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Thiol-dependent mycobacterial responses to oxidative and nitrosative stress Ung, Korine (Sim Ee)

Abstract

Mycothiol (MSH), produced in actinomycetes including mycobacteria, is functionally analogous to glutathione (GSH) in other organisms, replacing G S H as the main systemic protectant against oxidative stress. In this work, we investigated two possible control points in the regulation of MSH in response to oxidative stress: 1) transcriptional upregulation of MSH biosynthesis mediated by Rv0485 & Rv0818 (putative transcriptional regulators located directly upstream of some MSH biosynthesis genes); and 2) maintenance of the MSH:MS=SM redox balance upon oxidative stress. To monitor the changes in redox state and total MSH levels in Mycobacterium smegmatis mc²155 and M. bovis BCG cultures upon exposure to diamide (a thiol-specific oxidative agent), H₂O₂, or gaseous nitric oxide, we performed mycothiol assays and developed a novel, modified mycothiol assay to detect MSH oxidized as MS=SM. We found that diamide and H₂O₂-induced oxidative stress in M. bovis BCG induces partial depletion of MSH to the oxidized form MS=SM, while treatment with gNO does not. M. smegmatis, an environmental saprophyte, displays a greater tolerance to these oxidative stresses than M. bovis BCG, as reflected by the lesser magnitudes in changes in redox state and total MSH levels upon treatment. We also investigated gene expression of Rv0485 and Rv0818 in M. bovis BCG upon exposure to diamide and upon infection of J774A.1 murine macrophages, using quantitative real-time reverse-transcriptase PCR. We found that although expressions of Rv0485 and Rv0818 were unchanged in diamide-treated bacteria, they were increased about 8-fold in bacteria harvested 6 and 18 hours after macrophage infections, hi addition, we conducted protein-protein binding assays to investigate if Rv0818 protein binds to the SigH RNA polymerase subunit specifically under oxidizing conditions in vitro, as would be expected if Rv0818 is involved in the transcriptional regulation of msh biosynthesis genes upon oxidative stress. As an addendum to this thesis, we looked at two potential GSH-dependent genes in mycobacteria, ggtA & ggtB, which code for putative gammaglutamyltranspeptidases and might have a role in the recently described phenomenon of mycobacterial sensitivity to GSH and GSNO.

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