The Mechanism Of Type Three Secretion System Needle Protein And Clostridium Difficile Toxin B Induced Inflammasome Activation

The inflammasome, which is mainly present in macrophages and dendritic cells, plays an important role in innate immune system. The activation of inflammasome leads to the autocleavage of caspase-1, which is the active form. Caspase-1 activation causes two effects in macropahges/dendritic cells, the maturation of proinflammatory cytokines(IL-1β and IL-18) and the inflammatory cell death of the cell, which is defined as pyroptosis. Activation of inflammasome requires NLRs(Nod Like Receptors), which were characterized by the central NBD domain. There are 23 NLRs in human genome while even more in mice, most of them are with unknown functions.Among NLRs, NLRC4 is well studied. Many gram negative bacteria with type three secretion system (T3SS) activate NLRC4-inflammasome. The previous research proved that recognition of T3SS rod protein and flagellin by NAIP2 and NAIP5/6 respectively leads to the NLRC4 inflammasome activation. NAIP2 and NAIP5/6 belongs to NAIP sub-family. There are 4 NAIPs expressed in murine macrophages, NAIP1, NAIP2, NAIP5 and NAIP6. The function of NAIP1 is still unknown. Different from mouse, there is only one NAIP in human. Therefore, the function of NLRC4 in human is stil mysterious. Using Chromobacterium Violacium as a tool, we discovered that T3SS needle proteins could activate NLRC4 inflammsome in both mouse and human macrophages. The inflammatory activities of needle proteins are conserved among many T3SS bacterium. hNAIP and NAIP1 are responsibe for the recognition of needle proteins in human and mice, respectively. In human sourced macrophages, the infection of S.typhimurium and S.flexneri leads to the activation of caspase-1, based on the recognition of needle protein by hNAIP. In mice, NLRC4 inflammasome is more complicated, which NAIP is responsible for recognition of pathogens is determined by both the host cell type and pathogens. In mouse dendritic cell line DC2.4, in which NAIP1 is expressed at a relatively high level, S.flexneri infection triggered caspase-1 activation mainly depends on the recognition of the needle protein by NAIP1. Therefore, needle protein-hNAIP/NAIP1-NLRC4 plays an important role in the innate immune system in both human and mice. Besides NLRC4 inflammsome, there are three types of inflammasomes discovered, NLRP3 inflammasome, AIM2 inflammasome and NLRP1b inflammsome. It was reported that another protein named Pyrin could induce the formation of ASC-foci when overexpressed in 293T cells. Pyrin was encoded by Mefv, the gene related with the autoinflammatory disease FMF(Familial Mediterranean Fever). Therefore it was proposed long time ago that Pyrin inflammasome may be functional. However, its real function is still unknow. It was reported that TcdB from gram positive bacteria Clostridium difficile could trigger caspase-1 activation in BMM. TcdB is a glycotransferase which inactivates small GTPase. Using cell biology and mouse genetics as tools, we proved that Mefv was required for TcdB induced caspase-1 activation, which revealed the function of Pyrin inflammasome for the first time. The catalytic acitivy is required for TcdB to activate caspase-1. We further discovered that covalent modification leading to inactivation of Rho sub-family by other toxins/effectors could also activate Pyrin inflammasome. This suggests a new mechanism during the activation of Pyrin inflammasome, which is different from the known inflammsomes.