Mycobacterium tuberculosis protein ESAT-6 is a potent activator of the NLRP3/ASC inflammasome.

Interleukin-1β (IL-1β) represents one of the most important mediators of inflammation and host responses to infection. Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, induces IL-1β secretion at the site of infection, but the underlying mechanism(s) are poorly understood. In this work we show that Mtb infection of macrophages stimulates caspase-1 activity and promotes the secretion of IL-1β. This stimulation requires live intracellular bacteria expressing a functional ESX-1 secretion system. ESAT-6, an ESX-1 substrate implicated in membrane damage, is both necessary and sufficient for caspase-1 activation and IL-1β secretion. ESAT-6 promotes the access of other immunostimulatory agents such as AG85 into the macrophage cytosol, indicating that this protein may contribute to caspase-1 activation largely by perturbing host cell membranes. Using a high-throughput shRNA-based screen we found that numerous NOD-like receptors (NLRs) and CARD domain-containing proteins (CARDs) were important for IL-1β secretion upon Mtb infection. Most importantly, NLRP3, ASC and caspase-1 form an infection-inducible inflammasome complex that is essential for IL-1β secretion. In summary, we show that recognition of Mtb infection by the NLRP3 inflammasome requires the activity of the bacterial virulence factor ESAT-6, and the subsequent IL-1β response is regulated by a number of NLR/CARD proteins.This work was supported by grants from the National Foundation for Science-FCT project PPCDT/BIA-BCM/55327/2004, PIC/IC/82859/2007 to E.A., SFRH/BD/28173/2006 fellowship to B.B.M. and SFRH/BD/15238/2004 fellowship to P.M.-A. L.F.M. is a Young Investigator from the Human Frontier Science Program and receives support from FLAD and FCT (PTDC/SAU-MII/69280/2006 and PTDC/SAU-MII/78333/2006)