NAIP proteins are required for cytosolic detection of specific bacterial ligands in vivo.

NLRs (nucleotide-binding domain [NBD] leucine-rich repeat [LRR]-containing proteins) exhibit diverse functions in innate and adaptive immunity. NAIPs (NLR family, apoptosis inhibitory proteins) are NLRs that appear to function as cytosolic immunoreceptors for specific bacterial proteins, including flagellin and the inner rod and needle proteins of bacterial type III secretion systems (T3SSs). Despite strong biochemical evidence implicating NAIPs in specific detection of bacterial ligands, genetic evidence has been lacking. Here we report the use of CRISPR/Cas9 to generate Naip1(-/-) and Naip2(-/-) mice, as well as Naip1-6(Δ/Δ) mice lacking all functional Naip genes. By challenging Naip1(-/-) or Naip2(-/-) mice with specific bacterial ligands in vivo, we demonstrate that Naip1 is uniquely required to detect T3SS needle protein and Naip2 is uniquely required to detect T3SS inner rod protein, but neither Naip1 nor Naip2 is required for detection of flagellin. Previously generated Naip5(-/-) mice retain some residual responsiveness to flagellin in vivo, whereas Naip1-6(Δ/Δ) mice fail to respond to cytosolic flagellin, consistent with previous biochemical data implicating NAIP6 in flagellin detection. Our results provide genetic evidence that specific NAIP proteins function to detect specific bacterial proteins in vivo

Induced proximity of a TIR signaling domain on a plant-mammalian NLR chimera activates defense in plants

Plant and animal intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors detect pathogen-derived molecules and activate defense. Plant NLRs can be divided into several classes based upon their N-terminal signaling domains, including TIR (Toll-like, Interleukin-1 receptor, Resistance protein)- and CC (coiled-coil)-NLRs. Upon ligand detection, mammalian NAIP and NLRC4 NLRs oligomerize, forming an inflammasome that induces proximity of its N-terminal signaling domains. Recently, a plant CC-NLR was revealed to form an inflammasome-like hetero-oligomer. To further investigate plant NLR signaling mechanisms, we fused the N-terminal TIR domain of several plant NLRs to the N terminus of NLRC4. Inflammasome-dependent induced proximity of the TIR domain in planta initiated defense signaling. Thus, induced proximity of a plant TIR domain imposed by oligomerization of a mammalian inflammasome is sufficient to activate authentic plant defense. Ligand detection and inflammasome formation is maintained when the known components of the NLRC4 inflammasome is transferred across kingdoms, indicating that NLRC4 complex can robustly function without any additional mammalian proteins. Additionally, we found NADase activity of a plant TIR domain is necessary for plant defense activation, but NADase activity of a mammalian or a bacterial TIR is not sufficient to activate defense in plants

Playing with Fire: How NAIP Inflammasomes Sense and Respond to Bacterial Pathogens

The innate immune system is responsible for initiating the host immune response to infection. The study of microbial virulence has uncovered numerous mechanisms for microbes to evade innate immune detection. In contrast, relatively little is understood about the strategies employed by the host to prevent microbes from evading innate immunity. The NAIP innate immune receptors provide an intriguing case study to investigate these strategies. In mice, Naip has undergone gene duplication and drift, recombination, and pseudogenization1, all of which can be signatures of a co-evolutionary arms race with targeted pathogens2. This duplication and specialization has allowed NAIP paralogs to recognize several distinct bacterial proteins: NAIP5 binds bacterial flagellin (FlaA), and NAIP2 detects the inner rod protein (PrgJ) of the pathogen-associated type III secretion system (T3SS)3,4.I sought to address how gene duplication and drift enabled functional specialization by first defining which NAIP domains bind to bacterial ligands. I analyzed a panel of chimeric proteins, in which homologous domains of NAIP5 and NAIP2 were swapped, to determine which domains conferred the ability to recognize FlaA or PrgJ. A long-standing expectation in the field was that the auto-inhibitory C-terminal leucine-rich repeat (LRR) domain mediates ligand binding. Surprisingly, I found that the LRR was dispensable for ligand specificity. Instead, ligand recognition was mediated by several alpha-helical domains in the center of the protein. Strikingly, these domains are specifically evolving under positive selection, in which non-synonymous mutations are repeatedly selected to provide altered ligand binding surfaces. Separation of sensing and auto-inhibition functions into different domains may allow NAIPs to sample ligand recognition-altering mutations without disrupting steady-state auto-inhibition.These data suggested that NAIPs are engaged in a co-evolutionary arms race with bacteria over innate immune detection. However, bacterial ligands can evolve much more rapidly than mammalian NAIPs. To determine how NAIPs can successfully compete in such an arms race, I conducted alanine scanning screens of FlaA and PrgJ to comprehensively identify the ligand motifs recognized by NAIPs. Both NAIP5 and NAIP2 recognized multiple conserved surfaces, near the N- and C-termini, of their respective ligands. This multi-surface recognition strategy conferred NAIPs with robust detection of their bacterial ligands, as single point mutations in any recognition motif did not affect NAIP recognition. Rather, bacterial immune evasion required simultaneous mutation of multiple recognition motifs. However, highly mutated ligands that escaped immune detection also lost their native function, suggesting that multi-surface recognition serves to constrain bacterial immune evasion. To verify these biochemical results, we have determined the cryo-EM structure of NAIP5 bound to FlaA, an event which triggers oligomerization with the adapter protein NLRC4 into a large (>1 MDa) signaling complex. The structure reveals direct contacts between the NAIP5 ligand recognition domains and both recognition surfaces of FlaA. The extensive and largely hydrophobic contacts between NAIP5 and FlaA are consistent with a lack of “hot spot” binding sites and likely contribute to the robust recognition of NAIP5 for FlaA single point mutants. Additionally, our structure reveals how binding to FlaA triggers a conformational change in NAIP5 to expose its oligomerization surface, allowing the recruitment of NLRC4. The polymerization of NLRC45 and subsequent recruitment of the signaling effector, CASPASE-1, illustrates the switch-like mechanism by which the detection of a single ligand monomer is amplified into oligomerization-induced signaling.Collectively, this dissertation elucidates the biochemical mechanism of NAIP innate immune detection of bacterial ligands. Furthermore, it has provided surprising insights into strategies employed by innate immune receptors to compete with bacteria in an evolutionary arms race over host defense

Playing with Fire: How NAIP Inflammasomes Sense and Respond to Bacterial Pathogens

The innate immune system is responsible for initiating the host immune response to infection. The study of microbial virulence has uncovered numerous mechanisms for microbes to evade innate immune detection. In contrast, relatively little is understood about the strategies employed by the host to prevent microbes from evading innate immunity. The NAIP innate immune receptors provide an intriguing case study to investigate these strategies. In mice, Naip has undergone gene duplication and drift, recombination, and pseudogenization1, all of which can be signatures of a co-evolutionary arms race with targeted pathogens2. This duplication and specialization has allowed NAIP paralogs to recognize several distinct bacterial proteins: NAIP5 binds bacterial flagellin (FlaA), and NAIP2 detects the inner rod protein (PrgJ) of the pathogen-associated type III secretion system (T3SS)3,4.I sought to address how gene duplication and drift enabled functional specialization by first defining which NAIP domains bind to bacterial ligands. I analyzed a panel of chimeric proteins, in which homologous domains of NAIP5 and NAIP2 were swapped, to determine which domains conferred the ability to recognize FlaA or PrgJ. A long-standing expectation in the field was that the auto-inhibitory C-terminal leucine-rich repeat (LRR) domain mediates ligand binding. Surprisingly, I found that the LRR was dispensable for ligand specificity. Instead, ligand recognition was mediated by several alpha-helical domains in the center of the protein. Strikingly, these domains are specifically evolving under positive selection, in which non-synonymous mutations are repeatedly selected to provide altered ligand binding surfaces. Separation of sensing and auto-inhibition functions into different domains may allow NAIPs to sample ligand recognition-altering mutations without disrupting steady-state auto-inhibition.These data suggested that NAIPs are engaged in a co-evolutionary arms race with bacteria over innate immune detection. However, bacterial ligands can evolve much more rapidly than mammalian NAIPs. To determine how NAIPs can successfully compete in such an arms race, I conducted alanine scanning screens of FlaA and PrgJ to comprehensively identify the ligand motifs recognized by NAIPs. Both NAIP5 and NAIP2 recognized multiple conserved surfaces, near the N- and C-termini, of their respective ligands. This multi-surface recognition strategy conferred NAIPs with robust detection of their bacterial ligands, as single point mutations in any recognition motif did not affect NAIP recognition. Rather, bacterial immune evasion required simultaneous mutation of multiple recognition motifs. However, highly mutated ligands that escaped immune detection also lost their native function, suggesting that multi-surface recognition serves to constrain bacterial immune evasion. To verify these biochemical results, we have determined the cryo-EM structure of NAIP5 bound to FlaA, an event which triggers oligomerization with the adapter protein NLRC4 into a large (>1 MDa) signaling complex. The structure reveals direct contacts between the NAIP5 ligand recognition domains and both recognition surfaces of FlaA. The extensive and largely hydrophobic contacts between NAIP5 and FlaA are consistent with a lack of “hot spot” binding sites and likely contribute to the robust recognition of NAIP5 for FlaA single point mutants. Additionally, our structure reveals how binding to FlaA triggers a conformational change in NAIP5 to expose its oligomerization surface, allowing the recruitment of NLRC4. The polymerization of NLRC45 and subsequent recruitment of the signaling effector, CASPASE-1, illustrates the switch-like mechanism by which the detection of a single ligand monomer is amplified into oligomerization-induced signaling.Collectively, this dissertation elucidates the biochemical mechanism of NAIP innate immune detection of bacterial ligands. Furthermore, it has provided surprising insights into strategies employed by innate immune receptors to compete with bacteria in an evolutionary arms race over host defense

Molecular Basis for Specific Recognition of Bacterial Ligands by NAIP/NLRC4 Inflammasomes

NLR (nucleotide-binding domain [NBD]- and leucine-rich repeat [LRR]-containing) proteins mediate innate immune sensing of pathogens in mammals and plants. How NLRs detect their cognate stimuli remains poorly understood. Here, we analyzed ligand recognition by NLR apoptosis inhibitory protein (NAIP) inflammasomes. Mice express multiple highly related NAIP paralogs that recognize distinct bacterial proteins. We analyzed a panel of 43 chimeric NAIPs, allowing us to map the NAIP domain responsible for specific ligand detection. Surprisingly, ligand specificity was mediated not by the LRR domain, but by an internal region encompassing several NBD-associated α-helical domains. Interestingly, we find that the ligand specificity domain has evolved under positive selection in both rodents and primates. We further show that ligand binding is required for the subsequent co-oligomerization of NAIPs with the downstream signaling adaptor NLR family, CARD-containing 4 (NLRC4). These data provide a molecular basis for how NLRs detect ligands and assemble into inflammasomes

The structural basis of flagellin detection by NAIP5: A strategy to limit pathogen immune evasion.

Robust innate immune detection of rapidly evolving pathogens is critical for host defense. Nucleotide-binding domain leucine-rich repeat (NLR) proteins function as cytosolic innate immune sensors in plants and animals. However, the structural basis for ligand-induced NLR activation has so far remained unknown. NAIP5 (NLR family, apoptosis inhibitory protein 5) binds the bacterial protein flagellin and assembles with NLRC4 to form a multiprotein complex called an inflammasome. Here we report the cryo-electron microscopy structure of the assembled ~1.4-megadalton flagellin-NAIP5-NLRC4 inflammasome, revealing how a ligand activates an NLR. Six distinct NAIP5 domains contact multiple conserved regions of flagellin, prying NAIP5 into an open and active conformation. We show that innate immune recognition of multiple ligand surfaces is a generalizable strategy that limits pathogen evolution and immune escape

NAIP-NLRC4 Inflammasomes Coordinate Intestinal Epithelial Cell Expulsion with Eicosanoid and IL-18 Release via Activation of Caspase-1 and -8

Intestinal epithelial cells (IECs) form a critical barrier against pathogen invasion. By generation of mice in which inflammasome expression is restricted to IECs, we describe a coordinated epithelium-intrinsic inflammasome response in vivo. This response was sufficient to protect against Salmonella tissue invasion and involved a previously reported IEC expulsion that was coordinated with lipid mediator and cytokine production and lytic IEC death. Excessive inflammasome activation in IECs was sufficient to result in diarrhea and pathology. Experiments with IEC organoids demonstrated that IEC expulsion did not require other cell types. IEC expulsion was accompanied by a major actin rearrangement in neighboring cells that maintained epithelium integrity but did not absolutely require Caspase-1 or Gasdermin D. Analysis of Casp1-/-Casp8-/- mice revealed a functional Caspase-8 inflammasome in vivo. Thus, a coordinated IEC-intrinsic, Caspase-1 and -8 inflammasome response plays a key role in intestinal immune defense and pathology

NAIP proteins are required for cytosolic detection of specific bacterial ligands in vivo

NLRs (nucleotide-binding domain [NBD] leucine-rich repeat [LRR]–containing proteins) exhibit diverse functions in innate and adaptive immunity. NAIPs (NLR family, apoptosis inhibitory proteins) are NLRs that appear to function as cytosolic immunoreceptors for specific bacterial proteins, including flagellin and the inner rod and needle proteins of bacterial type III secretion systems (T3SSs). Despite strong biochemical evidence implicating NAIPs in specific detection of bacterial ligands, genetic evidence has been lacking. Here we report the use of CRISPR/Cas9 to generate Naip1(−/−) and Naip2(−/−) mice, as well as Naip1-6(Δ/Δ) mice lacking all functional Naip genes. By challenging Naip1(−/−) or Naip2(−/−) mice with specific bacterial ligands in vivo, we demonstrate that Naip1 is uniquely required to detect T3SS needle protein and Naip2 is uniquely required to detect T3SS inner rod protein, but neither Naip1 nor Naip2 is required for detection of flagellin. Previously generated Naip5(−/−) mice retain some residual responsiveness to flagellin in vivo, whereas Naip1-6(Δ/Δ) mice fail to respond to cytosolic flagellin, consistent with previous biochemical data implicating NAIP6 in flagellin detection. Our results provide genetic evidence that specific NAIP proteins function to detect specific bacterial proteins in vivo