Association of the pathophysiology and the immunity of tuberculosis with the Mycobacterium tuberculosis extracellular proteome

Abstract

Because of the devastating social and economic impact of tuberculosis on the countries of the world the need for an improved understanding of this disease is greater than ever. Chapter two of this research identified proteins with altered translocation patterns in a SecA2-/- mutant compared to wild-type M. tuberculosis. Superoxide dismutase (SodA), catalase-peroxidase (KatG), and Rv0393 contain no amino acid signal sequence, however, decreased levels of these proteins in the filtrate of SecA2-/- mutant cultures compared to the wild-type indicate a role for SecA2 in their export. Since the SecA2-/- mutant manifests truncated virulence in the mouse, and SodA and KatG are implicated in the detoxification of oxidative compounds produced by the macrophage, understanding the mechanisms by which this novel protein secretion pathway functions is of central importance. Based upon the hypothesis that the changing environment encountered by M. tuberculosis during the course of infection will translate to altered protein production by the bacilli, chapter three of this work identifies proteins with increased production under stressed conditions believed to more closely reflect conditions encountered by the bacilli during infection. M. tuberculosis was grown under standard, microaerophilic, anaerobic, and alternate carbon source conditions. 2DE and MS/MS analysis of the filtrates of these cultures revealed four proteins with increased abundance under stressed conditions: Acr, BfrB, Ppa, and Ssb. These proteins and others known to be induced under stressed conditions were produced and purified for use in immunological studies. In chapter four, the kinetics of the T cell response to these individual proteins during infection is determined overlaying leukocytes onto antigen-pulsed bone marrow derived dendritic cells. The response by T cells derived from the lungs and spleens of infected mice throughout 195 days of infection was specific to each protein and varied over the course of infection. The cytokines produced during the overlays were also examined and shown to be specific to each antigen and the length of infection. The protective potential of these proteins was determined in a vaccine study demonstrating that these proteins alone did not confer protection against subsequent challenge, but boosting BCG vaccination with these proteins augmented the protective efficacy of BCG alone significantly.