Molecular Genetics of Beta-Lactam Sensitivity and Resistance in Mycobacterium Tuberculosis
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CitationWivagg, Carl. 2012. Molecular Genetics of Beta-Lactam Sensitivity and Resistance in Mycobacterium Tuberculosis. Doctoral dissertation, Harvard University.
AbstractMycobacterium tuberculosis threatens global health. Its thick, impermeable cell wall renders it tolerant to high doses of many antibiotics. While we understand the biochemical functions of many cell wall-modifying enzymes, we often do not understand their physiological functions: their spatiotemporal roles in the cell cycle, their substrate preferences, or their individual effects on the macromolecular architecture of the cell wall complex. Mycobacterium tuberculosis possesses five peptidoglycan transpeptidases, five lytic transglycosylases, and numerous other peptidoglycan-modifying enzymes that lack precisely-understood roles. The lytic transglycosylases of Mycobacterium tuberculosis are collectively dispensable for in vitro growth. We sought to learn what other classes of peptidoglycan-degrading enzyme might substitute for the lytic transglycosylases or become essential in their absence. A high-throughput chemical screen was performed on a strain lacking all five lytic transglycosylases to identify compounds that specifically killed this strain and not wild-type Mycobacterium tuberculosis. Among the compounds identified were several members of the cephalosporin class of \(\beta\)-lactam antibiotics. It was shown that the cephalosporins had greater access to the periplasmic \(\beta\)-lactamase of Mycobacterium tuberculosis in the lytic transglycosylase-deficient strain and that this strain had enhanced sensitivity to several antibiotics with unrelated mechanisms of action. Together, greater periplasmic access and broadly heightened susceptibility in the deficient strain suggested a role for the lytic transglycosylases in maintaining the mycolic acid permeability barrier. To identify the specific penicillin-binding protein target of the cephalosporins, we isolated spontaneously-occurring resistant mutants. These strains contained polymorphisms in ponA2, a bifunctional penicillin-binding protein. The polymorphisms conferred sensitivity to heat stress, a phenotype associated with ponA2 loss of function. To clarify the relation between loss of function and cephalosporin resistance, a ponA2 deletion strain was created, which exhibited both cephalosporin resistance and sensitivity to carbapenems, another class of \(\beta\)-lactam. Restoration of the wild-type ponA2 allele suppressed both cephalosporin resistance and carbapenem sensitivity. Inactivation of other transpeptidases did not confer resistance to any \(\beta\)-lactams. The association of penicillin-binding protein inactivation with \(\beta\)-lactam resistance is unusual. One model to explain it is that upon deletion of ponA2, Mycobacterium tuberculosis compensates for its loss by upregulating a cephalosporin-resistant, meropenem-sensitive transpeptidase.
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