Mycobacterial EST12 activates a RACK1–NLRP3–gasdermin D pyroptosis–IL-1β immune pathway

Abstract

Pyroptosis, an inflammatory form of programmed cell death, has been implicated in eliminating pathogenic infections. However, macrophage pyroptosis–related proteins from Mycobacterium tuberculosis (M.tb) have largely gone unexplored. Here, we identified a cell pyroptosis–inducing protein, Rv1579c, named EST12, secreted from the M.tb H37Rv region of difference 3. EST12 binds to the receptor for activated C kinase 1 (RACK1) in macrophages, and the EST12-RACK1 complex recruits the deubiquitinase UCHL5 to promote the K48-linked deubiquitination of NLRP3, subsequently leading to an NLRP3 inflammasome caspase-1/11–pyroptosis gasdermin D–interleukin-1β immune process. Analysis of the crystal structure of EST12 reveals that the amino acid Y80 acts as a critical binding site for RACK1. An EST12-deficient strain (H37RvΔEST12) displayed higher susceptibility to M.tb infection in vitro and in vivo. These results provide the first proof that RACK1 acts as an endogenous host sensor for pathogens and that EST12-RACK1–induced pyroptosis plays a pivotal role in M.tb-induced immunity.

Aptamer Detection of Mycobaterium tuberculosis Mannose-Capped Lipoarabinomannan in Lesion Tissues for Tuberculosis Diagnosis

“…C57BL/6 mice were infected with M.tb H37Rv as described in Materials and Methods . We generated three polyclonal antibodies (pAbs) which reacted with a unique surface antigen ManLAM from M.tb H37Rv, the M.tb -specific antigens (Rv2645) ( Luo et al., 2015 ) and (Rv1579) ( Qu et al., 2020 ), respectively. We also applied the commercial polyclonal antibody against M.tb -specific Ag85B antigen.…”

“…After antigen retrieval, all the samples were incubated with hydrogen peroxide at room temperature for 10 min to inhibit the endogenous peroxidase activity. Next, samples were blocked with 100 μl 5% BSA at room temperature for 30 min, and 100 μl 300 nM biotin-labeled ssDNA aptamer or rabbit anti-Rv2645 (1:300) ( Luo et al., 2015 ), anti-Rv1579 (1:500) ( Qu et al., 2020 ) or commercial anti-Ag85B polyclonal antibody (Abcam, ab43019, 1:100) was added and incubated at 37°C for 1 h. Horseradish peroxidase (HRP)-conjugated streptavidin (YEASEN, 35105ES60, 1:500) or HRP-anti-rabbit IgG (Proteintech, SA00001-2, 1:500) was added and incubated for 30 min at 37°C. 3,3′-diaminobenzidine tetrahydrochloride (DAB) Chromogen Solution was added for 1 min, then sections were counterstained with hematoxylin, dehydrated, and then sealed with neutral resin glue.…”

The Roles of Inflammasomes in Host Defense against Mycobacterium tuberculosis

“…MTB activates the NLRP3 inflammasome via several constituents, including ESX-1 secretion system [ 34 ], Rv1579c (also called EST12) [ 144 ], Rv0878c (also called PPE13) [ 145 ], the cell wall component mannosylated lipoarabinomannan [ 60 ] and dsRNA [ 64 ]. MTB damages phagosomal and plasma membranes during phagocytosis of bacteria, leading to K + efflux and activation of NLRP3-dependent IL-1β release and pyroptosis [ 34 ].…”

“…Stimulation with M. bovis BCG complemented with region of difference 1 (RD1), which encodes a part of the ESX-1 secretion system, induces IL-1β release [ 63 ]. Rv1579c, secreted from MTB H37Rv RD3, interacts with the receptor for activated C kinase 1 (RACK1) via its amino acid Y80 at the C-terminus, then recruits ubiquitin C-terminal hydrolase L5 (UCHL5) to deubiquitinate NLRP3, and finally activate the NLRP3 inflammasome [ 144 ].…”

Deubiquitinases: Modulators of Different Types of Regulated Cell Death

“…Knockdown of UCHL5 completely inhibits EST12-mediated deubiquitination of NLRP3, which suppresses pyroptosis. This indicates that UCHL5 is required for the EST12/RACK/pyroptosis response in macrophages and the clearance of pathogens by the host [ 171 ].…”