Shigella Type III Secretion Protein MxiI Is Recognized by Naip2 to Induce Nlrc4 Inflammasome Activation Independently of Pkcδ

Recognition of intracellular pathogenic bacteria by members of the nucleotide-binding domain and leucine-rich repeat containing (NLR) family triggers immune responses against bacterial infection. A major response induced by several Gram-negative bacteria is the activation of caspase-1 via the Nlrc4 inflammasome. Upon activation, caspase-1 regulates the processing of proIL-1 and proIL-18 leading to the release of mature IL-1 and IL-18, and induction of pyroptosis. The activation of the Nlrc4 inflammasome requires the presence of an intact type III or IV secretion system that mediates the translocation of small amounts of flagellin or PrgJ-like rod proteins into the host cytosol to induce Nlrc4 activation. Using the Salmonella system, it was shown that Naip2 and Naip5 link flagellin and the rod protein PrgJ, respectively, to Nlrc4. Furthermore, phosphorylation of Nlrc4 at Ser533 by Pkc was found to be critical for the activation of the Nlrc4 inflammasome. Here, we show that Naip2 recognizes the Shigella T3SS inner rod protein MxiI and induces Nlrc4 inflammasome activation. The expression of MxiI in primary macrophages was sufficient to induce pyroptosis and IL-1 release, which were prevented in macrophages deficient in Nlrc4. In the presence of MxiI or Shigella infection, MxiI associated with Naip2, and Naip2 interacted with Nlrc4. siRNA-mediated knockdown of Naip2, but not Naip5, inhibited Shigella-induced caspase-1 activation, IL-1 maturation and Asc pyroptosome formation. Notably, the Pkc kinase was dispensable for caspase-1 activation and secretion of IL-1 induced by Shigella or Salmonella infection. These results indicate that activation of caspase-1 by Shigella is triggered by the rod protein MxiI that interacts with Naip2 to induce activation of the Nlrc4 inflammasome independently of the Pkc kinase. Author SummaryShigella are bacterial pathogens that are the cause of bacillary dysentery. An important feature of Shigella is their ability to invade the cytoplasm of host epithelial cells and macrophages. A major component of host recognition of Shigella invasion is the activation of the inflammasome, a molecular platform that drives the activation of caspase-1 in macrophages. Although Shigella is known to induce the activation of the Nlrc4 inflammasome, the mechanism by which the bacterium activates Nlrc4 is largely unknown. We discovered that the Shigella T3SS inner rod protein MxiI induces Nlrc4 inflammasome activation through the interaction with host Naip2, which promoted the association of Naip2 with Nlrc4 in macrophages. Expression of MxiI induced caspase-1 activation, Asc oligomerization, pyroptosis and IL-1 release which required Naip2, but not Naip5. Significantly, caspase-1 activation induced by Shigella infection was unaffected by deficiency of the Pkc kinase. This study elucidates the microbial-host interactions that drive the activation of the Nlrc4 inflammasome in Shigella-infected macrophages.