The role of Mycothiol Biosynthesis in Pathogenesis and Drug Susceptibility of Mycobacterium tuberculosis

Abstract

Antioxidant defense mechanisms are critical for the intracellular pathogen Mycobacterium tuberculosis survival within its hosts. The unique antioxidant molecule mycothiol (AcCys-GlcN-Ins, MSH), the major low-molecular-weight thiol in mycobacteria, was hypothesized to be critical for surviving oxidative stress. Using M. smegmatis as a model microorganism, a combination of biochemical and genetic studies had revealed that MSH is synthesized by five enzymes MshA, A2, B, C and D. While the previous work demonstrated that mutants of mshA, mshC, and mshD were defective in making MSH in M. smegmatis, the mshB mutant retained the ability to make MSH. For M. tuberculosis, only mutations in the mshA and mshD had revealed a defect in MSH production. Surprisingly, the M. tuberculosis mshA mutant showed no defect in growth in immunocompetent mice.;To extend these previous studies, I systematically attempted to generate precise null deletions in all identified MSH biosynthesis genes in M. smegmatis and M. tuberculosis. We found reproducible results as previous reports. Previous work had suggested that the mshB and mca genes encoded a redundant function. We successfully deleted each gene and then generated the double deletion mutants of mshB and mca in both M. smegmatis and M. tuberculosis. Analysis of both the M. smegmatis Deltamca Delta mshB and M. tuberculosis Deltamca Delta mshB revealed a complete loss in MSH production. The.;M. tuberculosis Deltamca Delta mshB was further analyzed and found to have a low level resistance to isoniazid and high level resistance to ethionamide, sensitive to hydrogen peroxide,dithiothreitol, menadione, plumbagin and acidic pH. In addition, we discovered that the M. tuberculosis Deltamca DeltamshB is sensitive to methylglyoxal, a toxic by product of glycolysis. Interestingly, the M. tuberculosis Delta mca DeltamshB showed a growth defect in bone marrow derived macrophage infections and a significant virulence defect following intravenous injections of C57BL/6 mice. All the phenotypes of the M. tuberculosis Deltamca DeltamshB could be restored to wild-type phenotypes when complemented with mshB.;These studies have comprehensively examined the genetic pathway of MSH biosynthesis and revealed the first attenuation phenotype of a M. tuberculosis MSH mutant.