Mechanism of Inducible Nitric Oxide Synthase Exclusion from Mycobacterial Phagosomes

Mycobacterium tuberculosis is sensitive to nitric oxide generated by inducible nitric oxide synthase (iNOS). Consequently, to ensure its survival in macrophages, M. tuberculosis inhibits iNOS recruitment to its phagosome by an unknown mechanism. Here we report the mechanism underlying this process, whereby mycobacteria affect the scaffolding protein EBP50, which normally binds to iNOS and links it to the actin cytoskeleton. Phagosomes harboring live mycobacteria showed reduced capacity to retain EBP50, consistent with lower iNOS recruitment. EBP50 was found on purified phagosomes, and its expression increased upon macrophage activation, paralleling expression changes seen with iNOS. Overexpression of EBP50 increased while EBP50 knockdown decreased iNOS recruitment to phagosomes. Knockdown of EBP50 enhanced mycobacterial survival in activated macrophages. We tested another actin organizer, coronin-1, implicated in mycobacterium-macrophage interaction for contribution to iNOS exclusion. A knockdown of coronin-1 resulted in increased iNOS recruitment to model latex bead phagosomes but did not increase iNOS recruitment to phagosomes with live mycobacteria and did not affect mycobacterial survival. Our findings are consistent with a model for the block in iNOS association with mycobacterial phagosomes as a mechanism dependent primarly on reduced EBP50 recruitment.
Author Summary Mycobacterium tuberculosis infects one third of the world's population, with the majority of infected individuals being asymptomatic while running a lifetime risk of developing active disease. The key to the success of M. tuberculosis as a recalcitrant human pathogen is its ability to parasitize macrophages and persist in these cells or their derivatives for long periods of time. We still do not have complete knowledge of the full repertoire of M. tuberculosis determinants that allow it to evade bactericidal mechanisms of the macrophage. Here we report the mechanism by which M. tuberculosis eludes effective elimination by nitric oxide, a radical with antimycobacterial properties that is generated by the inducible form of nitric oxide synthase. It was generally assumed that nitric oxide synthase, upon induction by the major anti-tuberculosis cytokine interferon gamma, simply homogeneously fills up the macrophage like a sack and generates nitric oxide throughout the cell. The present study shows that nitric oxide synthase is not randomly distributed in macrophages, and that its positioning in the cell is dictated by interactions with the scaffolding protein EBP50, shown here to be induced during macrophage activation. Thus, not only do the phagocytic cells increase the amount of nitric oxide synthase, but they also have a system to deliver and keep this enzyme in the vicinity of phagosomes. This is of significance, as nitric oxide is a highly reactive radical, and its generation somewhere else in the cell would lead to it being spent by the time it diffuses to the site of intended action, such as mycobacterium-laden phagosomes. It turns out, as this study shows, that M. tuberculosis interferes with the process of EBP50-guided positioning of the inducible nitric oxide synthase, thus avoiding delivery and accumulation of this enzyme and its noxious products near the phagosome where nitric oxide would have the best chance of inhibiting intracellular mycobacteria.