MOLECULAR AND CELL BIOLOGY OF PATHOGENIC MYCOBACTERIA

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M. marinum The objectives of the reasearch described in this project are to use M. marinum as a model for identifying genes required for survival and replication of M. tuberculosis (MTB) within the macrophage as well as for identifying genes required for MTB mediated cytotoxicity In previous studies, we established that M. marinum like M. tuberculosis survives in a unique phagosomal compartment which does not fuse with the lysosome. In order to determine how mycobacteria are able to survive in macrophages, we devised a novel strategy for isolating gfp fusions to mycobacterial genes expressed differentially in the phagosomal environment. In this method, infected cells were lysed and the released phagosomes FACS-sorted to obtain bacteria containing fusions to genes expressed intracellularly. These were then assayed for expression on solid media to obtain populations of fusions differentially expressed intracellularly. Using this methodology we identified 12 fusions to genes which are differentially expressed in macrophages. DNA sequence adjacent to GFP showed homology with genes in M. tuberculosis.. A further analysis of these genes and identification of their products should provide insight into how mycobacteria survive within the macrophage. During the process of isolating GFP fusions, we identified a construct , GFP13, which contained a promoter expressed constitutively at least 5 fold better than HSP60, the mycobacterial heat shock promoter.. The identification of this very strong promoter could provide a very useful tool for achieving high level expression of mycobacterial genes. M. ulcerans. Infection with M. ulcerans, the causative agent of Buruli ulcer , results in a severe necrotizing skin lesion with very little acute inflammatory response. In order to understand how M. ulcerans causes disease, we have purified and characterized a toxin, MULT, from M. ulcerans,. Structural studies of MULT reveal that it is a complex polyketide, a 12- membered ring macrolide. Complex polyketides include a large number of potent bioactive molecules such as antibiotics (erythromycin), immunosuppressants (FK506), antifungals (amphotericin) and cytostatins (Bafilomycin). Although complex polyketides are common among Streptomyces species, MULT is the first complex polyketide, and first macrolide isolated from Mycobacteria species. We have characterized the biological activities of MULT using both in vitro tissue culture and in vivo studies. In pg amounts, MULT causes mouse fibroblasts to arrest in G1 of the cell cycle. More remarkably, intradermal injection of MULT into a guinea pig produces lesions pathologically identical to those of Buruli ulcer.