The infection of mice using the ΔipaH-chromosome mutant, which lacks all of the chromosomal ipaH genes, leads to an increased production of the NF-κB responsive gene MIP-2 [28]. In this current study, we used HeLa cells to determine the effect of the ΔipaH-chromosome mutant on the NF-κB activation by measuring the kinetics of IκBα degradation. HeLa cells were infected with YSH6000 (S. flexneri WT) or ΔipaH-chromosome mutant, and then whole cell lysates were harvested at 20, 40, 60, and 80 min post-infection for the detection of IκBα. The degradation rate of IκBα in cells infected with the ΔipaH-chromosome mutant was higher than that of WT, indicating that one or more of the chromosomal IpaH proteins contributed to the dampening of IκBα degradation (Fig. 1A, left). To identify the IpaH proteins that were involved in suppressing NF-κB activation, we generated deletions mutants for each of the seven chromosomal ipaH genes. IpaH0722 was critical for the inhibition of NF-κB activation. The degradation rate of IκBα was higher in ΔipaH0722 infection compared to WT infection (Fig. 1A, right). IpaH0722, which corresponds to ORF SF0722 of Shigella flexneri 2a Sf301 strain, has a Cys residue in its C-terminal region that is required for E3 ubiquitin ligase activity [16], [28], [29].

In Cos-7 and HeLa cells, ectopically-expressed IpaH0722 localized to the cell membrane (Fig. 1B and S1A). A mouse lung infection model was used to evaluate the role of IpaH0722 in the pathogenesis of Shigella. The survival rates of mice infected with the ΔipaH0722 mutant Shigella were increased compared to mice infected with WT Shigella (Fig. 1C). Next, to investigate whether the Cys379 residue in the IpaH0722 C-terminal region and its E3 ubiquitin ligase activity contributed to the pathogenesis of Shigella, we substituted Cys379 residue (IpaH0722CA). When 293T cells expressing IpaH0722 or IpaH0722CA were infected with Shigella, the activation of NF-κB was decreased in the presence of IpaH0722 but not IpaH0722CA (Fig. 1D). In addition, to confirm the importance of the E3 ubiquitin ligase activity of IpaH0722 in Shigella infection, HeLa cells were infected with Shigella ΔipaH0722 harboring ipaH0722 (ΔipaH0722/ipaH0722) or ipaH0722CA (ΔipaH0722/ipaH0722CA). In cells infected with the WT strain or ΔipaH0722/ipaH0722 strain, the degradation rate of IκBα was reduced at 60 and 80 min after infection compared to the ΔipaH0722 or ΔipaH0722/ipaH0722CA strains (Fig. 1E). It is important to note that IpaH0722 had no effect on Elk-1 and JNK activation (Fig. 1D and E). We speculated that IpaH0722 plays a role in the inhibition of Shigella-induced NF-κB activation in an E3 ubiquitin ligase-dependent manner.

NF-κB activity can be stimulated by multiple signaling pathways that are triggered by various receptors in response to exogenous and endogenous stimuli, including TNFα-TNFR, TLRs, and NLRs [30]. To characterize IpaH0722-dependent dampening of NF-κB activation, we measured the levels of NF-κB activation in IpaH0722- or IpaH0722CA-expressing 293T cells that were stimulated with PMA, TNF-α, lipopolysacharide (LPS), or IL-1βusing NF-κB-luciferase reporter assays. IpaH0722 markedly impaired PMA-dependent, but not TNF-α-, LPS-, and IL-1β-dependent NF-κB activation in an E3 ubiquitin ligase activity-dependent manner (Fig. 2A). To confirm the specificity of IpaH0722-mediated induction of NF-κB activation, we investigated the expression of IL-8 in 293T cells stimulated with PMA using an IL-8 reporter assay. Consistent with the NF-κB findings, IpaH0722 preferentially inhibited the PMA-dependent IL-8 expression (Fig. 2B). Moreover, the preferential targeting of PMA- but not TNF-α-induced NF-κB activation was also demonstrated by ELISA assays (Fig. 2C). Although PMA was also known to induce MAPK activation, IpaH0722 failed to inhibit PMA-dependent AP-1 and Elk-1 activation (Fig. 2D). Taken together these results clearly indicated that IpaH0722 preferentially inhibited PMA-induced NF-κB activation by targeting factors that modulated the NF-κB signaling pathway.

It has recently reported that Salmonella SspH2, an IpaH cognate protein, localizes to the host cell membrane through its modification by cellular palmitoyl transferases. SspH2 has a putative S-palmitoylation motif in its N-terminal and undergoes modification by host cell palmitoyltransferases, resulting in its localization to the host cell membrane [31]. Since we found a putative S-palmitoylated motif in IpaH0722 N-terminal portion, we investigated whether this motif played a role in IpaH0722 localization to the cell membrane. In Cos-7 and HeLa cells, ectopically-expressed IpaH0722 localized to host cell membrane (Fig. 1B and S1A). IpaH0722 has two Cys residues in its palmitoylation motif (Cys14 and Cys18), we generated constructs in which the Cys residues were substituted with Ser singly and in tandem (IpaH0722-C14S, -C18S, and -C14S/C18S). These constructs were ectopically expressed in HeLa cells to evaluate the subcellular localization of IpaH0722. IpaH0722 WT and IpaH0722CA localized to the cell membrane, whereas IpaH0722-C14S, -C18S, and -C14S/C18S accumulated within the cytosol. Therefore, palmitoylation at Cys14 and Cys18 residues of IpaH0722 are critical for its localization to the plasma membrane (Fig. S1A).

We next investigated whether membrane localization of IpaH0722 was required for its inhibition of NF-κB. The various IpaH0722 constructs were ectopically expressed in 293T cells, which were stimulated with PMA, NF-κB activity was assessed using NF-κB-luciferase reporter assays. IpaH0722, but not IpaH0722CA, inhibited NF-κB activation (Fig. S1B). Moreover, since the disruption of the putative palmitoylation sites in IpaH0722 mutants did not alter their ability to reduce PMA-dependent NF-κB activation, we presumed that the localization of IpaH0722 to the plasma membrane was not required for NF-κB inhibition in our experimental setting (Fig. S1B).

PMA mimics the role of diacylglycerol (DAG) in the activation of the protein kinase C (PKC)-NF-κB pathway [32]. Therefore, we hypothesized that IpaH0722 selectively targeted the DAG–PKC–NF-κB pathway, which was likely due to the membrane localization of IpaH0722 once it was secreted by invading Shigella into epithelial cells. We sought to determine which of the PKC isoforms were involved in the activation of NF-κB. The PKC family of proteins is composed of 12 isoforms that act as lipid-activated Ser/Thr kinases [33]. The PKC family can be divided into four functional protein classes: conventional PKC (PKCα, β, and γ), novel PKC (PKCδ, ε, η, and θ), atypical PKC (PKCζ, λ/ι) and PKC related kinase (PKCμ). Conventional and novel PKCs have a DAG binding domain. Conventional PKCs require DAG and Ca²⁺ for their activation, whereas novel PKCs require only DAG for their activation. Atypical PKCs do not depend on DAG and Ca²⁺ for their activation. Since the activity of PKC is regulated by phosphorylation and its recruitment to the cell membrane, we investigated the levels of phosphorylated PKC during Shigella infection of HeLa cells. HeLa cells were infected with Shigella WT and cell lysates were harvested at 10, 20, 40, and 60 min after infection for the analysis of phosphorylated PKC by immunoblotting. As shown in Fig. 3A, Shigella infection augmented the phosphorylation of conventional or novel PKCs, such as PKCδ, at 10 and 20 min, and PKCμ at 20, 40, and 60 min post-infection.

To investigate whether PKC activation triggers NF-κB activation in Shigella infection, we exploited dominant negative forms (DN) of PKC and siRNA that targeted PKC. To investigate the role of PKC in the activation of NF-κB during Shigella infection, DN-PKCα and DN-PKCδ, ε, θ, were ectopically expressed in 293T cells. The DN-PKCδ, ε, θ, but not DN-PKCα, significantly decreased Shigella-induced NF-κB activation (Fig. 3B) suggesting that the PKCδ-NF-κB pathway plays a critical role during Shigella infection. Similarly, siRNA-mediated knockdown of PKCδ in 293T cells infected with Shigella decreased NF-κB activity to less than half of the control levels (Fig. 3C).

The invasion of epithelial by Shigella produces membrane ruffles through the remodeling of the F-actin cytoskeleton [34]–[36]. Immediately following bacterial invasion, the bacteria are rapidly surrounded by a vacuolar membrane that the bacteria disrupt to facilitate dissemination into the cytoplasm by inducing actin polymerization [34]–[36]. To further understand the mechanism of PKC activation by Shigella invasion, we examined PKC phosphorylation during Shigella infection. First we confirmed the importance of Shigella invasiveness. When HeLa cells were infected with Shigella WT or the T3SS deficient mutant S325, Shigella WT infection triggered the phosphorylation of PKCδ, however the S325 mutant did not (Fig. 3D). We next sought to determine whether vacuolar membrane disruption potentiated DAG–PKC–NF-κB signaling. We previously created an ΔipaB/inv mutant by introducing Yersinia invasin gene into the ΔipaB mutant [37]. The resulting ΔipaB/inv mutant was internalized into the endocytic vacuole and it was unable to disrupt the vacuolar membrane for dissemination into the cytoplasm (Fig. 3D). When HeLa cells were infected with Shigella WT or the ΔipaB/inv mutant, Shigella WT infection triggered the phosphorylation of PKCδ, however the ΔipaB/inv mutant did not (Fig. 3D). In contrast, both WT and ΔvirG mutant, which is unable to support intra- and inter-cellular movement, induced PKCδ phosphorylation (Fig. 3D). To further confirm the importance of vacuolar membrane disruption, we investigated PKC phosphorylation during Salmonella infection using a strain that triggers membrane ruffling but remain sequestered in the phagosome. When HeLa cells were infected with Shigella WT or the Salmonella WT, Shigella WT but not Salmonella WT infection triggered the phosphorylation of PKCδ (Fig. 3D). These data supported the notion that membrane rupture by Shigella in infected epithelial cells triggers the activation of the DAG-PKC-NF-κB pathway.

Because Shigella invasion of epithelial cells triggers the DAG–PKC–NF-κB signaling, we sought to determine the host factors in the PKC–NF-κB signaling pathway that were targeted by IpaH0722 E3 ubiquitin ligase. First, we determined which of the steps during the activation of PKC–NF-κB was targeted by IpaH0722. The NF-κB activity induced by PKC signaling factors was measured in 293T cells with ectopic expression of IpaH0722 or IpaH0722CA. The transiently transfected cells were stimulated with a subset of constitutively active PKC isoforms (PKCα, -δ, -ε, and θ) and NF-κB activity was determined. IpaH0722, but not IpaH0722CA, inhibited all PKC isoforms (PKCα, -δ, -ε, and -θ) and NF-κB activation, indicating that IpaH0722 targeted PKC itself or factors that lie directly downstream of PKC (Fig. 4A). In HeLa cells, similar levels of phosphorylated PKCδ were detected in response to Shigella WT and ΔipaH0722 infection (Fig. 4B). Moreover, no differences were detected in PKC phosphorylation in PMA plus ionomycin stimulated 293T cells expressing IpaH0722 or IpaH0722CA (Fig. 4C). In addition, IpaH0722 did not bind to PKC, and had no effect on protein stability of PKC, suggesting that IpaH0722 targets downstream of PKC rather than PKC itself (Fig. S2). We therefore focused on the CARMA-Bcl10-MALT1 (CBM) complex because the phosphorylation of CARMA1, 2, and 3 by PKC induces a conformational change that recruits Bcl10-MALT1 to CARMA (CBM signalosome), which is essential for the activation of downstream signaling [38], [39]. Therefore, we tested the effect of IpaH0722 on CBM complex formation, and found that IpaH0722 failed to inhibit CARMA1-, CARMA2-, CARMA3-, or Bcl10-induced NF-κB activity (Fig. 4D). Indeed, IpaH0722 did not bind to CARMA1-, CARMA2-, CARMA3-, or Bcl10, and had no effect on their protein stability (Fig. S3). Furthermore, since IpaH0722 did not affect PKC-mediated CARMA phosphorylation (Fig. 4E), we believe that IpaH0722 inhibited the PKC–NF-κB pathway but not via the CBM complex.

We subsequently investigated other NF-κB signaling factors, namely TRAF2, TRAF5, TRAF6, NIK, IKKα, IKKβ, or p65 as potential IpaH0722 targets [40]. To this end, 293T cells expressing NF-κB-luciferase reporter gene together with TRAF2, TRAF5, TRAF6, NIK, IKKα, IKKβ, or p65 were transfected with a vector encoding IpaH0722 or IpaH0722CA and luciferase activity was measured (Fig. 5A). IpaH0722, but not IpaH0722CA, inhibited NF-κB activity when the cells that expressed TRAF2 suggesting that TRAF2 is a target for IpaH0722 E3 ubiquitin ligase (Fig. 5A). In previous reports, PKC-mediated phosphorylation of TRAF2 was a prerequisite for NF-κB activation in epithelial cells [41], [42]. PKCδ and PKCε phosphorylated TRAF2 at residue Thr117 resulting in IKK recruitment and ultimately NF-κB activation [41], [42]. We therefore used siRNA-mediated TRAF2 knockdown cells to measure the effect of TRAF2 on activation of the PKC-NF-κB pathway. The results showed that TRAF2 knockdown decreased NF-κB activation in response to PMA stimulation and Shigella infection (Fig. S4A). To further confirm the effect of TRAF2 knockdown on NF-κB activation we utilized Traf2-knockout mouse embryonic fibroblasts (MEF) [43]. We transduced Traf2 into the Traf2-knockout MEFs by retrovirus infection to generate stably expressing Traf2 cell lines. The degradation of IκBα in Traf2 stably expressing MEF cells and Traf2-knockout MEF cells during Shigella infection was determined. As shown in Fig. S4B, the degradation rate of IκBα in Traf2 stably expressing MEFs was higher than that of Traf2-knockout MEFs at 20 and 40 min after infection suggesting that TRAF2 plays a role in Shigella-induced NF-κB activation.

Having shown that IpaH0722 targets TRAF2, we performed immunoprecipitation assays to determine whether IpaH0722 interacted with TRAF2. Cell lysates were harvested from 293T cells that expressed IpaH0722CA and TRAF2, TRAF5, TRAF6, NIK, IKKα, IKKβ, or p65. IpaH0722 precipitated with TRAF2, but not TRAF5, TRAF6, NIK, IKKα, IKKβ, or p65 (Fig. 5B). The interaction between IpaH0722 and TRAF2 was further confirmed by GST-pull down assay. Using whole cell lysates of HeLa and GST-IpaH0722, we were able to pull down endogenous TRAF2, but not TRAF6 (Fig. 5C).

In previous reports, structural analysis of IpaH proteins using IpaH1.4 or IpaH3 revealed that the LRR region of the IpaH family is required for substrate recognition, while the CTR region is essential for its E3 ubiquitin ligase activity [44], [45]. IpaH proteins share characteristic domains: an N-terminal 60–70 amino acid stretch, a LRR region, and an intervening region flanked by the LRR and the conserved CTR (Fig. S5A). We constructed a series of GST-tagged IpaH0722 truncations and performed GST-pull down assays using HeLa whole cell lysates. IpaH0722 truncations that contained the LRR regions bound to TRAF2; however, IpaH0722 truncations that contained the N-terminal, intervening region, or CTR failed to bind to TRAF2 (Fig. S5B).

Since IpaH0722 inhibited NF-κB activation in an E3 ubiquitin ligase-dependent manner, we tested whether IpaH0722 ubiquitinated TRAF2 using an in vitro ubiquitination assay. TRAF2, IpaH0722, or IpaH0722CA (purified from E. coli) were combined with E1, ATP, UbcH5b (an E2 ubiqitin conjugating enzyme) in assay medium and subjected to immunoblotting. TRAF2 was ubiquitinated by IpaH0722 but not IpaH0722CA (Fig. 6A). To confirm the fate of ubiquitinated TRAF2, we measured the stability of TRAF2 in 293T cells ectopically expressing IpaH0722 or IpaH0722CA and treated with cycloheximide (CHX; a protein synthesis inhibitor) for 2, 4, and 6 h. TRAF2 degradation was faster in IpaH0722 expressing cells compared to IpaH0722CA expressing cells, which confirmed that IpaH0722 targeted TRAF2 for ubiquitin-mediated protein degradation (Fig. 6B). We also measured TRAF2 degradation in 293T cells that were co-transfected with TRAF2 plus IpaH0722 or IpaH0722CA, and treated with MG132 (a proteasome inhibitor) or E64D plus pepstain A (lysosome inhibitors). As shown in Fig. 6C, MG132 treatment prevented TRAF2 degradation in the presence of IpaH0722, whereas E64D plus pepstatin A treatment did not prevent TRAF2 degradation. These data confirm that IpaH0722 targets TRAF2 for ubiquitination and proteasomal degradation.

Previous studies indicated that TRAF5 compensates for TRAF2 function [43], so we exploited Traf2/Traf5 double knockout MEFs to clarify the requirement of TRAF2 on NF-κB activation. We introduced Traf2 into the Traf2/Traf5 double knockout MEFs by retrovirus transduction. Under puromycin selection, we obtained stable TRAF2-expressing Traf2/Traf5 double knockout MEFs. Traf2/Traf5 double knockout MEFs and stable Traf2-expressing Traf2/Traf5 double knockout MEFs were infected with Shigella WT or ΔipaH0722, and IκBα degradation was measured over time. The degradation rate of IκBα in ΔipaH0722-infected stable TRAF2-expressing Traf2/Traf5 double knockout MEFs was higher compared to WT Shigella infection (Fig. 6D). In contrast, the degradation rates of IκBα in WT and ΔipaH0722-infected double knockout MEFs were almost equal (Fig. 6D). To evaluate the importance of the in vivo role of E3 ligase activity of IpaH0722 for Shigella virulence, we infected mice with a sublethal dose of Shigella WT, ΔipaH0722, ΔipaH0722/0722, or ΔipaH0722/0722CA. Mice infected with ΔipaH0722 or ΔipaH0722/0722CA mutants showed significantly reduced bacterial colonization when compared to that of WT or ΔipaH0722/0722 infection (Fig. S6A). Furthermore, histopathology showed that inflammatory responses, such as suppurative masses, neutrophil infiltration, and macrophage infiltration, were significantly higher in mice infected with ΔipaH0722 or ΔipaH0722/0722CA mutants when compared to that of WT or ΔipaH0722/0722 infections (Fig. S6B). These results supported our notion that the interaction between IpaH0722 and TRAF2 is an important mechanism for dampening NF-κB activation during Shigella infection of epithelial cells.

Shigella flexneri strain YSH6000 was used as the wild type and S325 (mxiA::Tn5) was used as a T3SS-deficient negative control. Salmonella typhimurium SB300 strain was used as the wild-type strain [58]. Construction of the non-polar mutant of ipaH-chromosome, ipaH0722, ipaB, and virG of S. flexneri YSH6000 were carried out as described previously [28], [37]. The ipaH0722-FLAG or ipaH0722CA-FLAG were cloned into pWKS130 to yield p-ipaH0722-FLAG and p-ipaH0722CA-FLAG. The resultant plasmids were introduced into the ΔipaH0722 strain. The ipaH0722 or ipaH0722CA coding sequences were amplified by PCR and cloned into pCMV-FLAG, pEGFP, pGEX-4T-1, pcDNA-Myc₆ (6× Myc), and pcDL-SRα-Myc vectors. cDNAs for human Traf2, Traf5, Traf6, Carma1, Carma2, Carma3, Bcl10, IKKα, IKKβ, p65, and a series of Pkc mutants were cloned into pCMV-FLAG, pEGFP, pcDNA-Myc₆, pcDL-SRα-Myc, and pcDNA-FLAG vectors. cDNA for mouse Traf2 was cloned into pGEX 4T-1. Site direct mutagenesis of ipaH0722 or Pkc was performed using the QuickChange site directed mutagenesis kit (Stratagene).

The anti-M2 FLAG monoclonal FLAG antibody (Sigma-Aldrich), anti-Myc 9B11, phospho-PKC (pan) (βII Ser660), phospho-PKD/PKCμ (Ser744/748), phospho-(Ser) PKC substrate, phospho-JNK antibody (Cell signaling), anti-actin (MILLIPORE), anti-PKCδ (C-20), PKCμ (C-20), TRAF2, TRAF6 antibody (Santa Cruz Biotechnology), and anti-IκBα (BD transduction) were obtained commercially. The anti-IpaH antibody was described previously [29]. PMA, LPS, ionomycin, E64D, and pepstatin A were obtained from Sigma. IL-1β and TNF-α were obtained from Peprotech. MG132 was obtained from Peptide Inst.

HeLa cells were cultured in Eagle's minimal essential medium (Sigma) supplemented with 10% fetal calf serum. Cos-7 and 293T cells were cultured in Dulbecco's modified Eagle medium (Sigma) supplemented with 10% fetal calf serum. The Traf2-deficient or Traf2/Traf5-deficient MEFs were maintained as described previously [43]. To construct Traf2^−/− or Traf2/Traf5^−/−cells stably expressing Traf2, cDNAs encoding these genes were subcloned into pMX-puro retroviral expression vectors. Retroviral supernatants were produced in Plat-E cells. Target cells were transduced with supernatants in the presence of DO-TAP (Roche) and then cloned under puromycin selection.

HeLa cells were infected with various strains of Shigella at a multiplicity of infection (moi) of 100. In the case of the Shigella strains expressing afimbrial adhesin (Afa), cells were infected at moi of 10. To adjust the intracellular bacterial number, ΔipaB/inv were set to 3 times moi of WT. Infection was initiated by centrifuging the plate at 700× g for 10 min. After incubation for 20 min at 37°C, the plates were washed three times with PBS, transferred into fresh medium containing gentamicin (100 µg/ml) and kanamycin (60 µg/ml) to kill extracellular bacteria. Salmonella infection was conducted as described previously [58]. After incubation for indicated time, the cells were washed with PBS and harvested into 2× Laemmli's sample buffer for immunoblotting. The density of each band was quantified by measuring the mean intensity using NIH image software version 1.63 and the expression levels were normalized to the levels of β-actin. All immunoblotting experiments were repeated multiple times and representative data of similar results are presented.

293T cells were seeded into 24-well plates. After 24 h, the cells were transfected with reporter plasmids (pNF-κB-luc, pIL-8-luc, pElK-1-luc, or pAP-1-luc), Renilla luciferase constructs (phRL-TK, Promega) using FuGENE6 transfection reagent (Roche). The IL-8 reporter plasmid was constructed by amplifying the 181 bp human IL-8 promoter sequence from the genomic DNA of HeLa cells, which was then inserted into the pMet-luc reporter plasmid (Clontech). Equal amounts of empty vector were used to control for the transfection process. After 24 h, cells were infected with Shigella WT (moi = 30) or treated with PMA (25 nM), TNF-α (10 ng/ml) or LPS (100 ng/ml) for 3 h. To investigate LPS stimulation, TLR4 and MD2 vectors were also transfected into the cells. Cell extracts were prepared and reporter activity was determined using the luciferase assay system (Promega). Results are presented as fold-change relative to the activity of uninfected or unstimulated cells. Data are representative of three independent experiments.

For GST fusion proteins, E. coli BL21 (DE3) strain harboring pGEX4T-1 derivatives were cultivated in L-broth supplemented with ampicillin (50 µg/ml) 3 h at 30°C. Expression was induced by the addition of 1 mM IPTG and incubation for 3 h at 30°C. Bacteria were disrupted by sonication and lysozyme treatment. Purification of the GST fusion proteins with glutathione-Sepharose 4B (GE Healthcare) was performed according to the manufacturer's protocol.

GST-IpaH0722 bound to glutathione sepharose 4B beads was mixed with HeLa cell lysates for 2 h at 4°C. After centrifugation, the beads were washed five times with 0.5% Triton X-100-PBS and subjected to immunoblotting. The immunoblotting experiments were repeated multiple times and representative data of similar results are presented.

293T cells were transiently transfected using FuGENE 6 (Roche). Cells were washed with PBS and lysed for 30 min at 4°C in lysis buffer containing 150 mM NaCl, 50 mM HEPES pH 7.5, 1 mM EDTA, 0.5% NP-40, and Complete protease inhibitor cocktail (Roche). Lysates were cleared by centrifugation and proteins were immunoprecipitated 2 h with anti-Myc (9B11) or anti-M2 FLAG and Protein G beads (Sigma) at 4°C. Immunoprecipitates were washed five times with lysis buffer and subjected to immunoblotting. The immunoblotting experiments were repeated multiple times and representative data of similar results are presented.

In vitro TRAF2 ubiquitination assays were performed in 40 µl reaction mixture containing reaction buffer (25 mM Tris-HCl [pH 7.5], 50 mM NaCl, 5 mM ATP, 10 mM MgCl₂, and 0.1 mM DTT), 1 µg TRAF2, 0.5 µg E1, 2 µg UbcH5b, and 2 µg ubiquitin purified from E. coli in the presence and absence of GST-IpaH0722 or GST-IpaH0722CA. Reactions were incubated at 37°C for 1 h and stopped by the addition of 5× Laemmli sample buffer. All immunoblotting experiments were repeated multiple times and representative data of similar results are presented.

At 24 h after transfection, 293T cells were treated with cycloheximide (50 µg/ml) (Wako) for the indicated times and cell lysates were harvested for immunoblotting. The density of each band was quantified by measuring the mean intensity using NIH image software version 1.63. The expression levels were normalized to the levels of β-actin. All immunoblotting experiments were repeated multiple times and representative data of similar results are presented.

Human PKCδ- or human TRAF2-specific siRNA were prepared by Sigma as follows: 5′-CGUGUGGACACGCCACAUUAU-3′ and 5′-AAUGUGGCGUGUCCACACGGA-3′ (PKCδ) or 5′-GGCCAGUCAACGACAUGAACA-3′ and 5′-UUCAUGUCGUUGACUGGCCUC-3′ (TRAF2). Cells were transfected using RNAiMax (Invitrogen). siRNA treated cells were utilized after 72 h for further analyses.

The pulmonary infection model in the mice was described previously [28]. In brief, five-week-old female Balb/c mice (CREA Japan) were housed in the animal facility of the Institute of Medical Science, University of Tokyo, in accordance with University guidelines. S. flexneri were suspended in sterile saline, and 20 µl of the bacterial suspension was administered intranasally at 1×10⁸ cfu (survival assay) or 1×10⁷ cfu (colonized bacterial number assay). For histological analysis, mice were sacrificed at 24 h after infection, and their lungs were harvested, fixed in 4% paraformaldehyde in PBS, and frozen in liquid nitrogen for sectioning. The sections were stained with hematoxylin and eosin and examined under a microscope. Viable bacteria in the lung tissue were counted by culturing homogenized tissue for 18 h on LB agar plates. Each data point is the mean of the values for 6 infected mice in each group.

All animal experiments were carried out in strict accordance with the University of Tokyo's regulations for Animal Care and Use protocol, which was approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo (approval number; PA11-92). The committee acknowledged and accepted both the legal and ethical responsibility for the animals, as specified in the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, 2006 (Japan). All surgery was performed under carbon dioxide euthanasia, and all efforts were made to minimize suffering.

Values are reported as means ± standard deviation (SD) or means ± standard error (SEM) of data obtained for independent experiments. Statistical analysis was performed Student's t-test or a one-way ANOVA. Kaplan-Meier survivor curves were generated. p-values<0.05 were considered significant.