Masters Degrees (Biochemistry)

Permanent URI for this collection

https://scholar.sun.ac.za/handle/10019.1/3910

Browse

Browsing Masters Degrees (Biochemistry) by browse.metadata.advisor "De Villiers, Marianne"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    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.
  • Thumbnail Image
    Item
    The expression, purification and characterization of escherichia coli NudL, a putative coenzyme A hydrolase
    (Stellenbosch : Stellenbosch University, 2021-04) Kellow-Webb, Sarah Maggie; Strauss, Erick; De Villiers, Marianne; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Coenzyme A (CoA) is an essential cofactor that is synthesized by a conserved five-step pathway from pantothenic acid (vitamin B5). The regulation of CoA levels is vital in supporting normal cellular function. In higher order organisms, CoA degradation and the recycling of CoA have proven efficient for the dynamic control of CoA levels, but in prokaryotes the contribution of CoA degradation to the regulation of CoA levels remains largely unstudied. Escherichia coli is known to make significant quantities of 4'-phosphopantetheine (PPanSH), a CoA biosynthesis intermediate, the excess of which is irreversibly exported to the extracellular environment. Early studies revealed that the source of the high PPanSH levels is both from the biosynthesis and the degradation of CoA. In E. coli, CoA degradation can be mediated via an indirect mechanism through acyl carrier protein (ACP) prosthetic group turnover, where AcpH releases the PPanSH moiety from the holo-ACP, or via a direct mechanism by an unidentified enzyme. One possible mechanism for direct degradation of CoA is by the action of a CoA hydrolase, a Nudix hydrolase subfamily member. Nudix hydrolases are a superfamily of housekeeping enzymes that regulate metabolic intermediates or remove toxic nucleotide metabolites. Nudix hydrolases specific for CoA are able to degrade CoA at the pyrophosphate moiety into 3',5'-ADP and PPanSH. E. coli has 13 predicted Nudix enzymes of which NudL is the only one yet to be characterized. NudL is encoded by the yeaB gene and contains both the Nudix box and NuCoA motif that points to its selectivity for CoA and strongly suggests that it is a CoA hydrolase. The first part of this study focused on optimizing the production of pure, soluble NudL protein for testing its putative CoA hydrolase activity. The protein was found to be unstable and significant challenges were experienced with soluble recombinant protein expression. NudL could therefore only be partially purified for the purpose of activity testing. The second part of this study focussed on characterizing NudL’s activity. Both the partially purified enzyme and E. coli lysate containing overexpressed NudL was found to be able to degrade CoA and form PPanSH. These results provide strong evidence that CoA hydrolysis into PPansH is mediated by NudL and confirms the presence of CoA hydrolase activity in E. coli lysate. CoA hydrolase activity was dependent on the presence of a metal cofactor and NudL appeared to prefer Mn²⁺ over Mg²⁺. Following this, the physiological relevance of NudL was investigated. The results of metabolomic studies revealed that NudL contributes significantly to the intracellular PPanSH levels as a knockout mutant lacking the enzyme had considerably lower intracellular PPanSH levels. An ∆acpH mutant also showed a similar trend, confirming that CoA degradation contributes to the intracellular PPanSH pool. Conditions which promote CoA degradation were also investigated. Transcription of the NudL- encoding gene yeaB did not appear to be influenced by oxidative stress; however, a significant growth defect was observed for the mutant strains when acetate was the carbon source, suggesting that CoA degradation by NudL is important during growth on acetate. The results of this study support a direct mechanism of CoA degradation in E. coli, specifically by the NudL enzyme, being the last E. coli Nudix hydrolase to be experimentally characterized. Further investigations are needed to understand how this enzyme is regulated. This may provide insight into how CoA levels are regulated in E. coli in general.
  • Thumbnail Image
    Item
    Investigation and characterisation of dephospho-coenzyme A kinase: a potential drug target
    (Stellenbosch : Stellenbosch University, 2018-12) Bouwer, Wilmarie; De Villiers, Marianne; Strauss, Erick; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Due to the rise in antimicrobial resistance, the coenzyme A (CoA) biosynthesis pathway has been identified as a metabolic process of interest as a potential new antimicrobial drug target that could aid in relieving the threat to global healthcare. The CoA biosynthesis pathway consists of five enzymes, each a potential novel drug target. The focus of this study was on dephospho-Coenzyme A kinase (DPCK), the last enzyme in the pathway, as it is thought to hold the most control over the flux through the CoA pathway and is largely unexplored. Particular focus was given to DPCK from Staphylococcus aureus and Plasmodium falciparum. Both these organisms have shown resistance to current drug treatments available in a clinical setting, thus causing resistant strains to become more prevalent and pose a greater threat to global healthcare. The aims of this study were to expand current knowledge on these two organisms’ DPCK enzymes, as neither of these enzymes have been characterised previously. The first aim of this study focussed on characterising S. aureus DPCK, whilst comparing the results to previously characterised DPCK enzymes from Corynebacterium ammoniagenes, Corynebacterium glutamicum, and Escherichia coli. From this we were able to determine the quaternary structure of DPCKs from S. aureus, C. ammoniagenes and C. glutamicum to be trimers in solution, whereas DPCK from E. coli was confirmed to be a monomer as previously reported in literature. Following this, we have determined that the specific activity of S. aureus DPCK is very low, in contrary to what is found for C. ammoniagenes DPCK. Techniques to aid in measuring the low activity of the enzyme were further explored. This led to the use of high performance liquid chromatography (HPLC) assays that incorporated all the CoA biosynthesis pathway enzymes to determine specific activity. We concluded that the presence of all of the other pathway enzymes, at physiological ratios, aided the specific activity of S. aureus DPCK. In the second aim of this study we wanted to determine the activity of P. falciparum DPCK, as this enzyme is the only CoA biosynthesis pathway enzyme that does not localise to the cytosol of the parasite. Further, little information is available on the activity of the parasite’s CoA biosynthesis enzymes as they are difficult to express recombinantly using bacterial expression vectors. We successfully determined conditions for that yielded soluble expression and purification conditions of P. falciparum DPCK, however the enzyme is not very stable in solution. This unfortunately prevented us from determining the activity of the enzyme. The results from this study have shed more light on methods to determine the activity of DPCK enzymes and have established soluble expression conditions for P. falciparum DPCK from a bacterial expression vector. Therefore, it can form the basis in future investigations on DPCK as a potential drug target.
  • Thumbnail Image
    Item
    Investigation of Coenzyme A levels in Plasmodium falciparum to ascertain the mode of action of new antimalarial candidates
    (Stellenbosch : Stellenbosch University, 2017-03) Scheepers, Melisse Sharne; De Villiers, Marianne; Strauss, Erick; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: The malaria parasite, Plasmodium falciparum, has become increasingly resistant to all commercially available drugs used in the treatment of malaria, and as such, the development of new antimalarial drugs with novel targets is of great importance. The coenzyme A (CoA) biosynthesis pathway is one such novel target since CoA and its precursor, pantothenate, have been shown to be essential for organism survival. N-phenethyl-α-methyl pantothenamide, a pantothenate analogue, has been shown in a previous study to inhibit growth in both bacteria and Plasmodium parasites, however the mode of action of this pantothenamide in Plasmodium is still unknown, and was thus investigated in this study. First, Plasmodiums’ requirement for pantothenate was investigated. We determined that parasites could survive without an extracellular source of pantothenate for up to eight days, however this contradicted what was found in literature, and was likely to be due to a Mycoplasma infection found late in the study. Secondly, it was investigated whether N-phenethyl-α-methyl pantothenamide can be metabolized to its CoA antimetabolites by the CoA biosynthetic enzymes present in P. falciparum. This was done by investigating the metabolism of the compound in both cell lysates and in in vivo P. falciparum cell cultures and it was found that PfPanK and PfDPCK is active in parasite lysates, while PfPPAT is inactive in parasite lysates. We could therefore not determine if the pantothenamide under investigation is being metabolized in the parasite by using lysates, but this is the first demonstration of the activity of PfDPCK in parasites lysate. Finally, we wanted to investigate the effect of tricyclic methylthiophenyl propanamide (TMP), a non-pantothenate analogue that inhibits the CoA biosynthesis pathway in other organisms, on P. falciparum proliferation. TMP was synthesized to use as a tool to investigate the mechanism of action of N-phenethyl-α-methyl pantothenamide to support that pantothenamides do not inhibit pantothenate kinase, as is known for TMP, but are rather metabolized downstream in the pathway. TMP was successfully synthesized and purified, however yields were too low to test TMP as an inhibitor of P. falciparum proliferation. Not only did the work done in this study shed more light on the mode of action of pantothenamides in P. falciparum, but also gave valuable insight into parasite biochemistry.