RESUMO
Mycobacterium tuberculosis can persist in macrophage phagosomes that acidify to a pH of approximately 4.5 after activation of the macrophage with gamma interferon. How the bacterium resists the low pH of the acidified phagosome is incompletely understood. A screen of 10,100 M. tuberculosis transposon mutants for mutants hypersensitive to pH 4.5 led to the discovery of 21 genes whose disruption attenuated survival of M. tuberculosis at a low pH (41). Here, we show that acid-sensitive M. tuberculosis mutants with transposon insertions in Rv2136c, Rv2224c, ponA2, and lysX were hypersensitive to antibiotics, sodium dodecyl sulfate, heat shock, and reactive oxygen and nitrogen intermediates, indicating that acid resistance can be associated with protection against other forms of stress. The Rv2136c mutant was impaired in intrabacterial pH homeostasis and unable to maintain a neutral intrabacterial pH in activated macrophages. The Rv2136c, Rv2224c, and ponA2 mutants were attenuated in mice, with the Rv2136c mutant displaying the most severe level of attenuation. Pathways utilized by M. tuberculosis for acid resistance and intrabacterial pH maintenance are potential targets for chemotherapy.
Assuntos
Ácidos/metabolismo , Parede Celular/metabolismo , Mutação , Mycobacterium tuberculosis/metabolismo , Estresse Oxidativo , Tuberculose/microbiologia , Ácidos/farmacologia , Animais , Antibacterianos/farmacologia , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Células Cultivadas , Feminino , Interações Hospedeiro-Patógeno , Humanos , Concentração de Íons de Hidrogênio , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Mutagênese Insercional , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genéticaRESUMO
Rv3671c, a putative serine protease, is crucial for persistence of Mycobacterium tuberculosis in the hostile environment of the phagosome. We show that Rv3671c is required for M. tuberculosis resistance to oxidative stress in addition to its role in protection from acidification. Structural and biochemical analyses demonstrate that the periplasmic domain of Rv3671c is a functional serine protease of the chymotrypsin family and, remarkably, that its activity increases on oxidation. High-resolution crystal structures of this protease in an active strained state and in an inactive relaxed state reveal that a solvent-exposed disulfide bond controls the protease activity by constraining two distant regions of Rv3671c and stabilizing it in the catalytically active conformation. In vitro biochemical studies confirm that activation of the protease in an oxidative environment is dependent on this reversible disulfide bond. These results suggest that the disulfide bond modulates activity of Rv3671c depending on the oxidative environment in vivo.