This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project aims at understanding the molecular and biochemical mechanisms of pathogenesis of mycobacterial infections. To elucidate the molecular mechanisms of mycobacterial pathogenesis, we use M. marinum (Mm) as a model organism to facilitate the research. Mm, a fish, amphibian, and opportunistic human pathogen, grows four times faster than Mtb and provides safety and ease in laboratory manipulations. We constructed a Mm transposon mutant library by using a mariner-based transposon and screened approximately 1000 mutants. Mutants were identified exhibiting defective phenotypes as follows: i) intracellular growth in resting or INF-g-activated macrophages; ii) cytotoxicity to macrophage; iii) contact-dependent hemolysis of red blood cell and cytolysis of macrophage; iv) resistance to defensin; and v) pigmentation variants that produced either no or red pigment. Sequencing of the transposon junctions has identified insertions within Mtb homologous genes that are implicated in virulence. Several of these are in a genomic region homologous to the RD1 locus of M. tuberculosis that has been implicated in secretion of virulence factors. Some of our mutants have quite abnormal secretion patterns. We are interested in working with the mass spec facility to determine I) the proteins that are differentially secreted between wildtype and mutant organisms; and II) the protein-protein interactions involved in the RD1 secretion system. Finally, mutants have been found in unknown open reading frames that affect cytokine secretion by infected macrophages. We would like to use mass spec to determine the nature of the protein and lipid alterations in these mutants.
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