Clostridium perfringens TpeL Induces Formation of Stress Fibers via Activation of RhoA-ROCK Signaling Pathway

Abstract

Clostridium perfringens TpeL belongs to a family of large clostridial glucosylating cytotoxins. TpeL modifies Rac1 and Ras subfamily proteins. Herein we report TpeL-induced formation of stress fibers via RhoA-Rho kinase (ROCK) signaling. A recombinant protein (TpeL1–525) derived from the TpeL N-terminal catalytic domain in the presence of streptolysin O (SLO) induced the formation of actin stress fibers in Madin–Darby canine kidney (MDCK) cells in a dose-dependent manner. The RhoA/ROCK pathway is known to control the formation of stress fibers. We examined the role of the RhoA/ROCK pathway in TpeL-induced formation of stress fibers. TpeL1–525-induced formation of stress fibers was inhibited by the ROCK inhibitor, Y27632 and Rho protein inhibitor, C3 transferase. TpeL1–525 activated RhoA and ROCK in a dose-dependent manner. C3 transferase blocked TpeL1–525-induced activation of RhoA and ROCK whereas Y27632 inhibited TpeL-induced activation of ROCK. These results demonstrate for the first time that TpeL induces the formation of stress fibers by activating the RhoA/ROCK signaling pathway.

Clostridium perfringens type C has been known as a virulence factor of necrotic enteritis, which is involved in diarrhea and dysentery in neonatal animals.^1,2) In humans, this bacteria induces necrotizing enterocolitis, which has been called “pig-bel.”²⁾ C. perfringens type C also causes necro-hemorrhagic enteritis in animals, such as calves, sheep, goats, and piglets,^3,4) and necrotic enteritis (NE) in poultry.^5,6) Type C isokates produce alpha-toxin, beta-toxin, beta2-toxin, and perfringolysin O. Beta-toxin possesses lethal and cytotoxic acitivities. The toxin has been linked to the virulence of C. perfringens type C.²⁾ Beta2-toxin is also responsible for its pathogenicity.⁷⁾

Many type C strains have been shown to elaborate the novel toxin, TpeL, which is a homolog of clostridial glucosylating toxins.⁸⁾ TpeL was intially found in the culture filtrate of C. perfringens type C strain MC18 from pig in Japan.⁸⁾ TpeL is thought to be a potential pathogeinc agent.⁸⁾ A recent study demonstrated that the inoculation of broilers with isolates positive for both tpeL, the gene encoding TpeL, and netB, the gene encoding NetB toxin, caused more severe gross lesions than isolates positive for netB only.⁹⁾ The role, if any, of TpeL in disease remains unclear. However, the production of TpeL may enhance the virulence of avian necrotic enteritis strains.

TpeL has cytotoxic effect, inducing cell rounding.⁸⁾ Its molecular mass, which was calculated from its deduced amino acid sequence, was 191 kDa, and a signal peptide region was absent from the open reading frame (ORF).⁸⁾ TpeL belongs to the large clostridial toxin (LCT) family,⁸⁾ which includes Clostridium difficile toxin A (TcdA) and B (TcdB), Clostridium sordellii lethal toxin (TcsL), and Clostridium novyi alpha-toxin (TcnA). The crystal structure of the catalytic domain of TcdB was reported.¹⁰⁾ Like other LCTs, TpeL has an N-terminal domain that is responsible for glucosyltransferase activity, a domain with autocatalytic activity, and a putative transmembrane domain that is involved in the delivery of the enzymatic domain into the cytosol.¹¹⁾ However, TpeL is dissimilar from other LCTs due to its truncated C-terminal domain, a putative receptor binding domain.^8,11,12) LCTs are thought to bind to the membrane receptor of sensitive cells.^13,14) After endocytosis, cellular inositolhexaphosphate (InsP₆)-dependent cysteine protease cleavage, and transport across the endosomal membrane, the catalytic domain internalizes the cytolasm from early endosomes. Small guanosine 5′-triphosphatases (GTPases) are glucosylated in the cytoplasm and, therefore, inactivated. TpeL is the sole LCT that can use both uridine 5′-diphosphate (UDP)-glucose and UDP-N-acetylglucosamine as donor substrates.^11,12,15) TpeL modifies the Rac1 and Ras subfamily by glucosylation in order to concern its cytotoxic activities. Blokade of the Ras signaling pathway was previously shown to be essential for TpeL-caused apoptosis.¹¹⁾

Pathogenic bacteria use a number of mechanisms to perturb the cellular actin cytoskeleton during the infection. These bacteria can operate the actin stress fiber formation.^16,17) Actin stress fibers, one of the main cytoskeleton, are composed of bundles of ca. 10–30 F-actin filaments.¹⁸⁾ Stress fibers play a role in many cellular functions, including cell adhesion, mobility, contraction and morphogenesis. The small GTPase Rho and its effector Rho-associated kinase (ROCK) are well-known to be responsible for formation of stress fiber.¹⁹⁾ Rho cycles between inactive guanosine 5′-diphosphate (GDP)-bound and active GTP-bound states. Once RhoA is activated, it, in turn, activates a lot of downstream targets. ROCK, one of downstream effectors of RhoA, is a serine/threonine kinase. ROCK, through the phosphorylation and deactivation of the myosin binding subunit of myosin phosphatase (MYPT1), and direct phosphorylation of myosin light chain (MLC), enhances the actin binding. One common action of effector and toxin produced by bacteria is reorganization of the cellular actin cytoskeleton to benefit pathogenic bacteria.²⁰⁾

TpeL has been shown to exert cytotoxic effects on Vero and HeLa cells.^8,11) This toxin also blocked Ras signaling pathways in rat phenochromocytoma PC12 cells.¹¹⁾ However, our understanding of the biological activity of TpeL remains unclear. Full-lenght TpeL is higly labile and hard to purify from the culture filtrate of C. perfringens type C. We previously prepared the recombinant glucosyltransferase domain, TpeL1–525 (covering amino acids 1–525)¹²⁾ in order to clarify the biological activity of TpeL. We also used the streptolysin O (SLO) delivery systems to transport the catalytic domain into cells.^12,21) We examined the intracellular events that occurred during the intoxication process by TpeL. We here demonstrated that TpeL induced the formation of stress fibers in cells.

MATERIALS AND METHODS

Materials

Recombinant TpeL1–525 was expressed and purified as described previously.¹²⁾ Y27632 and streptolysin O (SLO) were obtained from Sigma (St. Louis, MO, U.S.A.). Rac1 inhibitor (NSC23766) was purchased from Millipore (Tokyo, Japan). Rac1 and Ras signals were either detected with the glucosylation-sensitive antibodies Mab 102 (BD Biosciences, Tokyo, Japan) and Mab 27H5 (Cell Signaling, Tokyo, Japan), respectively, or with the glucosylation-insentive antibodies Mab 23A8 (Millipore, Tokyo, Japan) and Mab Y13-256 (Merck Millipore, Tokyo, Japan), respectively. The Rho kinase (ROCK) activity immunoblot kit was purchased from Cell Biolabs (San Diego, CA, U.S.A.). The Rho activation assay biochem kit and cell permeable C3 exoenzme were obtained from Cytoskeleton (Denver, CO, U.S.A.). Horseradish peroxidase-labeled anti-rabbit immunoglobulin G (IgG), horseradish peroxidase-labeled anti-mouse IgG, and an enhanced chemiluminescence kit were obtained from GE Healthcare (Tokyo, Japan). Alexa Fluor 488-conjugated phalloidin and 4′,6′-diamino-2-phenylindole (DAPI) were obtained from Molecular Probes (Eugene, OR, U.S.A.). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco BRL (New York, NY, U.S.A.). A monoclonal anti-TpeL antibody was prepared as previously described.⁸⁾ All other chemicals were of the highest grade available from commercial sources.

Cell Culture and Cytotoxicity Assay

Madin–Darby canine kidney (MDCK) cells were obtained from the Riken Cell Bank (Tsukuba, Japan). They were cultured in DMEM supplemented with 10% fetal calf serum (FCS), 100 U of penicillin/mL, 100 µg of streptomycin/mL, and 2 mM glutamine (FCS-DMEM). All incubation steps were performed at 37°C in a 5% CO₂ atmosphere. In the cytotoxicity assays, cells (5×10⁴) were inoculated in 48-well plates. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt conversion assay (Promega, Madison, WI, U.S.A.) as described previously.¹²⁾

Toxin Treatment

TpeL1–525 was transported into MDCK cells using SLO as a delivery system.¹²⁾ Cells were plated on 48-well plates and incubated at 37°C in a 5% CO₂ incubator overnight in FCS-DMEM. Cells were incubated with 0.1 mL of Hank’s buffered salt solution (HBSS) without Ca²⁺ containing 30 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (Hepes), pH 7.2, for 15 min at 37°C. SLO (100 ng/mL) was then added together with various concentrations of TpeL1–525 as indicated for 15 min at 37°C and 5% CO₂. To reseal, 0.25 mL of ice-cold HBSS containing 30 mM Hepes and 2 mM Ca²⁺, pH 7.2, was added. After a 1 h incubation at 37°C, HBSS was replaced with full growth medium.²¹⁾ Cells were examined for morphological alterations by phase-contrast microscopy 2 h after the inoculation. In some experiments, heat-inactivated TpeL1–525 was prepared by heating at 95°C for 10 min.

Fluorescence Staining of Actin Stress Fibers and Microscopic Examination

Cells were fixed with 4% paraformaldehyde for 5 min and washed three times for 10 min each in phosphate buffered saline (PBS) containing 0.02% Triton X-100. The cells were permeablilized with 0.1% Triton X-100 for 5 min and washed with PBS containing 0.02% Triton X-100 three times. After blocking with 3% bovine serum albumin (BSA) in PBS for 30 min, the cells were incubated with Alexa Fluor-488-phalloidin and DAPI for 60 min at room temperature. All images were captured with a Nikon A1 laser scanning confocal microscope (Tokyo, Japan).^22,23) The fluorescence intensity of F-actin was quantified using Nikon image software (NIS-Elements AR3.22), which involved assigning a value for the intensity of fluorescence to every pixel in a defined area.

Immunoblotting

A protein assay was performed using the method of Bradford.²⁴⁾ Proteins were separated on 12.5% polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (PVDF, Millipore) for 2 h at 250 mA. This was followed by blocking with Tris-buffered saline containing 5% BSA and 1% Tween20 (blocking buffer) for 1 h. Blots were incubated for 2 h with the appropriate primary antibody in the blocking buffer, then for 1 h with a horseradish peroxidase-conjugated secondary antibody, and finally with the reagents from an enhanced chemiluminescence analysis kit.

ROCK Activity Assay

ROCK activity was measured using the ROCK activity immunoblot kit according to the manufacturer’s instructions with a recombinant myosin phosphatase target subunit 1 (MYPT1) as the substrate (Cell Biolabs). Cell cultures in 6-cm dishes were treated with SLO only or SLO plus TpeL1–525, as described above. Cells were rinsed with 5 mL of ice-cold phosphate-buffered saline and scraped off in 300 µL of lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM ethylenediaminetetraacetic acid (EDTA), 40 µg/mL aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, 20 µg/mL leupeptin, and 80 µg/mL benzamidine) per dish. The cells were disrupted by freeze-thawing (5 min in liquid nitrogen, 10 min at 37°C; 3 cycles), then centrifugated for 10 min at 10000×g. Supernatants were used as cell lysates. Cell lysates were incubated in 50 µL of kinase/ATP/substrate solution at 30°C for 30 min with gentle agitation. Reducing sodium dodecyl sulfate (SDS)–sample buffer was added to the solution to stop the reaction and it was then boiled for 5 min. The phosphorylation of MYPT1 was assessed by Western blot using an antibody for phospho-MYPT1.

RhoA Activity Assay

The activation of RhoA was measured according to the manufacturer’s instructions (Cytoskeleton). Cell cultures in 6-cm dishes were treated with SLO only or SLO plus TpeL1–525, as described above. Cells were rinsed with 5 mL of ice-cold phosphate-buffered saline (PBS) and scraped off in 300 µL of cell lysis buffer containing protease inhibitors per dish. Cells were disrupted by freeze-thawing (5 min in liquid nitrogen, 10 min at 37°C; 3 cycles), then centrifugated for 10 min at 10000×g. Supernatants were used as cell lysates. The cell lysate from each sample was then incubated with rhotekin rho-binding domain (RBD) agarose beads at 4°C for 1 h with gentle agitation. The beads were washed with ice-cold PBS and bound Rho was eluted by boiling each sample in reducing SDS-sample buffer. RhoA proteins were detected by Western blotting using a RhoA antibody.

Statistical Analysis

One-way ANOVA, followed by Bonferroni’s multiple-comparison posttest, was used to compare the means. Data are expressed as the means±standard deviations (S.D.). A p value of 0.05 or less was considered statistical significant.

RESULTS

TpeL1–525-Induced Formation of Stress Fibers

To clarify the biological activity of TpeL, MDCK cells were incubated with low concentrations of TpeL1–525 in the presence of SLO at 37°C. At concentrations over 1.6 nM, TpeL1–525 caused the formation of stress fibers in a dose-dependent manner in the presence of SLO (Figs. 1A, B). Under this experimental condition, we investigated whether TpeL1–525 in the presence of SLO glucosylated Rac1 and Ras on MDCK cells. We determined the glucosylation status of Rac1 and Ras with antibodies recognizing either total Rac1 and Ras levels or only the non-modified versions of the GTPase. As shown in Fig. 2A, TpeL1–525 caused a decrease in the cellular level of non-glucosylated Rac1 in a dose-dependent manner. Total Rac1 levels did not decrease in cells treated with SLO plus TpeL1–525. On the other hand, TpeL1–525 did not modify Ras under the conditions (Fig. 2B). When TpeL1–525 was delivered at a concentration of 1.6 to 16 nM to the cells by SLO, cell viability did not decrease (Fig. 1S). The formation of stress fibers induced by TpeL1–525 was completely neutralized by the monoclonal anti-TpeL antibody (Fig. 2S), and heat-inactivated TpeL1–525 did not induce the formation of stress fibers.

Fig. 1. TpeL1–525-Induced Formation of Stress Fibers

(A) MDCK cells were incubated with SLO (100 ng/mL) alone or a combination of various amounts of TpeL1–525 with SLO (100 ng/mL) at 37°C for 15 min. After 120 min of resealing, cells were fixed, permeabilized, and then stained with DAPI and Alexa Fluor 488-phalloidin. Actin (green) and the nucleus (blue) were viewed with a confocal microscope. Experiments were repeated three times, and a representative result is shown. Bar, 10 µm. (B) Quantification of stress fibers. The fluorescence intensity of F-actin was quantified as described in Materials and Methods. The results represent the means±S.D. obtained from at least four independent experiments. Data were analyzed using a one-way ANOVA with Bonferroni’s mutiple-comparison posttest. * p<0.05, ** p<0.01.

Fig. 2. TpeL1–525-Induced Glucosylation of Rac1 and Ras

MDCK cells were incubated with SLO (100 ng/mL) alone or a combination of various amounts of TpeL1–525 with SLO (100 ng/mL) at 37°C for 15 min. After 120 min of resealing, the cells were lysed in SDS-sample buffer, followed by SDS-PAGE and immunoblotting for the detection of Ras and Rac with glucosylation-insensitive antibodies (input control; total Rac or Ras, respectively) or with glucosylation-sensitive antibodies (non-glucosylated Rac or Ras, respectively). One representative experiment from three is shown. Non-glucosylated signals were normalized to total signals for quantification. Non-glucosylated band level of untreated cells was set to 1. The results represent the means±S.D. obtained from at least four independent experiments. Data were analyzed using a one-way ANOVA with Bonferroni’s mutiple-comparison posttest. * p<0.05, ** p<0.01.

Effects of Inhibitors on the TpeL1–525-Induced Formation of Stress Fibers

The small GTPase RhoA and its effector Rho kinase (ROCK) have been shown to play a critical role in the formation of stress fibers.^18,25) To confirm whether RhoA was involved in the formation of stress fibers in MDCK cells, we firstly treated the cells with RhoA-specific inhibitor-exoenzyme C3 cell-permeable transferase, followed by the TpeL1–525 treatment in the presence of SLO (Fig. 3A, 3B). The TpeL1–525-induced formation of stress fibers was inhibited by C3 transferase, which indicated the requirement of RhoA in the formation of stress fibers by TpeL1–525. We then pretreated MDCK cells with the specific ROCK inhibitor Y27632 to examine whether ROCK contributed to the TpeL1–525-induced formation of stress fibers. As shown in Figs. 3A and B, Y27632 inhibited the formation of stress fibers by TpeL1–525. These results suggested that TpeL1–525 induced the formation of stress fibers through the RhoA/ROCK pathway.

Fig. 3. Effects of Various Inhibitors on the TpeL1–525-Induced Formation of Stress Fibers

(A) MDCK cells were pretreated with inhibitors (C3 transferase 2 µg/mL, 2 h; Y27632 10 µM, 1 h) and then incubated with SLO (100 ng/mL) alone or a combination of TpeL1–525 (8 nM) with SLO (100 ng/mL) at 37°C for 15 min. After 120 min of resealing, cells were fixed, permeabilized, and then stained with DAPI and Alexa Fluor 488-phalloidin. Actin and the nucleus were viewed with a confocal microscope. The experiments were repeated three times, and a representative result is shown. Bar, 10 µm. (B) MDCK cells were pretreated with inhibitors (C3 transferase 2 µg/mL, 2 h; Y27632 10 µM) and then incubated with SLO (100 ng/mL) alone or a combination of TpeL1–525 (8 nM) with SLO (100 ng/mL) at 37°C for 15 min. After 120 min of resealing, the fluorescence intensity of F-actin was quantified as described in Materials and Methods. The results represent the means±S.D. obtained from four independent experiments. Data were analyzed using a one-way ANOVA with Bonferroni’s mutiple-comparison posttest. * p<0.01.

Activation of RhoA by TpeL1–525

We investigated whether RhoA was involved in the formation of stress fibers by TpeL1–525 in MDCK cells. RhoA activity was determined by pull down assay using the Rho-GTP binding domain of Rhotekin, which preferentially associates with active RhoA-GTP. As shown in Fig. 4A, TpeL1–525 in the presence of SLO increased RhoA activity in a dose-dependent manner more than SLO alone. The C3 enzyme inhibited the activation of Rho activity by TpeL1–525 (Fig. 4B). These results demonstrated that TpeL1–525-induced activation of RhoA was involved in the formation of stress fibers.

Fig. 4. TpeL1–525-Induced Activation of RhoA

(A) MDCK cells were incubated with SLO (100 ng/mL) alone or a combination of various amounts of TpeL1–525 with SLO (100 ng/mL) at 37°C for 15 min. (B) MDCK cells were pretreated with inhibitors (C3 transferase 2 µg/mL, 2 h) and then incubated with SLO (100 ng/mL) alone or a combination of TpeL1–525 (8 nM) with SLO (100 ng/mL) at 37°C for 15 min. After 30 min of resealing, cells were lysed and subjected to RhoA activity assays. Signal intensities from immunoblots were recorded densitometrically. Active RhoA signals were normalized to total RhoA signals for quantification. The active RhoA level of untreated cells was set to 1. The results represent the means±S.D. obtained from four independent experiments. Data were analyzed using a one-way ANOVA with Bonferroni’s mutiple-comparison posttest. * p<0.05, ** p<0.01.

Activation of ROCK by TpeL1–525

We examined the role of ROCK signaling on the TpeL1–525-induced formation of stress fibers. ROCK activity was analyzed by assessing the phosphorylation of MYPT1. The treatment of MDCK cells with TpeL1–525 in the presence of SLO led to the activation of ROCK in a dose-dependent manner (Fig. 5A). Furthermore, Y27632 and the C3 enzyme blocked the TpeL1–525-induced activation of ROCK (Fig. 5B). These results indicated that TpeL1–525 activated ROCK via RhoA.

Fig. 5. TpeL1–525-Induced Activation of ROCK

(A) MDCK cells were incubated with SLO (100 ng/mL) alone or a combination of various amounts of TpeL1–525 with SLO (100 ng/mL) at 37°C for 15 min. (B) MDCK cells were pretreated with an inhibitor (C3 transferase 2 µg/mL, 2 h; Y27632 10 µM, 1 h) and then incubated with SLO (100 ng/mL) alone or a combination of TpeL1–525 (8 nM) with SLO (100 ng/mL) at 37°C for 15 min. After 30 min of resealing, cells were lysed and subjected to ROCK activity assays. Signal intensities from immunoblots were recorded densitometrically. Phosho-MYPT1 signals were normalized to total MYPT1 signals for quantification. The phospho-MYPT1 level of untreated cells was set to 1. The results represent the means±S.D. obtained from four independent experiments. Data were analyzed using a one-way ANOVA with Bonferroni’s mutiple-comparison posttest. * p<0.05, ** p<0.01.

Effect of Rac1 Inhibitor on TpeL1–525-Induced Action

As Rac1 has been reported to act upstream of RhoA in order to regulate cell actin organization,²⁶⁾ we examined the effect of Rac1 inhibitor (NSC23766) on TpeL1–525-induced formation of stress fiber. As shown in Figure 6A, Rac1 inhibitor slightly enhanced the formation of stress fibers by TpeL1–525. Similarly, the inhibitor modestly augmented the activation of RhoA induced by TpeL1–525 (Fig. 6B).

Fig. 6. Effect of Rac1 Inhibiton on TpeL1–525-Induced Action

MDCK cells were pretreated with Rac1 inhibitor (50 µM, 1 h) and then incubated with SLO (100 ng/mL) alone or a combination of TpeL1–525 (8 nM) with SLO (100 ng/mL) at 37°C for 15 min. (A) After 120 min of resealing, the fluorescence intensity of F–actin was quantified as described in Materials and Methods. The results represent the means±S.D. obtained from four independent experiments. (B) After 30 min of resealing, cells were lysed and subjected to RhoA activity assays. Signal intensities from immunoblots were recorded densitometrically. Active RhoA signals were normalized to total RhoA signals for quantification. The active RhoA level of untreated cells was set to 1. The results represent the means±S.D. obtained from four independent experiments.

DISCUSSION

In the present study, we demonstrated that TpeL induced the formation of stress fibers in MDCK cells. TpeL was previously shown to be the cytotoxic to Vero cells and induced apoptosis in HeLa cells.^8,11) It has not been reported whether other LCTs can induce formation of stress fiber. The results of the present study indicated that the low-dose TpeL induced the formation of stress fibers in host cells.

When cells were treated with TpeL, glucosylation of the Rac1 and Ras subfamily but not RhoA was confirmed.^11,12,15) Guttenberg et al.¹¹⁾ reported that inhibiting the Ras signaling pathway by TpeL was essential for the induction of apoptosis. However, in the present study, we showed that the treatment of cells with low concentrations of TpeL that were insufficient to evoke cytopathic effects resulted in an increase in the formation of stress fibers in a dose-dependent manner. The TpeL-induced formation of stress fibers was inhibited by C3 transferase and Y27632, therefore, TpeL activated RhoA and ROCK. The treatment of cells with C3 transferase inhibited the activation of RhoA and ROCK by TpeL. Furthermore, Y27632 blocked the activation of ROCK by TpeL. These results clearly demonstrated that TpeL induced the formation of stress fibers through the RhoA/ROCK signaling pathway in cells. However, the mechanism by which TpeL activates RhoA remains unclear. TpeL glucosylated Rac1, but not RhoA. On the other hand, Rac1 is able to down-regulate Rho activity. The downregulation of Rac1 activity was previously shown to be accompanied by an increase in RhoA activity.²⁷⁾ Rac1 was reported to act upstream of RhoA in order to regulate cell actin organization.²⁶⁾ Here, we observed that Rac1 inhibitor slightly enhanced the formation of stress fiber induced by TpeL1–525. On the basis of our findings and data from the literatures, the inactivation of Rac1 by TpeL may activate the RhoA/ROCK pathway. Further studies are needed to validate the precise mechanism underlying the TpeL-induced activation of RhoA/ROCK signaling.

We reported that TpeL glucosylated Rac1, as well as Ras family consisting of Ha-Ras, RalA and Rap1B, but not RhoA and Cdc42. In animal cells, it has been recognized that Ras and Rho GTPases regulate an overlapping set of cellular responses. However, the elucidation of the molecular basis for the cross-talk between Ras family and Rho has awaited the delineation of the signaling events controlled by each family.²⁸⁾ As the biochemical details are still poorly understood, there is much experimental work that points to the importance of combinatorial acitvities controlled by these two families in celluar responses.²⁸⁾

Pathogenic bacteria modified the actin cytoskeletal organization in eukaryotic host cells, which finally contributed to virulence. For example, Tam et al.²⁰⁾ reported that the translocation of VopF produced by Vibrio cholera altered the actin cytoskeletal organization in target cells, resulting in the efficient bacterial colonization of the bowels. Moreover, Vibrio parahaemolyticus VopL is a bacterial pathogenic agent that accelerates the assembly of F-actin filaments during an enteric infection in the host.²⁹⁾ A previous study reported that the production of TpeL may enhance the virulence of poultry necrotizing enterocolitis strains.⁹⁾ In the present study, we speculated that C. perfringens may use TpeL to disrupt actin homeostasis in intestinal epithelial cells during the infection, which suggests enterotoxic effects in the intestine. Hence, TpeL provides a newly pathogenic mechanism and is also a valuable tool for studying infectious processes involving the organization of actin.

In conclusion, TpeL stimulated the formation of stress fibers and this was mediated by activation of the RhoA/ROCK signaling pathway. The results offer a novel insight into the role of TpeL in host–pathogen interactions.

Acknowledgments

We thank M. Satoh and H. Nakamura for their technical assistance. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, 2013

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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