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

Saturday, February 18, 2017
Exhibit Hall (Hynes Convention Center)
Sarah Ahlbrand, University of Maryland, College Park, MD
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.