Human NAIP/NLRC4 and NLRP3 inflammasomes detect Salmonella type III secretion system activities to restrict intracellular bacterial replication

Abstract Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that uses two distinct type III secretion systems (T3SSs), termed Salmonella pathogenicity island (SPI)-1 and SPI-2, to deliver virulence factors into the host cell. The SPI-1 T3SS enables Salmonella to invade host cells, while the SPI-2 T3SS facilitates Salmonella’s intracellular survival. In mice, a family of cytosolic immune sensors, including NAIP1, NAIP2, and NAIP5/6, recognizes the SPI-1 T3SS needle, inner rod, and flagellin proteins, respectively. Ligand recognition triggers assembly of the NAIP/NLRC4 inflammasome, which mediates caspase-1 activation, IL-1 family cytokine secretion, and pyroptosis of infected cells. In contrast to mice, humans encode a single NAIP that broadly recognizes all three ligands. The role of NAIP/NLRC4 or other inflammasomes during Salmonella infection of human macrophages is unclear. We find that although the NAIP/NLRC4 inflammasome is essential for detecting SPI-1 T3SS ligands in human macrophages, it is partially required for responses to infection, as Salmonella also activated the NLRP3 and CASP4/5 inflammasomes. Importantly, we demonstrate that combinatorial NAIP/NLRC4 and NLRP3 inflammasome activation restricts Salmonella replication in human macrophages. In contrast to SPI-1, the SPI-2 T3SS inner rod is not sensed by human or murine NAIPs, which is thought to allow Salmonella to evade host recognition and replicate intracellularly. Intriguingly, we find that human NAIP detects the SPI-2 T3SS needle protein. Critically, in the absence of both flagellin and the SPI-1 T3SS, the NAIP/NLRC4 inflammasome still restricted intracellular Salmonella replication. These findings reveal that recognition of Salmonella SPI-1 and SPI-2 T3SSs and engagement of both the NAIP/NLRC4 and NLRP3 inflammasomes control Salmonella infection in human macrophages. Author summary Salmonella enterica serovar Typhimurium is a gastrointestinal bacterial pathogen that causes diarrheal disease and is a major cause of morbidity and mortality worldwide. Salmonella uses molecular syringe-like machines called type III secretion systems (T3SSs) to inject virulence factors into host cells. These T3SSs enable Salmonella to infect and survive within host cells such as macrophages. However, host cells contain a family of cytosolic immune receptors, termed NAIPs, that recognize T3SS and flagellin components. Upon detecting these components, NAIPs recruit the adaptor protein NLRC4 to form signaling complexes called inflammasomes. Inflammasomes activate host proteases called caspases that mount robust immune responses against the invading pathogen. While mice encode multiple NAIPs that have been extensively studied, much remains unknown about how the single human NAIP mediates inflammasome responses to Salmonella in macrophages. Our study reveals that while NAIP is necessary to detect individual T3SS ligands in human macrophages, it is only partially required for inflammasome responses to Salmonella infection. We found that the NLRP3 and CASP4/5 inflammasomes are also activated, and the combination of NAIP- and NLRP3-mediated recognition limits intracellular Salmonella replication in human macrophages. Our results demonstrate that human macrophages employ multiple inflammasomes to mount robust host defense against Salmonella infection.