Identification and characterization of a Mycobacterium tuberculosis gene that enhances mycobacterial survival within macrophages
AdvisorFriedman, Richard L.
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PublisherThe University of Arizona.
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AbstractThe virulence of Mycobacterium tuberculosis (Mtb) depends on its ability to multiply and survive within host macrophages. In screening for Mtb genes that play a role in the intracellular survival, a Mtb gene ( eis) was identified that enhanced survival of Mycobacterium smegmatis in both human monocytes and in the human macrophage-like cell line U-937 when introduced on the multi-copy plasmid pOLYG. When a single chromosomal copy of eis was introduced into M. smegmatis, using an integrative vector, the construct still exhibited increased intracellular survival in U-937 cells. The eis gene was found in the genomic DNA of various M. tuberculosis strains and of Mycobacterium bovis BCG but not in that of M. smegmatis or 10 other mycobacterial species. Western blot analysis showed that the eis gene could produce a 42-kD protein product in both M. smegmatis and M. tuberculosis. The role of the eis gene in M. tuberculosis intracellular survival and multiplication was investigated by inactivation of the eis gene via allelic exchange. A mutated eis allele ( eis::hyg) was delivered at the eis locus, using the suicide vector pMJ10, in both Mtb strains H37Rv and H37Ra. Complemented mutants were also constructed by reintroducing a wild-type eis into the chromosome attB site. Southern and Western blot analysis demonstrated that the eis gene was disrupted and no Eis protein was produced, and that complemented strains regained the ability to produce Eis. Wild-type M. tuberculosis, eis knockout mutant and complemented strain were then evaluated for their capacity to survive and multiply within U-937 cells. The eis mutant survived and multiplied, as its parental strain, in U-937 cells over a 7-day period. These findings suggest that eis may not be required for the short-term multiplication of M. tuberculosis in U-937 cells. Further in vivo study needs to be done to clarify the role of eis in the pathogenesis of tuberculosis.
Degree ProgramGraduate College
Microbiology and Immunology