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.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat
