To aid understanding of the basic pathogenesis of tuberculosis, a rapidly resurgent disease worldwide, Mycobacterium marinum, a rapidly growing human pathogen will be used as a model system to elucidate virulence factors. Genetic systems for transformation and transposition have already been established in this organism, and will be exploited to isolate mutants deficient in invasion into eukaryotic cells and in intracellular replication. Attempts will be made to establish homologous recombination in this organism so that genes for putative virulence factors can be insertionally inactivated in order to study their effects. Mutants will also be tested in the Rana pipiens model for chronic disease which has been established. A Cosmid bank containing M. marinum genes will be introduced into the nonpathogen, M. smegmatis which is incapable of persistence and replication in eukaryotic cells in vitro and of persistence and disease in vivo. Pools of transformed M. smegmatis will be tested for persistence in vitro as well as in R. pipiens. Additionally, M. marinum RNAs that are expressed specifically in the eukaryotic cell milieu will be isolated by differential centrifugation of the mycobacteria from the host cells followed by subtractive hybridization of cDNA from that of broth grown mycobacteria. Finally, the cell biology of mycobacterial invasion, persistence and intracellular trafficking will be examined using a combination of video, immunoelectron and confocal microscopy. The role of the complement and Fc receptors in invasion and intracellular trafficking will be examined by the use of epithelial cells transfected with the appropriate receptors.
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