UBC Theses and Dissertations
The impact of cell wall alterations in Mycobacterium tuberculosis on macrophage interactions and virulence Lynett, Jennifer Theresa
While Mycobacterium tuberculosis is known to infect and persist within macrophages, the characterization of the mycobacterial gene products that are involved during host cell interactions remains incomplete. For the present study, we screened a transposon library of M. tuberculosis using a positive selection strategy to identify mutants with an enhanced binding affinity for macrophages. Our expectation was that this approach would identify genes that are involved in the maintenance of the cell wall architecture and could provide novel insights into the virulence of the organism. The initial library screen identified five mutants with transposon disruptions within genes that encode enzymes associated with lipid synthesis, a putative membrane protein and proteins with unknown functions. One mutant selected for further study contained a transposon insertion within the fadD23 gene. The mutant was found to be devoid of sulfolipid production, impaired for survival within macrophages and unable to cause disease in aerosol-infected mice. A second mutant was also characterized where the transposon insertion mapped to the Rv1505c gene which codes for a conserved hypothetical protein. This mutant also exhibited a significantly reduced rate of intracellular replication within macrophages and failed to actively replicate in vivo. However, the interpretation of the in vivo findings with the Rv1505c mutant were complicated by the fact that genetic complementation of the mutant with the wild type gene failed to demonstrate a restoration of virulence within the mouse model of infection. A second screen of the transposon library for alterations in colonial morphology resulted in the identification of a mutant with a disruption in the mtrB gene which encodes the sensor kinase of a two-component regulatory system. Studies in other mycobacterial species have also found that the inactivation of this locus leads to alterations in cell wall structure. We found that the absence of mtrB affected the distribution of the phosphatidylinositol mannosides and the phthiocerol dimyococerosates lipids of the cell envelope. In addition, the mutant was also compromised for virulence within the murine model of infection.