Clostridium difficile infection (CDI) is the leading cause of nosocomial infection in hospitalized patients receiving long-term antibiotic treatment. An excessive host inflammatory response is believed to be the major mechanism underlying the pathogenesis of C. difficile infection, and various proinflammatory cytokines such as IL-1β are detected in patients with C. difficile infection. IL-1β is known to be processed by caspase-1, a cysteine protease that is regulated by a protein complex called the inflammasome, which leads to a specialized form of cell death called pyroptosis. The function of inflammasome activation-induced pyroptosis is to clear or limit the spread of invading pathogens via infiltrated neutrophils. Here, we focused on inflammasome activation induced by intact C. difficile to re-evaluate the nature of inflammasome activation in CDI pathogenesis, which could provide information that leads to an alternative therapeutic strategy for the treatment of this condition in humans. First, we found that caspase-1-dependent IL-1β production was induced by C. difficile pathogens in macrophages and increased in a time-dependent manner. Moreover, intracellular toxigenic C. difficile was essential for ATP-P2X7 pathway of inflammasome activation and subsequent caspase-1-dependent pyroptotic cell death, leading to the loss of membrane integrity and release of intracellular contents such as LDH. Notably, we also observed that bacterial components such as surface layer proteins (SLPs) were released from pyroptotic cells. In addition, pro-IL-1β production was completely MyD88 and partially TLR2 dependent. Finally, to investigate the role of the caspase-1-dependent inflammasome in host defense, we found that colonic inflammasome activation was also induced by CDI and that caspase-1 inhibition by Ac-YVAD-CMK led to increased disease progression and C. difficile load. Taken together, the present results suggest that MyD88 and TLR2 are critical component in pro-IL-1β production and intracellular C. difficile following the ATP-P2X7 pathway of inflammasome activation and pyroptosis, which play important roles in host defense through the utilization of inflammation-mediated bacterial clearance mechanisms during C. difficile infection.

Clostridium difficile, an obligate anaerobic gram-positive bacillus, generates spores and is commonly found colonizing the human gut. Patients with C. difficile infection (CDI) often exhibit clinical manifestations of pseudomembranous colitis or antibiotic-associated diarrhea. Surface layer proteins (SLPs) are the most abundant proteins in the C. difficile cell wall, suggesting that they might involve in immune recognition. Our previous results demonstrated that C. difficile triggers inflammasome activation. Here, we found SLPs as well as C. difficile induced inflammasome activation, and in a dose-dependent manner. In addition, the cholesterol-rich microdomains on the cell membrane (also referred to as lipid rafts) are thought to be crucial for bacterial adhesion and signal transduction. We demonstrated that lipid rafts participated in C. difficile SLPs binding to the cell membrane. Fluorescence microscopy showed that membrane cholesterol depletion by methyl-β-cyclodextrin (MβCD) reduced the association of SLPs with the cell surface. The coalescence of SLPs in the cholesterol-rich microdomains was confirmed in C. difficile-infected cells. Furthermore, the inflammasome activations induced by SLPs or C. difficile were abrogated by MβCD. Our results demonstrate that SLPs recruit the lipid rafts, which may be a key step for C. difficile colonization and inducing inflammasome activation.

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