Characterising the role of metabolic enzymes in the pathogenesis of Coxiella burnetii

Abstract

Coxiella burnetii is a Gram-negative intracellular pathogen that replicates in a highly acidic and oxidative lysosome-derived vacuole known as the Coxiella-containing vacuole (CCV). The relatively recent identification of axenic medium that supports C. burnetii replication and the development of improved genetic tools has advanced knowledge of this pathogen and facilitated the identification of virulence factors. A previously conducted transposon mutant library screen using a human cell line identified a number of genes that appeared to be required for intracellular replication, including genes encoding metabolic enzymes such as nadB and sdrA. To investigate the role of nadB and sdrA in C. burnetii replication, and to link the metabolism of C. burnetii with pathogenesis, a number of approaches were used, such as advanced genetic and biochemical techniques, including targeted and untargeted metabolomics. Firstly, each transposon mutant was clonally isolated to avoid wild type contamination. Genetic complementation of both nadB and sdrA mutants and subsequent intracellular growth assays using both epithelial HeLa cells and macrophage-like THP-1 cells conclusively demonstrated that both nadB and sdrA are required for efficient C. burnetii replication and CCV formation. Analysis of the protein sequence revealed that NadB has a functionally conserved arginine residue at a position of 275 and this arginine was mutated to leucine using site-directed mutagenesis. Purification of both GST-NadB and R275L GST-NadB showed that GST-NadB had L-aspartate oxidase activity, an enzyme catalysing the first reaction of de novo NAD synthesis, whereas R275L NadB had lost enzymatic activity. Complementation of nadB mutant with a plasmid encoding this inactive R275L NadB was unable to rescue the intracellular replication defect, confirming the requirement of NAD de novo synthesis for intracellular replication of C. burnetii. Steady state metabolite analysis also showed key changes in the level of abundance of metabolites in the NAD biosynthetic pathway in the nadB mutant as compared to parent C. burnetii and the complemented nadB mutant, demonstrating the role of NadB in de novo NAD synthesis. This suggests that this pathway is an ideal target for development of therapeutics, and inhibition of this pathway using a compound non-toxic to mammalian cells reduced C. burnetii replication significantly. The role of SdrA in C. burnetii metabolism and pathogenesis was also further investigated in this thesis. SdrA is a putative short chain dehydrogenase containing a conserved glycine residue at position 12 that serves as an NADP binding site facilitating NADP(H) regeneration, a key process in resistance to oxidative stress in C. burnetii. This Gly residue was replaced by Ala using site directed mutagenesis and purified recombinant 6xHis-G12A was enzymatically inactive when compared to wild type 6xHis-SdrA that converted NADP+ to NADP(H) in vitro. Complementing the sdrA mutant with a plasmid encoding enzymatically inactive 3xFLAG-tagged G12A_SdrA failed to restore intracellular growth, confirming the link between NADP(H) regeneration and C. burnetii replication. The sdrA mutant showed greater susceptibility to oxidative stress in vitro induced by treatment with hydrogen peroxide, whereas both parent C. burnetii and the complemented sdrA mutant were less sensitive. Supplementation of a commonly available anti-oxidant, L-ascorbate, into the host cell growth medium partially restored the intracellular growth defect of the sdrA mutant. Metabolite profiling using GC-MS revealed significant changes in the level of abundance of metabolites of central carbon metabolism in the sdrA mutant as compared to parent C. burnetii NM II and the complemented mutant. Finally, stable isotope labelling studies using [13C] label glucose showed a change in flux through central carbon metabolic pathways in the sdrA mutant demonstrating the presence of oxidative stress. Overall, this thesis has demonstrated the crucial role of NadB and SdrA in C. burnetii metabolism and pathogenesis.