<i>Shigella</i> Type III Secretion Protein MxiI Is Recognized by Naip2 to Induce Nlrc4 Inflammasome Activation Independently of Pkcδ

<div><p>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 <i>Salmonella</i> 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 <i>Shigella</i> 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 <i>Shigella</i> infection, MxiI associated with Naip2, and Naip2 interacted with Nlrc4. siRNA-mediated knockdown of Naip2, but not Naip5, inhibited <i>Shigella</i>-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 <i>Shigella</i> or <i>Salmonella</i> infection. These results indicate that activation of caspase-1 by <i>Shigella</i> is triggered by the rod protein MxiI that interacts with Naip2 to induce activation of the Nlrc4 inflammasome independently of the Pkcδ kinase.</p></div

Pkcδ is not required for inflammasome activation caused by <i>Shigella</i> infection.

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

<i>Shigella</i> induces Naip2-dependent Asc pyroptosome formation in macrophages.

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

Expression of <i>Shigella</i> rod protein MxiI induces activation of the Nlrc4 inflammasome in macrophages.

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

<i>Shigella</i> MxiI interacts with Naip2 and promotes the interaction of Naip2 with Nlrc4.

<p>(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 <i>Shigella</i> 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.</p

MCM2 (FL and mutants) interacts with PP2A.

<p>(<b>A</b>) The <i>Mcm2-FL</i>- or mutant-transfected 3T3 cells (left) and <i>gp70</i>/<i>Mcm2-FL-</i> or <i>gp70</i>/mutants-transfected 3T3 cells (right) were treated with 1 µM doxorubicin for 24 h. Cell lysates were subjected to a pull-down assay to detect the binding of MCM2-FL or the mutants to PP2A. (<b>B</b>) 3T3 cells were pre-incubated with 10 nM okadaic acid (OA) and 10 µM NU7026 for 2 h, and treated with 1 µM doxorubicin for 24 h. The apoptotic cell ratio was determined with annexin V-staining. Asterisk (*) indicates <i>p</i><0.01 for control vs. mutant-transfected cells. Data represent the mean and 95% CI of 3 independent experiments. (<b>C</b>) Western blot analysis of 3T3 cells to detect phospho-DNA-PK. Note the significantly increased levels of DNA-PK-p2053 in OA-treated 3T3 cells, and the complete abrogation by NU7026.</p

<i>In vivo</i> anti-tumor effects of <i>gp70</i> expression and DNA-damage on the C3H-derived cells in SCID mice.

<p>Two weeks after transplantation, mice were inoculated (i.p.) with FLV. Seven days later, the mice were treated with 1.5 mg/kg of doxorubicin or PBS. (<b>A</b>) Quantitative RT-PCR analysis of <i>gp70</i> mRNA expression in the liver of SCID mice with multiple foci of leukemic infiltration. The samples from FLV-infected mice exhibit higher levels of <i>gp70</i> than those from uninfected mice (*<i>p</i><0.01). Data represent the mean and 95% CI of from 10 mice in each group and are representative of 2 independent experiments. (<b>B</b>) Microscopic features of TUNEL-positive cells in hepatic nodules and (<b>C</b>) TUNEL-positive cell ratio in each group of mice. Note the significant increase in apoptotic 8047 cells in mice with FLV infection and doxorubicin treatment (*<i>p</i><0.01 compared with the tumor cells of “FLV (−), doxorubicin (−) mice”). Data represent the mean and 95% CI of from 10 mice in each group and are representative of 2 independent experiments. (<b>D</b>) Subcellular localization of MCM2 in 8047 cells of the liver demonstrated by immunohistochemistry. Images were captured with a microscope at 1,000× magnification power. Note the nuclear and/or cytoplasmic localization of MCM2 in the 8047 cells from each group of mice. (<b>E</b>) The cell counts for cytoplasmic localization of MCM2. Cell counts are shown as the number of cells per 10 high-power fields (HPF). [# <i>p</i><0.01 compared with tumor cells of “FLV (−), doxorubicin (−)” mice; *<i>p</i><0.001 compared with “FLV (−) doxorubicin (−)” mice and <i>p</i><0.05 compared with “FLV (+), doxorubicin (−)” mice]. Data represent the mean and 95% CI of from 10 mice in each group and are representative of 2 independent experiments. (<b>F</b>) Kaplan-Meier survival curves for 8047-transplanted SCID mice with/without FLV-infection and doxorubicin-treatment. Note the significant elongation of survival time in mice with FLV-infection [<i>p</i><0.01 compared with “FLV (−), doxorubicin (−)” and “FLV (−), doxorubicin (+)” mice] and in mice with FLV-infection and doxorubicin-treatment [<i>p</i><0.001 compared with “FLV (−), doxorubicin (−)” and “FLV (−), doxorubicin (+)” mice, <i>p</i><0.01 compared with “FLV (+), doxorubicin (−)” mice]. The survival curves represent data from 10 mice in each group.</p

Direct interaction of MCM2 with gp70.

<p>(<b>A</b>) Schematic diagram of full-length MCM2 (MCM2-FL) and MCM2 deletion mutants, MCM2-ΔC (aa 1–703), MCM2-ΔN (aa 156–703), MCM2-N (aa 1–155) and MCM2-C (aa 704–904). The NLS domains are shown in black, and the Zn-finger domains are gray. 3T3 cells were transfected with <i>HA</i>-tagged <i>Mcm2</i> mutants along with <i>FLAG</i>-tagged <i>gp70</i>, and either left untreated (<b>B</b>) or treated with 1 µM doxorubicin for 24 h (<b>C</b>). The expression of the MCM2 mutants (<b>B</b>, <b>C</b>, left upper) and FLAG-gp70 (<b>B</b>, <b>C</b>, left middle) was confirmed in 3T3 cells. Cell lysates were subjected to a pull-down assay to detect the binding of MCM2-FL or MCM2 mutants to FLAG-gp70 (<b>B</b>, <b>C</b>, right panel).</p

The C-terminal portion of MCM2 is important for apoptosis enhancement.

<p>3T3 cells were co-taransfected with <i>gp70</i> and <i>Mcm2-FL</i> or the mutants (<b>A</b>, <b>B</b>) or transfected with <i>Mcm2-FL</i> or the mutants (<b>C</b>, <b>D</b>) and treated with 1 µM doxorubicin for 24 h. Cell survival (<b>A</b>, <b>C</b>) and apoptotic cell ratios (<b>B</b>, <b>D</b>) were determined using the MTT assay and annexin V-staining, respectively. Asterisks (*) indicate <i>p</i><0.01 for control vs. mutant-transfected cells. In all panels, data represent the mean and 95% CI of 3 independent experiments. Western blot analysis of <i>gp70</i>/<i>Mcm2-FL-</i> and <i>gp70</i>/mutant-transfected 3T3 cells (<b>E</b>) and <i>Mcm2-FL-</i> and mutant-transfected 3T3 cells (<b>F</b>) after treatment with 1 µM doxorubicin for 24 h. The levels of DNA-PK, phospho-DNA-PK (pS2053), P53, phospho-P53, and cleaved caspase-3 are elevated in the groups with elevated apoptotic ratios. (<b>G</b>) 3T3 cells co-transfected with <i>gp70</i>/<i>Mcm2-FL</i> or <i>gp70</i>/mutants and (<b>H</b>) 3T3 cells transfected with <i>Mcm2-FL</i> or the mutants were pre-incubated with 10 µM NU7026, a DNA-PK-inhibitor, for 2 h and treated with 1 µM doxorubicin for 24 h. DNA-PK-pS2053 levels are substantially reduced in cells treated with the DNA-PK-inhibitor (<b>G</b> and <b>H</b>, bottom) compared to the levels in the absence of NU7026 (<b>E</b> and <b>F</b>, respectively). Whole cell lysates from <i>gp70-</i> and <i>Mcm2-FL</i>-transfected 3T3 cells after doxorubicin treatment are shown as a positive control (PC, <b>G</b> and <b>H</b>, bottom). Apoptotic cell ratios were determined with annexin V-staining (<b>G</b> and <b>H,</b> upper graph). In both panels, data represent the mean and 95% CI of three independent experiments.</p

Schematic illustration of the structure of MCM2 and its functions in the cytoplasm and nucleus.

<p>(<b>A</b>) The various functional domains of the MCM2 protein are shown, and the domains and regions required for the activities are indicated. (<b>B</b>) Schematic of the novel role of MCM2 in apoptosis enhancement. Normally, MCM2 is recruited into the nucleus for participation in DNA replication. As a result, cellular proliferation is upregulated (proliferation signal). However, when gp70 is present in the cytoplasm, it binds to MCM2 and inhibits its nuclear entry. Furthermore, cytoplasmic gp70-MCM2-complex interacts with PP2A and inhibits its interaction with DNA-PK. Consequently, hyperphosphorylated DNA-PK enhances DNA-damage-induced apoptosis via a P53-related pathway (apoptosis signal).</p