| Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, infects over 2 billion people worldwide and kills as many as 3 million each year. While vaccination offers the theoretical potential to eradicate the disease, the reality has been disappointing: Bacille Calmette-Guerin (BCG) is currently the only available vaccine against Mtb but is of limited efficacy against pulmonary tuberculosis, the most common form of the disease. An ideal vaccine is likely to involve inoculation with an attenuated, but more immunogenic, mutant strain of Mtb. Development of such strains will require a broader understanding of the interaction between Mtb and the innate immune system, particularly with respect to the Mtb genes that modulate host immunity.;This thesis describes a genome-wide, "forward genetics" approach to identify Mtb genes that modulate host immune responses. Using a library of 10,100 Mtb mutants, we infected macrophages expressing promoter-reporter constructs in which the promoters of target host genes were used to drive expression of fluorescent reporter proteins. Following infection with each of the Mtb mutants, fluorescence induction in these macrophages was monitored as a surrogate for the native immune response. Target host genes were composed of a dozen factors involved in the primary immune response. We confirmed the results of our screen in vitro by performing infections in both bone-marrow-derived macrophages and dendritic cells and measuring the endogenous immune response by ELISA and qPCR. As proof-of-principle, we successfully complemented one of the mutants and rescued its immune phenotype. Finally, we obtained in vivo validation of the screen by performing murine aerosol infections with two of these mutants and quantifying bacterial burden, lung mRNA expression, and histopathology of the animals.;We identified over 30 mutants whose effect on the primary immune response has now been broadly defined with respect to diverse aspects of innate immunity. Vaccine challenge studies using these mutants would allow important correlations to be derived between the primary immune response and vaccine protection. Ultimately, mutations of the genes that we have identified could be generated in auxotrophic strains of Mtb to yield more effective vaccine candidates. |
