ESAT-6 of Mycobacterium tuberculosis downregulates cofilin1, leads to filamentous actin enrichment and reduces the phagosome acidification in infected macrophages, which are partially reversed by a single methionine mutation

Abstract

Mycobacterium tuberculosis (M. tuberculosis) persists within macrophages by evading phagosome maturation. In this study, we considered the role of actin dynamics in this process. Macrophages infected with virulent M. tuberculosis showed high F-actin/G-actin ratio, accompanied by reduced expression of the actin-depolymerizing protein cofilin1 and increased levels of its inactive phosphorylated form. Overexpression of a constitutively active cofilin1 variant reduced F-actin accumulation and enhanced phagosome acidification. Similar effects were observed with sorafenib, a PI3K-dependent activator of cofilin1, which decreased F-actin levels and promoted phagosome acidification in infected macrophages. Ectopic expression of the mycobacterial virulence factor ESAT-6 in macrophages led to cofilin1 downregulation. ESAT-6 also increased F-actin, altered cell morphology and impaired phagosome acidification in infections with avirulent M. tuberculosis strain. As cofilin1 is positively regulated by reactive oxygen species (ROS), we examined the role of methionine in ESAT-6-mediated ROS suppression. Mutation of methionine 93 in ESAT-6 increased intracellular ROS, enhanced phagosome acidification, and decreased F-actin levels. These findings reveal that M. tuberculosis impairs phagosome acidification by modulating host actin dynamics at least partially through ESAT-6–mediated suppression of cofilin1 and ROS. Enhancing cofilin1 activity may represent a potential therapeutic strategy to restore phagosome function and improve bacterial clearance, more specifically during the initial establishment of infection.