Virulence mechanisms of mycobacteria - New insights from studies of BCG vaccine strains and cell wall biosynthesis

Virulence mechanisms of Mycobacterium tuberculosis remain largely unknown. Currently, only two well-established virulence determinants have been identified: the RD1 protein secretion system and the cell wall lipids. Studies of RD1 secretion system originated from the observation that the RD1 locus is deleted in all strains of Mycobacterium bovis Bacillus Calmette--Guerin (BCG), an attenuated, live vaccine that has been used in humans since 1920s. However, reintroduction of RD1 into BCG does not restore full virulence to BCG; therefore other mechanisms exist to fully account for the attenuation of BCG. The mycobacterial cell wall is a unique and complex structure that distinguishes mycobacteria from other bacteria. Although not fully understood, there is increasing evidence that the complex cell wall plays a major role in the virulence and pathogenesis of M. tuberculosis. However, the genetic and biochemical pathways leading to the biosynthesis of individual cell wall lipids remain incompletely understood.;The second part of my thesis deals with cell wall biosynthesis. I observed that some BCG strains cannot synthesize the virulence lipids phthiocerol dimycocerosates and phenolic glycolipids, providing an explanation for heterogeneity in residual BCG virulence and reactogenicity. I have also identified Lsr2, a previously uncharacterized protein found in all mycobacteria, as a novel regulator of lipid biosynthesis. Characterization of the Lsr2 protein revealed DNA-bridging properties functionally analogous to H-NS, a bacterial nucleoid associated protein. And like H-NS, Lsr2 likely functions as a global repressor for a number of genes including those involved in virulence. This finding opens new avenues for identifying novel virulence genes in M. tuberculosis .;My thesis consists of two parts. In the first part, I describe the metabolic deficiency of BCG vaccine strains. I show that BCG exhibits dysregulation of glutamine synthetase, a key enzyme in nitrogen metabolism. This defect, together with mutations that affect the production (e.g., alanine dehydrogenase) or the expression (e.g., serine deaminase) of catabolic enzymes renders BCG with deficient growth under nitrogen-limited conditions in vitro, which may also contribute to the attenuation of BCG in vivo. These findings shed new light into the mechanisms of attenuation of BCG.