The Type I NADH Dehydrogenase of Mycobacterium tuberculosis Counters Phagosomal NOX2 Activity to Inhibit TNF-α-Mediated Host Cell Apoptosis

Abstract

The capacity of infected cells to undergo apoptosis upon insult with a pathogen is an ancient innate immune defense mechanism. Consequently, the ability of persisting, intracellular pathogens such as the human pathogen Mycobacterium tuberculosis (Mtb) to inhibit infection-induced apoptosis of macrophages is important for virulence. The nuoG gene of Mtb, which encodes the NuoG subunit of the type I NADH dehydrogenase, NDH-1, is important in Mtb-mediated inhibition of host macrophage apoptosis, but the molecular mechanism of this host pathogen interaction remains elusive. Here we show that the apoptogenic phenotype of MtbΔ nuoG was significantly reduced in human macrophages treated with caspase-3 and -8 inhibitors, TNF-α-neutralizing antibodies, and also after infection of murine TNF ^−/− macrophages. Interestingly, incubation of macrophages with inhibitors of reactive oxygen species (ROS) reduced not only the apoptosis induced by the nuoG mutant, but also its capacity to increase macrophage TNF-α secretion. The MtbΔ nuoG phagosomes showed increased ROS levels compared to Mtb phagosomes in primary murine and human alveolar macrophages. The increase in MtbΔ nuoG induced ROS and apoptosis was abolished in NOX-2 deficient ( gp91 ^−/− ) macrophages. These results suggest that Mtb, via a NuoG-dependent mechanism, can neutralize NOX2-derived ROS in order to inhibit TNF-α-mediated host cell apoptosis. Consistently, an Mtb mutant deficient in secreted catalase induced increases in phagosomal ROS and host cell apoptosis, both of which were dependent upon macrophage NOX-2 activity. In conclusion, these results serendipitously reveal a novel connection between NOX2 activity, phagosomal ROS, and TNF-α signaling during infection-induced apoptosis in macrophages. Furthermore, our study reveals a novel function of NOX2 activity in innate immunity beyond the initial respiratory burst, which is the sensing of persistent intracellular pathogens and subsequent induction of host cell apoptosis as a second line of defense.

Author Summary

Mycobacterium tuberculosis, the causative agent of tuberculosis, is highly adapted to survive in macrophages of its human host. Host cell suicide is an ancient host cell defense mechanism employed by organisms to wall off invading pathogens. M. tuberculosis manipulates infected cells to inhibit host cell death but the molecular mechanism of this interaction has not been elucidated. Here we describe that M. tuberculosis uses an enzyme complex (NDH-1) usually needed for energy generation in order to neutralize the NOX-2 enzyme-mediated production of toxic oxygen radicals (ROS) by the host cell. We demonstrate that an M. tuberculosis mutant deficient in NDH-1 accumulates ROS inside the macrophage which induces the secretion of an inflammatory cytokine (TNF-α) and subsequent host cell death. The increase of ROS is dependent upon functional NOX-2, since host cells missing a NOX-2 component do not undergo cell death upon infection with the mutant. We propose that a novel function of the host cell NOX-2 complex is to allow sensing of intracellular pathogens by the host cell in order to commit suicide and thus limit bacterial survival.