Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation

Inflammasome mediated by central nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) protein is critical for defense against bacterial infection. Here we show that type III secretion system (T3SS) needle proteins from several bacterial pathogens, including Salmonella typhimurium, enterohemorrhagic Escherichia coli, Shigella flexneri, and Burkholderia spp., can induce robust inflammasome activation in both human monocyte-derived and mouse bone marrow macrophages. Needle protein activation of human NRL family CARD domain containing 4 (NLRC4) inflammasome requires the sole human neuronal apoptosis inhibitory protein (hNAIP). Among the seven mouse NAIPs, NAIP1 functions as the mouse counterpart of hNAIP. We found that NAIP1 recognition of T3SS needle proteins was more robust in mouse dendritic cells than in bone marrow macrophages. Needle proteins, as well as flagellin and rod proteins from five different bacteria, exhibited differential and cell type-dependent inflammasome-stimulating activity. Comprehensive profiling of the three types of NAIP ligands revealed that NAIP1 sensing of the needle protein dominated S. flexneri-induced inflammasome activation, particularly in dendritic cells. hNAIP/NAIP1 and NAIP2/5 formed a large oligomeric complex with NLRC4 in the presence of corresponding bacterial ligands, and could support reconstitution of the NLRC4 inflammasome in a ligand-specific manner.Innate immunity in mammals relies on a group of germline-encoded pattern recognition receptors (PRRs) to sense conserved pathogen-associated molecular patterns (PAMPs) and defend against microbial infections (1). Cytosolic nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) proteins, characterized by an N-terminal caspase recruitment domain or a pyrin domain, a central NOD, and a C-terminal leucine-rich repeat domain, are a family of PRRs with increasingly appreciated function in innate immune defense (2, 3). The NLR family contains 23 members in humans and 34 members in mice, many of which are known or thought to form large oligomeric inflammasome complexes in response to particular stimulation. The inflammasome is present mostly in macrophage and dendritic cells, and functions as a signaling platform for caspase-1 autoprocessing and activation (4). Activated caspase-1 further cleaves IL-1β and IL-18 into mature forms, and also induces macrophage inflammatory death, or pyroptosis (5), both of which play important roles in restricting microbial infection (6).The physiological function of most NLRs is not established, and only very few NLRs have defined ligands and stimulation signals. The NLRC4 inflammasome senses a wide spectrum of bacterial infections, including Legionella pneumophila, Salmonella typhimurium, Pseudomonas aeruginosa, enteropathogenic Escherichia coli (EPEC), and Shigella flexneri. NLRC4-dependent IL-1β production by intestinal phagocytes can discriminate pathogenic from commensal bacteria, contributing to immune defense against enteric bacterial infections (7). The NLRC4 inflammasome senses cytosolic flagellin as well as the rod component of bacterial type III secretion system (T3SS) (8–10). T3SS translocates effector proteins into host cells and is a general virulence mechanism for many Gram-negative pathogens (11).How does NLRC4 sense the two different bacterial molecules? Recent identification of the NAIP family of inflammasome receptors provides significant insights into this question (12, 13). NAIPs are a family of NLRs with seven paralogs in mice (NAIP1–7) but only one family member in humans (hNAIP). NAIP5/6 and NAIP2 bind directly to bacterial flagellin and T3SS rod components, respectively (12, 13), mediating caspase-1 activation through direct interaction with NLRC4 (13). Intriguingly, the NLRC4 inflammasome in human U937 monocytes does not respond to flagellin and T3SS rod protein, but instead is activated by T3SS needle protein CprI in Chromobacterium violaceum infection (13).Here we report that T3SS needle proteins can activate NLRC4 inflammasome in both human and mouse macrophages, and identify hNAIP and its mouse ortholog NAIP1 as responsible for recognizing cytosolic T3SS needle proteins. Recognition of the needle protein by hNAIP/NAIP1 stimulates formation of the large hetero-oligomeric hNAIP/NAIP1-NLRC4 inflammasome complex in 293T cell reconstitution. Further profiling of the inflammasome-stimulation activities of flagellin and T3SS rod and needle proteins from five bacterial pathogens reveals that each NAIP-bacterial ligand pair contributes differently to NLRC4-mediated innate immune detection of a particular bacterial infection. This extensive profiling also reveals a dominant role of NAIP1 recognition of T3SS needle protein in inflammasome detection of S. flexneri infection.