Inflammasomes are cytosolic multiprotein complexes that sense microbial infection and trigger cytokine production and cell death. However, the molecular components of inflammasomes, and what they sense, remain poorly defined. Here we demonstrate that 35 amino acids from the Cterminus of flagellin triggered inflammasome activation in the absence of bacterial contaminants or secretion systems. To further elucidate the host flagellin-sensing pathway, we generated mice deficient in Naip5. Naip5-deficient mice failed to activate the inflammasome in response to the 35 amino acids of flagellin or in response to Legionella pneumophila infection. Taken together, these data clarify the molecular basis for the cytosolic response to flagellin.Inflammasomes are cytosolic multiprotein complexes that are critical regulators of inflammation, and are required for proteolytic activation of the cysteine protease caspase-1 (refs. 1-3). Caspase-1 (A000492; http://www.signaling-gateway.org/molecule/query?afcsid=A000492) is itself required for the proteolytic processing and release of inflammatory cytokines such as interleukin 1β (IL-1β) and IL-18, as well as for induction of a necrotic-like cell death called pyroptosis1-3. The molecular components and structures of inflammasomes remain poorly defined. It is believed that multiple distinct inflammasomes may exist, each containing a key scaffold protein of the NLR (nucleotide-binding domain, leucine-rich repeat) superfamily that confers specificity for particular microbial products. For example, NLR proteins of the NLRP1 family (also called NALP1) appear to activate the inflammasome in response to anthrax lethal toxin4 and bacterial muramyl dipeptide5. In contrast, the NLR protein NLRP3 (also called NALP3 or cryopyrin) has been proposed to sense a wide range of stimuli including bacterial RNA6, viral DNA7, uric acid crystals8, muramyl dipeptide9,10, nigericin11, amyloid-beta12, and other irritants13-16. There is at present no molecular explanation for how a single NLR protein can NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript be activated by all these microbial products and the precise molecular nature of what is sensed by any inflammasome remains undefined.The inflammasome containing the NLR protein IPAF (also called NLRC4) is one of the best characterized inflammasomes, and has been proposed by several groups to respond to the presence of flagellin in the cytosol17-19. Flagellin-deficient mutants of Salmonella typhimurium and Legionella pneumophila are defective in IPAF-dependent inflammasome activation, and flagellin, purified from or expressed in bacteria, triggers IPAF-dependent caspase-1 activation when delivered to the cytosol of macrophages by use of a pore-forming toxin (listeriolysin O (LLO)) or transfection reagents17-21. It was proposed that during natural infections, flagellin triggers inflammasome activation upon secretion into the host cytosol via bacterial type III/IV secretion systems17-21. However, doubts have been expressed as to w...

Inflammasomes are cytosolic multiprotein complexes that assemble in response to infectious or noxious stimuli and activate the CASPASE-1 protease. The inflammasome containing the nucleotide binding domainleucine-rich repeat (NBD-LRR) protein NLRC4 (interleukin-converting enzyme protease-activating factor [IPAF]) responds to the cytosolic presence of bacterial proteins such as flagellin or the inner rod component of bacterial type III secretion systems (e.g., Salmonella PrgJ). In some instances, such as infection with Legionella pneumophila, the activation of the NLRC4 inflammasome requires the presence of a second NBD-LRR protein, NAIP5. NAIP5 also is required for NLRC4 activation by the minimal C-terminal flagellin peptide, which is sufficient to activate NLRC4. However, NLRC4 activation is not always dependent upon NAIP5. In this report, we define the molecular requirements for NAIP5 in the activation of the NLRC4 inflammasome. We demonstrate that the N terminus of flagellin can relieve the requirement for NAIP5 during the activation of the NLRC4 inflammasome. We also demonstrate that NLRC4 responds to the Salmonella protein PrgJ independently of NAIP5. Our results indicate that NAIP5 regulates the apparent specificity of the NLRC4 inflammasome for distinct bacterial ligands.

Health is a multidimensional landscape. If we just consider the host, there are many outputs that interest us: evolutionary fitness determining parameters like fecundity, survival and pathogen clearance as well as medically important health parameters like sleep, energy stores and appetite. Hosts use a variety of effector pathways to fight infections and these effectors are brought to bear differentially. Each pathogen causes a different disease as they have distinct virulence factors and niches; they each warp the health landscape in unique ways. Therefore, mutations affecting immunity can have complex phenotypes and distinct effects on each pathogen. Here we describe how two components of the fly's immune response, melanization and phagocytosis, contribute to the health landscape generated by the transcription factor ets21c (CG2914) and its putative effector, the signaling molecule wntD (CG8458). To probe the landscape, we infect with two pathogens: Listeria monocytogenes, which primarily lives intracellularly, and Streptococcus pneumoniae, which is an extracellular pathogen. Using the diversity of phenotypes generated by these mutants, we propose that survival during a L. monocytogenes infection is mediated by a combination of two host mechanisms: phagocytic activity and melanization; while survival during a S. pneumoniae infection is determined by phagocytic activity. In addition, increased phagocytic activity is beneficial during S. pneumoniae infection but detrimental during L. monocytogenes infection, demonstrating an inherent trade-off in the immune response.