Development and use of genetic reporters to identify Mycobacterium tuberculosis exported proteins important for virulence

Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease that kills nearly two million people each year globally. To cause disease, M. tuberculosis must survive and replicate in macrophages in the lung within phagosomal compartments. Macrophages typically neutralize bacterial invaders, but M. tuberculosis has developed defenses against macrophage attack. The relatively recent ability to construct M. tuberculosis mutants has greatly advanced our understanding of the protein transport systems and the importance of exported proteins to virulence of this pathogen. Exported proteins, which we define here as those exposed on the surface or exported out of the bacterium, make excellent bacterial virulence factors as they are best localized to counteract host defenses against infection. The main objective of the research described in this dissertation was to develop genetic tools and then use them to identify M. tuberculosis exported proteins important to pathogenesis. For many of the proteins identified in this research, these studies provide the first experimental evidence of export. In the chapters that follow, three different genetic export reporter systems are described. We constructed a new reporter system to specifically identify M. tuberculosis proteins exported by the twin arginine translocation pathway. We also developed a new reporter system with the unique ability to report on protein export during M. tuberculosis growth in host cells. This system was then modified into a new reporter transposon used to simultaneously identify exported proteins of M. tuberculosis and collect mutants lacking these proteins. The resulting library of M. tuberculosis mutants was successfully screened for mutants defective for growth in macrophages as a strategy to identify new exported virulence factors. Future investigations are sure to focus more directly on how proteins exported by this pathogen interact with the host and cause disease.