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

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Abstract
Mycobacterium tuberculosis and other pathogenic mycobacteria export abundant quantities of proteins into their extracellular milieu when growing either axenically or within phagosomes of host cells. One major extracellular protein, the enzyme glutamine synthetase, is of particular interest because of 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/glutamine structure in the mycobacterial cell wall. In this study, we investigated the influence of glutamine synthetase inhibitors on the growth of pathogenic and nonpathogenic mycobacteria and on the poly-l-glutamate/glutamine cell wall structure. The inhibitor l-methionine-S-sulfoximine rapidly inactivated purified M. tuberculosis glutamine synthetase, which was 100-fold more sensitive to this inhibitor than a representative mammalian glutamine synthetase. Added to cultures of pathogenic mycobacteria, l-methionine- S-sulfoximine rapidly inhibited 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. Remarkably, the inhibitor selectively blocked the growth of pathogenic mycobacteria, 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 phagocytes (THP-1 cells), the pathogen9s primary host cells. Paralleling and perhaps underlying its bacteriostatic effect, the inhibitor markedly reduced the amount 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 concept that extracellular proteins of M. tuberculosis and other pathogenic mycobacteria are worthy targets for new antibiotics. Such proteins constitute readily accessible targets of these relatively impermeable organisms, which are rapidly developing resistance to conventional antibiotics.