huidige antimikrobiese teenmiddels. Die indentifisering en ontwikkeling van nuwe antimikrobiese teenmiddels en teenmiddelteikens geniet dus tans ‘n hoë prioriteit. ‘n Potensiële teenmiddelteiken wat hernude aandag ontvang is die koënsiem A (KoA) biosintese padweg. KoA is ‘n essensiële ko-faktor wat noodsaaklik is vir lewe in alle organismes, insluitend menslike patogene, en is daarom ‘n aanloklike teiken vir die ontwikkeling van nuwe antimikrobiese teenmiddels. Die KoA biosintese padweg van Staphylococcus aureus, wat verantwoordelik is vir menigte hospitaal-verwante infeksies, was die fokus van hierdie studie. Alhoewel verskeie studies al gefokus het op die padweg in S. aureus as moontlike teenmiddelteiken, is daar steeds baie ontbrekende inligting wat uitgelig moet word. Een gaping in ons kennis omtrent die KoA biosintese ensieme (PanK, CoaBC, PPAT en DPCK) is dat die ensiemvlakke onder fisiologiese kondisies tans onbekend is. Die doel van hierdie studie was dus gerig op die ontwikkeling van ‘n immunologiese tegniek wat geimplementeer kan word om die vlakke van hierdie ensieme tydens verskillende groeifases van S. aureus, wat opgegroei word onder fisiologiese groeitoestande, te kwantifiseer. Die vier KoA biosintese ensieme van S. aureus is rekombinant uitgedruk en gesuiwer deur gebruik te maak van gevestigde metodiek. Poliklonale teenliggaampies is volgende geproduseer in konyne deur die immunisering van hierdie diere met die onderskeidelike ensieme van die KoA biosintese padweg van S. aureus wat geadsorbeer is aan suurbehandelde, naakte Salmonella minnesota R595. Hierdie tegniek is gebruik in die verlede vir die suksesvolle produksie van teenliggampies teen ‘n groot verskeidenheid antigene, veral in omstandigehede waar daar slegs klein hoeveelhede van die antigeen beskikbaar was. ‘n Kompetisie “enzyme-linked immunosorbent assay” (ELISA) met uitstekende standaardkurwes is ontwikkel vir die kwantifisering van elk van die onderskeidelike ensieme met hierdie teenliggaampies. Verder het kruisreaktiwiteitstudies, uitgevoer deur middel van ELISA en western blot analises, getoon dat die anti-SaPanK, anti-SaPPAT and anti-SaDPCK teenliggampies geringe kruisreaktiwiteit toon. In ‘n poging om die hoeveelheid kruisreaktiwiteit van elke teenliggampie-antigeen paar te kwantifiseer, is daar egter bevind dat slegs die kruisreaktiwiteit van die anti-SaPPAT teenliggaampies die finale, geoptimiseerde toets beïnvloed. In hierdie studie is‘n oorspronklike, hoogs sensitiewe ELISA metode ontwikkel vir die deteksie en kwantifisering van die ensieme van die KoA biosintese padweg in S. aureus. Hierdie toetse verskaf ‘n koste-effektiewe metode vir die kwantifisering van ensiemvlakke en het dus die potensiaal om insig te lewer oor die vlakke van die verskillende ensieme onder fisiologiese toestande en uiteindelik bydrae te lewer tot die ontwikkeling van nuwe antimikrobiese teenmiddels.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/110356
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