Background: A major impediment in global efforts to control tuberculosis (TB) is the complex long-term nature of the host-pathogen interactions leading to latent TB infection. It is estimated that one third of the world's population has latent TB infection. Given the alarming rate of spread of human immunodeficiency virus (HIV) infection, this latent population of organisms represents a significant reservoir for later reactivation and continued transmission of tubercle bacilli. A novel in vivo granuloma model involving the encapsulation of Mycobacterium tuberculosis (M. tb) in semi-diffusible hollow fibers which are implanted subcutaneously in mice has shown great promise as a model for latent TB infection. Objectives and Methods: The objectives of this KO8 proposal are three-fold and involve the use of the in vivo hollow fiber model. Objective 1 is to characterize the adaptive mechanisms employed by M. tb during persistence in granulomas in vivo by global gene expression analysis. Objective 2 is to identify M. tb virulence genes required for persistence in artificial granulomas in mice using transposon mutagenesis, and to confirm these findings in the standard mouse model of chronic pulmonary TB. Objective 3 is to characterize the immunogenicity of persistent M. tb in the hollow fiber model in vivo. Specifically, the global gene expression profile of mouse spleens will be analyzed in order to gain insight into host immune mechanisms involved in the containment of bacillary growth within the hollow fiber model, as well as theformation of granuloma-like lesions surrounding the fibers and mouse spleen enlargement. The role of cytokines essential to the maintenace of mycobacterial latency will be addressed by the use of mice deficient in certain cytokines, including TNF-alpha. The protective efficacy of continuous immune presentation of the entire spectrum of M. to-secreted soluble factors against aerosol challenge with M. tb will be evaluated using the hollow fiber model in vivo. Relevance: Characterization of mycobacterial virulence factors and host immune mechanisms involved in the maintenance of latent TB infection may identify novel molecular targets for anti-TB drug therapy, and permit the rational design of attenuated vaccine strains which can persist in host tissues without causing disease.
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