Shigella type III secretion protein MxiI is recognized by Naip2 to induce Nlrc4 inflammasome activation independently of Pkcδ

Abstract

Recognition of intracellular pathogenic bacteria by members of the nucleotide-binding domain and leucine-rich repeat containing (NLR) family triggers immune responses against bacterial infection. A major response induced by several Gram-negative bacteria is the activation of caspase-1 via the Nlrc4 inflammasome. Upon activation, caspase-1 regulates the processing of proIL-1β and proIL-18 leading to the release of mature IL-1β and IL-18, and induction of pyroptosis. The activation of the Nlrc4 inflammasome requires the presence of an intact type III or IV secretion system that mediates the translocation of small amounts of flagellin or PrgJ-like rod proteins into the host cytosol to induce Nlrc4 activation. Using the Salmonella system, it was shown that Naip2 and Naip5 link flagellin and the rod protein PrgJ, respectively, to Nlrc4. Furthermore, phosphorylation of Nlrc4 at Ser533 by Pkcδ was found to be critical for the activation of the Nlrc4 inflammasome. Here, we show that Naip2 recognizes the Shigella T3SS inner rod protein MxiI and induces Nlrc4 inflammasome activation. The expression of MxiI in primary macrophages was sufficient to induce pyroptosis and IL-1β release, which were prevented in macrophages deficient in Nlrc4. In the presence of MxiI or Shigella infection, MxiI associated with Naip2, and Naip2 interacted with Nlrc4. siRNA-mediated knockdown of Naip2, but not Naip5, inhibited Shigella-induced caspase-1 activation, IL-1β maturation and Asc pyroptosome formation. Notably, the Pkcδ kinase was dispensable for caspase-1 activation and secretion of IL-1β induced by Shigella or Salmonella infection. These results indicate that activation of caspase-1 by Shigella is triggered by the rod protein MxiI that interacts with Naip2 to induce activation of the Nlrc4 inflammasome independently of the Pkcδ kinase.

Figure 1. Expression of Shigella rod protein MxiI induces activation of the Nlrc4 inflammasome in macrophages.

WT or Nlrc4 ^−/− BMDMs were nucleofected with MSCV-IRES-GFP (GFP) or MSCV-IRES-GFP encoding Shigella MxiI (MxiI-GFP). After 20 hrs, the percentage of GFP-positive viable cells in the total cell population was analyzed by fluorescence microscopy (A) and the production of IL-1β in cell free supernatants by ELISA (B). * p<0.0001. (A and B) Results represent mean ± SD and are representative of three independent experiments.

Figure 2. Shigella MxiI interacts with Naip2 and promotes the interaction of Naip2 with Nlrc4.

(A)T7-tagged MxiI was co-expressed with HA-tagged Naip2 or Naip5 or control empty vector in caspase-1-deficient BMDMs. Cell lysates were immunoprecipitated with anti-T7 antibody and the interaction between MxiI and Naip2/5 was analyzed by immunoblotting with anti-HA antibody. (B) T7-tagged Nlrc4 or empty vector was co-expressed with HA-tagged Naip2, Naip5, or empty vector in caspase-1-deficient BMDMs. After 16 hrs cells were infected with Shigella WT or S325 mutant for 2 hr at a bacteria/macrophage ratio of 10∶1. Cell lysates were immunoprecipitated with anti-T7 beads and the interaction between Nlrc4 and Naip2/5 was analyzed by immunoblotting with anti-HA antibody. (C) T7-tagged MxiI or empty vector was co-expressed with HA-tagged Naip2. Cell lysates were immunoprecipitated with anti-HA beads and the interaction of Naip2 with MxiI and endogenous Nlrc4 was analyzed by immunoblotting with anti-T7 or anti-Nlrc4 antibody. (A–C) Results are representative of three independent experiments.

Figure 3. Shigella induces caspase-1 activation via Nlrc4, Asc and Naip2 in macrophages.

(A–C). BMDM were nucleofected with siRNA targeting Naip2 or Naip5 for 48 hrs and then infected, or not, with Shigella WT or S325 mutant for additional 1–3 hrs at a bacteria/cell ratio of 10∶1. (A) Knockdown efficiency was evaluated by RT-PCR after 48 hrs in uninfected cells. (B and C) The activation of caspase-1 was evaluated by detecting cleaved caspase-1 (p20) by immunoblotting, and the level of casp1 p20 was further quantified by densitometry (B). Cytokines production was analyzed by ELISA in cell free supernatants (B–C). * p<0.05. **p<0.001; NS, not significant. (A–C) Results represent mean ± SD and are representative of three independent experiments.

Figure 4. Shigella induces Naip2-dependent Asc pyroptosome formation in macrophages.

WT (A–D), Asc ^−/− (A–D), Naip2-deficient (siRNA) or Naip5-deficient (siRNA) (C) BMDM were infected with Shigella WT or S325 mutant for up to 90 min (A–D). Cells were fixed and analyzed by confocal microscopy (A–D) and the percentage of cells containing Asc pyrotopsomes was evaluated (D). Caspase-1 activation was detected using FLICA reagent (A) (green), Asc localization with anti-Asc antibody (red in A, green in B and C) and nuclei with DAPI (A) (blue). Arrows denotes Asc pyroptosomes. * p<0.01. Results represent mean ± SD and are representative of three independent experiments.

Figure 5. Shigella or MxiI expression induces Naip2-dependent Asc dimerization in macrophages.

WT (A and C), Asc ^−/− (A and C), casp1 ^−/− (B), Naip2-deficient (siRNA) or Naip5 deficient (siRNA) (C) BMDM were infected with Shigella WT or S325 Shigella mutant, Salmonella WT or ΔfliA Salmonella mutant at the indicated bacteria/cell ratio for up to 60 min (A and C), or nucleofected with pCMV producing, or not, MxiI (B). The dimerization of Asc in the insoluble fraction (pellet) was evaluated by immunoblotting with anti-Asc antibody (A–C) and the production of IL-1β in cell free supernatants was analyzed by ELISA (A and C). Middle panel shows the presence of Asc in total cell lysate. Results represent mean ± SD. Representative of three independent experiments. *p<0.002 (A), *p<0.05 (C).

Figure 6. Pkcδ is not required for inflammasome activation caused by Shigella infection.

(A) BMDMs from WT and Prkcd ^−/− mice were stimulated with LPS and the expression of Pkcδ was evaluated by immunoblotting. (B–D) BMDMs from WT and Prkcd ^−/− mice were infected with Shigella WT or S325 Shigella mutant (B–E) or Salmonella (C and E) at a bacteria/macrophage ratio of 10∶1 for various time points (B) or at the indicated bacteria/macrophage ratio (C) for 1 hr, or with indicated bacteria/macrophage ratios for 2 hrs (D) or 30 min (E). The production of cytokines in cell free supernatant was analyzed by ELISA (B–D) and the activation of caspase-1 was evaluated by detecting cleaved caspase-1 (p20) by immunoblotting (E). *p<0.02. Results represent mean ± SD. Results are representative of at least three independent experiments.