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

Abstract

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.

Figure 1.

CRISPR/Cas9 targeting strategy. (A) Naip gene cluster in B6 mice. Guide RNA sequence for CRISPR/Cas9 targeting and protospacer-adjacent motif (PAM) are indicated. (B) The Naip1 locus in WT and Naip1^−/− mice. (C) The Naip2 locus in WT and Naip2^−/− mice. (D) The Naip1 and Naip5 loci in WT and Naip1-6^Δ/Δ mice (left) and the Naip2 locus in WT and Naip1-6^Δ/Δ (right).

Figure 2.

Response of BMMs from WT and mutant mice to retrovirally transduced bacterial ligands. (A) Representative flow plots of WT, Nlrc4^−/−, Naip1-6^Δ/Δ, Naip1^−/−, Naip2^−/−, and Naip5^−/− BMMs transduced with control vector (IRES-GFP only) or with vectors encoding needle-IRES-GFP, inner rod-IRES-GFP or flagellin-IRES-GFP. Gates show the percentage of GFP-positive cells. (B) Quantification of retroviral transduction experiments. Data are representative of two independent experiments (n = 3), mean ± SD; one-way ANOVA, Dunnett’s post test: ***, P < 0.001.

Figure 3.

Requirement for individual NAIPs for pyroptosis during macrophage infection. (A–C) LDH release assay of supernatants of WT, Nlrc4^−/−, Naip1-6^Δ/Δ, Naip1^−/−, Naip2^−/−, and Naip5^−/− BMMs infected with WT LT2 or flagellin-deficient (fliC fljB) S. Typhimurium (MOI = 5) for 6 h, with or without 5 µg/ml polyIC 24 h before infection (note different y-axis scales for WT and flagellin-deficient data; A), WT LP02 or flagellin-deficient L. pneumophila (MOI = 5) for 4 h (B), or P. aeruginosa (MOI = 20) PA103ΔU Fla⁺ for 2 h after 3-h pretreatment with 50 ng/ml LPS (C). Data are representative of two independent experiments (n = 3), mean ± SD; one-way ANOVA, Dunnett's post test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4.

NAIP1 and NAIP2 detect T3SS needle and rod protein, respectively. (A) LDH release assay of WT, Nlrc4^−/−, Naip1-6^Δ/Δ, Naip1^−/−, Naip2^−/−, and Naip5^−/− BMMs treated with 4 µg/ml PA and 200 ng/ml LFn-needle for 4 h after 24-h pretreatment with 5 µg/ml polyIC. (B) Rectal temperatures of WT, Nlrc4^−/−, Naip1-6^Δ/Δ, Naip1^−/−, Naip2^−/−, and Naip5^−/− mice injected with 0.8 µg/g body weight of PA and 80 ng/g LFn-needle intravenously (retroorbitally). (C) As in B, except hematocrit was measured at 60 min after injection. (D) As in A, except BMMs were treated with 4 µg/ml PA and 10 ng/ml LFn-rod for 4 h. n.d., not detectable. (E) As in B, except mice were injected with 0.8 µg/g body weight of PA and 1 ng/g LFn-rod intravenously (retroorbitally). (F) As in E, except hematocrit was measured at 60 min after injection. (C and F) Horizontal lines indicate median. Data are representative of two independent experiments (n = 3), mean ± SD; one-way ANOVA, Dunnett’s post test: **, P < 0.01; ***, P < 0.001.

Figure 5.

NAIP specifically senses flagellin protein in vitro and in vivo. (A) LDH release assay of WT, Nlrc4^−/−, Naip1-6^Δ/Δ, Naip1^−/−, Naip2^−/−, and Naip5^−/− BMMs treated with 4 µg/ml PA and 4 µg/ml LFn-flagellin for 4 h. n.d., not detectable. (B) Rectal temperatures of WT, Nlrc4^−/−, Naip1-6^Δ/Δ, Naip1^−/−, Naip2^−/−, and Naip5^−/− mice injected with 0.8 µg/g body weight of PA and 0.8 µg/g LFn-flagellin intravenously (retroorbitally). (C) As in B, except hematocrit was measured at 30 min after injection. (D) As in B, except hematocrit was measured at 90 min after injection. At this time point, WT, Naip1^−/−, and Naip2^−/− mice had already succumbed to the treatment. (C and D) Horizontal lines indicate median. Data are representative of two independent experiments (n = 3), mean ± SD; one-way ANOVA, Dunnett’s post test: ***, P < 0.001.