A GAIN-OF-FUNCTION SCREEN REVEALS M.TUBERCULOSIS GENES MEDIATING INFLAMMASOME INHIBITION

Host responses resulting in production of the pro-inflammatory cytokine IL-1β promote Mycobacterium tuberculosis (Mtb) clearance. Mtb infection of macrophages and dendritic cells activates the NLRP3 inflammasome to stimulate IL-1β secretion. Interestingly, we have recently shown that Mtb inhibits the activation of the host cell AIM2 inflammasome suggesting a novel immune evasion mechanism. AIM2 inhibition depends on the presence of a functional ESX-1 secretion system, suggesting that a secreted effector is involved in this process. In order to identify the Mtb genes important in this process we utilized a gain-of-function genetic screen in M. smegmatis (Msm). We screened a library consisting of 312 independent Msm clones each containing an episomal cosmid with a ~40kbp insert of Mtb genomic DNA. The screening was performed using bone marrow derived dendritic cells (BMDC) generated from NLRP3 knock-out mice in order to eliminate NLRP3-inflammasome activation. BMDCs were infected individually with the 312 Msm clones and IL-1β production was measured using ELISA. We found that a total of 25 clones exhibited significant reduction of IL-1β secretion compared to BMDCs infected with wild-type Msm. These results demonstrate that the Mtb genome contains genes important for inhibition of host cell AIM2-inflammasome activation. In future studies we will identify the specific gene(s) within the Mtb DNA insert of the selected cosmids that mediate AIM2 inhibition. The generation of specific loss-of-function mutants in Mtb will enable studies of the molecular mechanism of AIM2-inflammasome inhibition by Mtb and its importance for the virulence of the bacteria.