The RhoA-ROCK pathway is essential for T3SS2-dependent stress fiber formation.

<p>(A) Schematic model of stress fiber formation via the RhoA-ROCK pathway and the inhibitors/siRNAs used in this study HeLa cells were treated with 10 μμM ROCK inhibitor Y27632 for 1 h (B) or 2 μμg/mL RhoA inhibitor I for 2 h (C) prior to infection. Next, the cells were infected with POR-2 (a <i>tdhAS</i>- and T3SS1-deficient strain) for 3 h, fixed, and stained to detect F-actin with Alexa-488 phalloidin. (D) Silencing of RhoA by siRNA transfection. At 24 h after siRNA transfection, HeLa cells were infected with POR-2 for 3 h. The cells were then fixed and stained with Alexa-488 phalloidin. The silencing effect on RhoA protein expression was confirmed by western blot analysis. A scrambled target sequence siRNA was used as a negative control (siControl).</p

Interaction between the Type III Effector VopO and GEF-H1 Activates the RhoA-ROCK Pathway

<div><p><i>Vibrio parahaemolyticus</i> is an important pathogen that causes food-borne gastroenteritis in humans. The type III secretion system encoded on chromosome 2 (T3SS2) plays a critical role in the enterotoxic activity of <i>V. parahaemolyticus</i>. Previous studies have demonstrated that T3SS2 induces actin stress fibers in various epithelial cell lines during infection. This stress fiber formation is strongly related to pathogenicity, but the mechanisms that underlie T3SS2-dependent actin stress fiber formation and the main effector have not been elucidated. In this study, we identified VopO as a critical T3SS2 effector protein that activates the RhoA-ROCK pathway, which is an essential pathway for the induction of the T3SS2-dependent stress fiber formation. We also determined that GEF-H1, a RhoA guanine nucleotide exchange factor (GEF), directly binds VopO and is necessary for T3SS2-dependent stress fiber formation. The GEF-H1-binding activity of VopO via an alpha helix region correlated well with its stress fiber-inducing capacity. Furthermore, we showed that VopO is involved in the T3SS2-dependent disruption of the epithelial barrier. Thus, VopO hijacks the RhoA-ROCK pathway in a different manner compared with previously reported bacterial toxins and effectors that modulate the Rho GTPase signaling pathway.</p></div

GEF-H1, a VopO binding partner, is necessary for VopO-induced stress fiber formation.

<p>(A) Identification of GEF-H1 as a VopO binding partner using a GST pull-down assay. Purified GST-VopO was mixed with a HeLa cell lysate, and the proteins retained on the glutathione beads were separated by SDS-PAGE. The proteins were detected with anti-GEF-H1, anti-LARG, anti-Ect2, anti-RhoA, and anti-β-actin antibodies. Lane 1, total HeLa cell lysate (INPUT); lane 2, eluate from glutathione beads alone; lane 3, eluate from GST-VopO bound to glutathione beads; lane 4, cell lysate associated with glutathione beads; and lane 5, cell lysate associated with GST-VopO bound to glutathione beads. (B) Direct binding of VopO to GEF-H1. Lane 1, glutathione beads alone; lane 2, purified GST-VopO; lane 3, recombinant 3×FLAG-tagged GEF-H1 (INPUT); lane 4, recombinant 3×FLAG-tagged GEF-H1 associated with glutathione beads; and lane 5, 3×FLAG-tagged GEF-H1 associated with GST-VopO bound to glutathione beads. Proteins were detected with anti-FLAG or anti-GST antibodies. (C) Diagram showing the recombinant truncated GEF-H1 proteins and summaries of their VopO-binding properties. (D) VopO-binding activity of truncated GEF-H1 proteins. After a pull-down assay using purified GST-VopO and recombinant 3×FLAG-tagged truncated GEF-H1 proteins, as shown in (C), GEF-H1 protein was detected using anti-FLAG antibody. (E) Effects of silencing GEF-H1, LARG, or Ect2 via siRNA on T3SS2-dependent stress fiber formation. At 24 h after the siRNA transfection, the cells were infected with POR-2 for 3 h. After infection, the cells were fixed and stained to detect F-actin (green) and DNA (blue). The silencing effects on the expression of each protein were confirmed by western blotting. A scrambled target sequence was used as a negative control (siControl).</p

Identification of the stress fiber formation-inducing T3SS2 effector VopO.

<div><p>(A) HeLa cells were infected with POR-2, POR-2<i>∆vcrD2</i> (a T3SS1- and T3SS2-deficient strain), POR-2<i>∆vopO</i> (a <i>vopO</i> mutant strain derived from POR-2), or POR-2<i>∆vopO/pvopO</i> (a strain complemented with the <i>vopO</i> gene) for 3 h, or were treated with 10 mM nocodazole for 1 h. After infection or nocodazole treatment, the cells were stained to detect F-actin (green) and cellular and bacterial DNA (blue).</p> <p>(B) G-LISA was used to evaluate the relative RhoA activation level in cells infected with isogenic <i>V. parahaemolyticus</i> mutant strains for 150 min or treated with 10 mM nocodazole for 30 min. The asterisks indicate results that differ significantly from those obtained using the parent strain (POR-2) (*<i>p</i> < 0.05). The error bars indicate the standard errors for experiments performed in triplicate.</p> <p>(C) Phosphorylated myosin light chain (pMLC) levels were evaluated via western blot analysis of cells infected with isogenic <i>V. parahaemolyticus</i> mutant strains for 3 h or treated with 10 mM nocodazole for 1 h. The infected cell lysates were probed with anti-pMLC or anti-MLC antibodies.</p> <p>(D) The GTP-bound (active) RhoA levels were evaluated using a rhotekin pull-down assay in cells transfected with a GFP-fused VopO-expression construct (GFP-vopO) or empty GFP vector (GFP). The precipitates (GTP-RhoA) and total cell lysates (Total-RhoA) were probed with an anti-RhoA antibody.</p> <p>(E) The pMLC levels in cells transfected with a GFP-fused VopO-expression construct (GFP-vopO) or empty GFP vector (GFP) were evaluated by western blot analysis.</p> <p>(F) Visualization of GFP (green), F-actin (red), and cellular DNA (blue) in cells transfected with a VopO-expression construct (GFP-vopO) or empty GFP vector (GFP). The arrowheads indicate GFP-VopO-expressing cells.</p></div

A predicted α-helix region in VopO (H2) is required for both GEF-H1 binding and stress fiber formation.

<p>(A) Diagram showing the recombinant truncated VopO proteins and summaries of their stress fiber-inducing activity, as determined by transfection and infection assays, and their GEF-H1-binding properties. (B) Identification of the GEF-H1-binding domain of VopO. The GEF-H1-binding activity of truncated VopO was evaluated using a GST pull-down assay. The 3xFLAG GEF-H1 and truncated GST-VopO proteins were detected using anti-FLAG or anti-GST antibodies, respectively. The asterisk indicates GST or a breakdown product. (C) Stress fiber-inducing activity of truncated VopO proteins in transfected cells. HeLa cells were transfected with GFP-fused truncated VopO constructs. The cells were stained to detect F-actin (red) and cellular DNA (blue). (D) HeLa cells were infected with <i>vopO</i> deletion mutants that expressed truncated VopO proteins and the stress fiber-inducing activity was evaluated by staining to detect F-actin (green) and cellular DNA (blue). (E) Percentage of cells exhibiting actin stress fibers after infection with <i>vopO</i> deletion mutants expressing truncated VopO proteins. One hundred cells from several fields were analyzed by microscopy in each experiment to determine whether stress fiber formation was induced. The means ± standard errors are presented for experiments conducted in triplicate. The asterisks indicate statistically significant differences (*<i>p</i> < 0.05).</p

Microarray analysis of VtrA and VtrB regulon in <i>V. parahaemolyticus</i>.

a<p>Fold change in gene transcripts between the wild-type and <i>ΔvtrA</i> or <i>ΔvtrB</i> mutant as determined by microarray analysis. Statistically significant changes (≥2-fold difference with <i>P</i><0.05) are highlighted in bold as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008678#s4" target="_blank"><i>Materials and Methods</i></a>.</p

VtrA and VtrB have a critical role in <i>V. parahaemolyticus</i>-induced enterotoxicity.

<p>A. VtrA and VtrB are essential for T3SS2-dependent enterotoxicity. The enterotoxic activity levels of isogenic mutants of POR-2 (<i>ΔtdhASΔvcrD1</i>) and complemented strains in rabbit ileal loops were examined. Bar 1, POR-2 (<i>ΔtdhASΔvcrD1</i>); bar 2, POR-2<i>ΔvtrA</i> (<i>ΔtdhASΔvcrD1ΔvtrA</i>); bar 3, POR-2<i>ΔvtrA</i> expressing <i>vtrA</i> (POR-2<i>ΔvtrA</i>+p<i>vtrA</i>); bar 4, POR-2<i>ΔvtrA</i> expressing <i>vtrB</i> (POR-2<i>ΔvtrA</i>+p<i>vtrB</i>); bar 5, POR-2<i>ΔvtrB</i> (<i>ΔtdhASΔvcrD1ΔvtrB</i>); bar 6, POR-2<i>ΔvtrB</i> expressing <i>vtrA</i> (POR-2<i>ΔvtrB</i>+p<i>vtrA</i>); bar 7, POR-2<i>ΔvtrB</i> expressing <i>vtrB</i> (POR-2<i>ΔvtrB</i>+p<i>vtrB</i>); bar 8, POR-2<i>ΔvtrAΔvtrB</i> (<i>ΔtdhASΔvcrD1ΔvtrAΔvtrB</i>); bar 9, POR-2<i>ΔvtrAΔvtrB</i> expressing <i>vtrA</i> (POR-2<i>ΔvtrAΔvtrB</i>+p<i>vtrA</i>); bar 10, POR-2<i>ΔvtrAΔvtrB</i> expressing <i>vtrB</i> (POR-2<i>ΔvtrAΔvtrB</i>+p<i>vtrB</i>); bar 11, <i>ΔvcrD1ΔvcrD2</i> (<i>ΔtdhASΔvcrD1ΔvcrD2</i>); bar 12, non-infected (NI) control. Results were measured as the amount of accumulated fluid (in milliliters) per length (in centimeters) of ligated rabbit small intestine. Error bars represent standard deviations for results from triplicate experiments. Asterisks indicate significant differences from the results obtained with the parental strain (<i>P</i><0.05). B. VtrA and VtrB are essential for <i>V. parahaemolyticus</i>-induced enterotoxicity. The enterotoxic activity of isogenic mutants of wild-type <i>V. parahaemolyticus</i> (WT) and complemented strains in rabbit ileal loops were examined. Bar 1, wild-type (WT); bar 2, WT<i>ΔvtrA</i>; bar 3, WT<i>ΔvtrA</i> expressing <i>vtrA</i> (WT<i>ΔvtrA</i>+p<i>vtrA</i>); bar 4, WT<i>ΔvtrA</i> expressing <i>vtrB</i> (WT<i>ΔvtrA</i>+p<i>vtrB</i>); bar 5, WT<i>ΔvtrB</i>; bar 6, WT<i>ΔvtrB</i> expressing <i>vtrA</i> (WT<i>ΔvtrB</i>+p<i>vtrA</i>); bar 7, WT<i>ΔvtrB</i> expressing <i>vtrB</i> (WT<i>ΔvtrB</i>+p<i>vtrB</i>); bar 8, WT<i>ΔvtrAΔvtrB</i>; bar 9, WT<i>ΔvtrAΔvtrB</i> expressing <i>vtrA</i> (WT<i>ΔvtrAΔvtrB</i>+p<i>vtrA</i>); bar 10, WT<i>ΔvtrAΔvtrB</i> expressing <i>vtrB</i> (WT<i>ΔvtrAΔvtrB</i>+p<i>vtrB</i>); bar 11, NI control. Error bars represent standard deviations for results from triplicate experiments. Asterisks indicate significant differences from the results obtained with the parental strain (<i>P</i><0.05).</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

VtrB expression is under the control of VtrA.

<p>A. Neither <i>vtrA</i> nor <i>vtrB</i> was involved in the transcription of <i>vtrA</i>. <i>V. parahaemolyticus</i> strains carrying the <i>vtrA-lacZ</i> transcriptional fusion vector were assayed for β-galactosidase activity. The bars show the average of three separate experiments, and the standard deviations are indicated by error bars. B. Transcription of <i>vtrB</i> was decreased in <i>vtrA</i> deletion strains. <i>V. parahaemolyticus</i> strains carrying the <i>vtrB-lacZ</i> transcriptional fusion vector were assayed for β-galactosidase activity. The bars show the average of three separate experiments, and the standard deviations are indicated by error bars. C. Deletion of <i>vtrA</i> caused a decrease in the production of VtrB. Immunoblot analysis of VtrA and VtrB protein expression in <i>vtrA</i> and <i>vtrB</i> mutant strains are shown. Lane 1, wild-type <i>V. parahaemolyticus</i> (WT); lane 2, <i>vtrA</i> mutant strain (WT<i>ΔvtrA</i>); lane 3, <i>vtrB</i> mutant strain (WT<i>ΔvtrB</i>); lane 4, <i>vtrA</i> and <i>vtrB</i> double mutant strain (WT<i>ΔvtrAΔvtrB</i>). Blots were probed with anti-VtrA (upper panel) and anti-VtrB (lower panel) polyclonal antibodies. D. Effects of <i>vtrA</i> and <i>vtrB</i> expression on <i>vtrB</i> transcription in <i>E. coli</i>. <i>E. coli</i> MC4100 carrying <i>vtrB-lacZ</i> transcriptional fusion vector were assayed for β-galactosidase activity. The bars show the average of three separate experiments, and the standard deviations are indicated by error bars. E. Binding of purified VtrA DNA binding domain to the upstream region of <i>vtrB</i> is shown by an electrophoretic mobility shift assay. Each lane contains the same amount of upstream region of <i>vtrB</i> (30 nM) and various concentrations (0, 1.5, 2.25, 3.0, 4 µM) of VtrA DNA binding domain (upper panel) or VtrB DNA binding domain (lower panel). The molecular ratios are indicated in the top line.</p

VtrA and VtrB are not necessary for T3SS1-dependent cytotoxicity but necessary for T3SS2-dependent cytotoxicity.

<p>A. <i>vtrA</i> and <i>vtrB</i> are not necessary for T3SS1-dependent cytotoxicity. Caco-2 cells were infected for 6 h with isogenic strains of POR-3 (<i>ΔtdhASΔvcrD2</i>). Bar 1: POR-3 (<i>ΔtdhASΔvcrD2</i>); bar 2: POR-3<i>ΔvtrA</i>; bar 3: POR-3<i>ΔvtrB</i>; bar 4: <i>ΔvcrD1ΔvcrD2</i> (<i>ΔtdhASΔvcrD1ΔvcrD2</i>). Cytotoxicity was evaluated by the amount of LDH released. Error bars represent standard deviations for results from triplicate experiments. B. <i>vtrA</i> and <i>vtrB</i> are essential for T3SS2-dependent cytotoxicity. Caco-2 cells were infected for 6 h with isogenic mutant strains of POR-2 (<i>ΔtdhASΔvcrD1</i>). Bar 1: POR-2 (<i>ΔtdhASΔvcrD1</i>); bar 2: POR-2<i>ΔvtrA</i> (<i>ΔtdhASΔvcrD1ΔvtrA</i>); bar 3: POR-2<i>ΔvtrA</i> expressing <i>vtrA</i> (POR-2<i>ΔvtrA</i>+p<i>vtrA</i>); bar 4: POR-2<i>ΔvtrA</i> expressing <i>vtrB</i> (POR-2<i>ΔvtrA</i>+p<i>vtrB</i>); bar 5: POR-2<i>ΔvtrB</i> (<i>ΔtdhASΔvcrD1ΔvtrB</i>); bar 6; POR-2<i>ΔvtrB</i> expressing <i>vtrA</i> (POR-2<i>ΔvtrB</i>+p<i>vtrA</i>); bar 7: POR-2<i>ΔvtrB</i> expressing <i>vtrB</i> (POR-2<i>ΔvtrB</i>+p<i>vtrB</i>); bar 8: POR-2<i>ΔvtrAΔvtrB</i> (<i>ΔtdhASΔvcrD1ΔvtrAΔvtrB</i>); bar 9: POR-2<i>ΔvtrAΔvtrB</i> expressing <i>vtrA</i> (POR-2<i>ΔvtrAΔvtrB</i>+p<i>vtrA</i>); bar 10: POR-2<i>ΔvtrAΔvtrB</i> expressing <i>vtrB</i> (POR-2<i>ΔvtrAΔvtrB</i>+p<i>vtrB</i>); bar 11: <i>ΔvcrD1ΔvcrD2</i> (<i>ΔtdhASΔvcrD1ΔvcrD2</i>). Cytotoxicity was evaluated by the amount of LDH released. Error bars represent standard deviations for results from triplicate experiments. Asterisks indicate significant differences from the results obtained with the parent strain (*<i>P</i><0.05). C. Overexpressing of <i>vtrA</i> and <i>vtrB</i> promoted T3SS2-dependent cytotoxicity. Caco-2 cells were infected for 1.5–6 h with <i>V. parahaemolyticus</i>. Cytotoxicity was evaluated by the amount of LDH released. POR-2 (<i>ΔtdhASΔvcrD1</i>) with control vector (pSA19CP-MCS) (filled squares, solid line), POR-2 expressing <i>vtrA</i> (filled circles, solid line), POR-2 expressing <i>vtrB</i> (filled triangles, solid line), <i>ΔvcrD1ΔvcrD2</i> (<i>ΔtdhASΔvcrD1ΔvcrD2</i>) with control vector (pSA19CP-MCS) (open squares, dashed line), <i>ΔvcrD1ΔvcrD2</i> expressing <i>vtrA</i> (open circles, dashed line), and <i>ΔvcrD1ΔvcrD2</i> expressing <i>vtrB</i> (open triangles, dashed line). Error bars represent standard deviations for results from triplicate experiments. Asterisks indicate significant differences from the results obtained with the parent strain (*<i>P</i><0.05).</p