Graduate Thesis Or Dissertation

 

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  • Mycobacterium avium is an intracellular pathogen that is associated with disseminated infection, especially in patients with acquired immunodeficiency syndrome (AIDS). It appears that patients with AIDS acquire M. avium mostly through the intestinal tract, and that bacteria enter the intestinal wall at the terminal ileum. Previous studies have found that the rate at which M. avium invades intestinal cells in vitro is increased by pre-exposure to hyperosmolar or hypoxic conditions. Acting on the hypothesis that a repressor of invasion exists that is active in the presence of oxygen, transposon mutagenesis and positive selection, using serial invasion of intestinal epithelial cells in an amikacin protection assay, were used to isolate a putatively hyperinvasive mutant clone of M. avium strain A5, dubbed HI5. In a murine oral infection model, the mutant was found to have attenuated dissemination to in vivo. Using a polarized monolayer grown on a filter membrane as a model of the intestinal wall, HI5 was found to also be attenuated in translocation in vitro. Using RAW264.7 cells, the mutant was found to have comparable rates of entry into and survival inside macrophages over 4 days. Expression studies in a DNA macroarray and real-time PCR failed to detect a difference in gene expression between the wild-type A5 and mutant HI5 strains. Later invasion studies showed HI5 not to be hyperinvasive, but in fact resistant to the amikacin used in the invasion assay. The transposon mutation was originally believed to be in a gene similar to Rv1722 from M. tuberculosis, but this too was found to be in error. Taken together, these results suggest a dissemination deficient mutant has been isolated in this study, but the mechanism of invasion regulation remains to be discovered.
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