An inhibitor of exported Mycobacterium tuberculosis glutamine synthetase selectively blocks the growth of pathogenic mycobacteria in axenic culture and in human monocytes: Extracellular proteins as potential novel drug targets

Mycobacterium tuberculosis and other pathogenic mycobacteria export abundan t quantities of proteins into their extracellular milieu when growing eithe r axenically or within phagosomes of host cells. One major extracellular pr otein, the enzyme glutamine synthetase, is of particular interest because o f its Link to pathogenicity. Pathogenic mycobacteria, but not nonpathogenic mycobacteria, export large amounts of this protein. Interestingly, export of the enzyme is associated with the presence of a poly-L-glutamate/glutami ne structure in the mycobacterial cell wall. In this study, we investigated the influence of glutamine synthetase inhibitors on the growth of pathogen ic and nonpathogenic mycobacteria and on the poly-L-glutamate/glutamine cel l wall structure. The inhibitor L-methionine-S-sulfoximine rapidly inactivated purified M. tu berculosis glutamine synthetase, which was 100-fold more sensitive to this inhibitor than a representative mammalian glutamine synthetase. Added to cu ltures of pathogenic mycobacteria, L-methionine-S-sulfoximine rapidly inhib ited extracellular glutamine synthetase in a concentration-dependent manner but had only a minimal effect on cellular glutamine synthetase, a finding consistent with failure of the drug to cross the mycobacterial cell wall. R emarkably, the inhibitor selectively blocked the growth of pathogenic mycob acteria, all of which release glutamine synthetase extracellularly, but had no effect on nonpathogenic mycobacteria or nonmycobacterial microorganisms , none of which release glutamine synthetase extracellularly. The inhibitor was also bacteriostatic for M, tuberculosis in human mononuclear phagocyte s (THP-1 cells), the pathogen's primary host cells. Paralleling and perhaps underlying its bacteriostatic effect, the inhibitor markedly reduced the a mount of poly-L-glutamate/glutamine cell wall structure in M. tuberculosis. Although it is possible that glutamine synthetase inhibitors interact with additional extracellular proteins or structures, our findings support the c oncept that extracellular proteins of M. tuberculosis and other pathogenic mycobacteria are worthy targets for new antibiotics. Such proteins constitu te readily accessible targets of these relatively impermeable organisms, wh ich are rapidly developing resistance to conventional antibiotics.