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. 2008 Jan;4(1):e3.
doi: 10.1371/journal.ppat.0040003.

Yersinia controls type III effector delivery into host cells by modulating Rho activity

Edison Mejía  1 , James B Bliska, Gloria I Viboud
Affiliations

Yersinia controls type III effector delivery into host cells by modulating Rho activity

Edison Mejía et al. PLoS Pathog. 2008 Jan.

Abstract

Yersinia pseudotuberculosis binds to beta1 integrin receptors, and uses the type III secretion proteins YopB and YopD to introduce pores and to translocate Yop effectors directly into host cells. Y. pseudotuberculosis lacking effectors that inhibit Rho GTPases, YopE and YopT, have high pore forming activity. Here, we present evidence that Y. pseudotuberculosis selectively modulates Rho activity to induce cellular changes that control pore formation and effector translocation. Inhibition of actin polymerization decreased pore formation and YopE translocation in HeLa cells infected with Y. pseudotuberculosis. Inactivation of Rho, Rac, and Cdc42 by treatment with Clostridium difficile toxin B inhibited pore formation and YopE translocation in infected HeLa cells. Expression of a dominant negative form of Rac did not reduce the uptake of membrane impermeable dyes in HeLa cells infected with a pore forming strain YopEHJT(-). Similarly, the Rac inhibitor NSC23766 did not decrease pore formation or translocation, although it efficiently hindered Rac-dependent bacterial uptake. In contrast, C. botulinum C3 potently reduced pore formation and translocation, implicating Rho A, B, and/or C in the control of the Yop delivery. An invasin mutant (Y. pseudotuberculosis invD911E) that binds to beta1 integrins, but inefficiently transduces signals through the receptors, was defective for YopE translocation. Interfering with the beta1 integrin signaling pathway, by inhibiting Src kinase activity, negatively affected YopE translocation. Additionally, Y. pseudotuberculosis infection activated Rho by a mechanism that was dependent on YopB and on high affinity bacteria interaction with beta1 integrin receptors. We propose that Rho activation, mediated by signals triggered by the YopB/YopD translocon and from engagement of beta1 integrin receptors, stimulates actin polymerization and activates the translocation process, and that once the Yops are translocated, the action of YopE or YopT terminate delivery of Yops and prevents pore formation.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effect of Caspase-1 Inhibitor or Glycine on YopB/D-Mediated LDH Release
HeLa cells were left untreated or treated with 50 μM or 100 μM caspase-1 inhibitor Ac-YVAD-cmk one hour before infection (A) or through out the infection with 5mM glycine (B). After 3 h infection with a yopEHJ mutant (YP27) or yopEHJB mutant (YP29), culture supernatants were removed and tested for LDH release using CytoTox 96 assay kit (Promega). The percentage of LDH release was calculated by dividing the amount of LDH release from infected cells by the amount of LDH release from uninfected cells lysed by a freeze-thaw cycle. Error bars represent the standard deviation of the mean values obtained from three infected wells.
Figure 2
Figure 2. Effect of ToxB or Cytochalasin D on Pore Formation and Yop Translocation
HeLa cells were left untreated, exposed to 40ng/ml C. difficile Toxin B (ToxB), or 3.9μM cytochalasin D (CD) for 2 hours prior to infection. Cells on coverslips were infected with yopEHJ mutant (YP27) or yopEHJB mutant (YP29) for 3 h, and stained with DEAD-LIVE kit, as described in Material and Methods. Cells with disrupted membranes exhibit a red nuclei staining (A). LDH release was determined in the culture supernatants 3h post infection (B). Wild type (YP126) and yopB mutant (YP18) were used to infect Hela cells for 2 hours. Triton X-100 cell lysates were centrifuged, and soluble and insoluble fractions (containing translocated Yops and bacterial Yops, respectively) were analyzed by immunoblotting using anti-YopE antibodies. Anti-β actin antibody was used as a loading control of the soluble fraction. Anti-rabbit antibodies conjugated with IR800 or IR680 were used as secondary antibodies, and the infrared signal was detected using the Li-Cor Odyssey infrared scanner. The intensity of each band was calculated using the software provided by the Odyssey IR imaging system, and the YopE/β-actin ratios were plotted on a graph (C).
Figure 3
Figure 3. Effect of Invasin and yadA Inactivation on Pore Formation and Translocation
HeLa cells were infected with Y. pseudotuberculosis strains yopEHJ (YP27), yopEHJB (YP29), yopEHJ,yadA,inv/ppsaABC (YP50/pAY66), yopEHJB,yadA,inv/ppsaABC (YP51/pAY66), yopEHJ,yadA,inv/pMMB67HE YadA (YP50/pYadA), or yopEHJ,yadA,invD911E (YP50invD911E). LDH was determined in the culture supernatants as described in Figure 1 (A). Culture supernatants were collected from triplicate wells 5h post infection and assayed using an IL-8 ELISA (Antigenix America) (B). Y. pseudotuberculosis wild type (YP126), yopB mutant (YP18), yopHJ,yadA,inv/psaABC (YP54/pAY66), yopHJ,yadA,inv/pMMB67HE YadA (YP54/pYadA), or yopHJ,yadA,invD911E (YP54invD911E) strains were used to infect HeLa cells for 2 hours, and translocated YopE was analyzed by immunoblotting as described in Figure 2. Soluble fractions correspond to translocated YopE, and insoluble fractions correspond to bacteria-associated YopE (C). YopE-mediated cytotoxicity was analyzed by phase contrast microscopy at 15, 30 and 60 min post infection in cells infected with wild type (YP126) strain, YP54/pAY66 or YP54invD911 (D).
Figure 4
Figure 4. Effect of Src Kinase Inhibitor PP2 on Pore Formation and Yop Translocation
HeLa cells were exposed to 10 μM PP2 in DMSO or to DMSO alone one hour prior to infection. HeLa cells infected with YP27 (yopEHJ) or YP29 (yopEHJB) were assessed for pore formation as indicated for Figure 1 A. Wild type (YP126) and yopB mutant (YP18) were used to infect treated HeLa cells for 2 hours. YopE translocation was determined as described in Figure legend 2B.
Figure 5
Figure 5. Rac Inactivation Inhibits Bacterial Uptake but Not Pore Formation or Translocation
Hela cells were treated for 6h with Rac inhibitor NSC23766 (100μM) in 5% serum-DMEM, or with 5% serum-DMEM alone. NSC23766-treated and untreated cells were infected with YP27 (yopEHJ) or the uptake-deficient strain YP50/pAY66 (yopEHJ,yadA,inv/psaABC). The percentage of internalized bacteria was assessed one hour after infection by double staining immunofluorescence, as described in Material and Methods (A). LDH released by uninfected cells or by cells infected with YP27 or YP29 (yopEHJB), in the presence or absence of the Rac inhibitor, was assessed as described in Figure 1 (B). The amount of translocated YopE in the cell lysate of cells infected with wild type (YP126) or yopB (YP18) was analyzed by immunoblotting as described Figure 2 (C).
Figure 6
Figure 6. TAT-C3 Treatment Inhibits Pore Formation, Actin Halos, and Translocation in HeLa Cells
Hela cells were treated for 4h with 10μg/ml, 20μg/ml, or 40μg/ml TAT-C3 in serum-free DMEM or with serum-free DMEM alone. Cells were infected with yopEHJ (YP27) or yopEHJB (YP29), in the presence or absence TAT-C3, and LDH released was tested after 3 hours, as described in Figure 1 (A). YopE and YopH translocation into Hela cells infected with wild type (YP126) or translocation-deficient yopB (YP18) strain, in the presence and absence of TAT-C3, was analyzed by immunoblotting, as described in Figure 2 (B). Cells seeded on coverslips were treated with 40μg/ml TAT-C3, or left untreated, and infected with yopEHJ (YP27) or yopEHJB (YP29). After 10 min infection cells were washed and fixed, and subjected to immunofluorescence. Actin was visualized by staining with Rhodamine–phalloidin. Images were acquired by confocal microscopy. Results were expressed as the percentage of bacteria inducing a halo of actin polymerization. A minimum of 250 bacteria was counted (C).
Figure 7
Figure 7. Rho Activation Requires YopB and High Affinity Interaction of the Bacteria with β1 Integrin
HeLa cells grown in 10cm diameter dishes were infected with yopEHJ (YP27) for 5, 10, 15, and 20 min. GTP-bound active Rho was pulled-down from cell lysates with a GST-fusion protein harboring the Rho binding domain of rhotekin. The precipitates were subjected to immunobloting using an anti-Rho monoclonal antibody. The amount of total Rho was determined in a 20μl aliquot (approx. 3%) of the cell lysates. Results were expressed in arbitrary units (AU) as the ratio between pulled-down GTP-Rho and total Rho (A). HeLa cells were left uninfected or were infected with yopEHJ (YP27), yopEHJB (YP29), yopEHJ,yadA,invD911E (YP50invD911E), and yopEHJB,yadA,invD911E (YP51invD911E) for 15min. The amount of GTP-Rho in each of the lysates was determined as described above (B).
Figure 8
Figure 8. Model for the Requirement of Rho Activation and Actin Polymerization for Pore Formation and Efficient Translocation
Upon binding of invasin or YadA to β1 integrin receptor, TTSS is activated, and YopB/D insert in the host cell plasma membrane (in cholesterol-rich domains present in the lipid rafts). YadA/invasin-mediated high affinity binding to β1 integrin receptor activates Rac, and Rho. Membrane-associated YopB/D, stimulates signaling that cooperates with β1 integrins to fully activate Rho. Actin polymerization, resulting from Rho activation, presumably induces lipid raft clustering at the site of the bacterial contact. More injectisomes can then interact with lipid rafts, and more effector Yops are translocated. As soon as enough Yops are translocated, the process is reverted by the inhibitory action of YopE and YopT on the Rho GTPases. Depicted are the inhibitory action on pore formation and translocation of an invasin mutant that binds to β1 integrin with low affinity (InvD911E), the Src inhibitor PP2, the RhoGTPases pan inhibitor ToxB, the specific Rho inhibitor C3, the specific Rac inhibitor NSC23766, and the actin polymerization inhibitor cytochalasin D.
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References

    1. Cornelis GR. Molecular and cell biology aspects of plague. Proc Natl Acad Sci U S A. 2000;97:8778–8783. - PMC - PubMed
    1. Viboud GI, Bliska JB. Yersinia outer proteins: role in modulation of host cell signaling responses and pathogenesis. Annu Rev Microbiol. 2005;59:69–89. - PubMed
    1. Barbieri JT, Riese MJ, Aktories K. Bacterial toxins that modify the actin cytoskeleton. Annu Rev Cell Dev Biol. 2002;18:315–344. - PubMed
    1. Black DS, Bliska JB. The RhoGAP activity of the Yersinia pseudotuberculosis cytotoxin YopE is required for antiphagocytic function and virulence. Mol Microbiol. 2000;37:515–527. - PubMed
    1. Von Pawel-Rammingen U, Telepnev MV, Schmidt G, Aktories K, Wolf-Watz H, et al. GAP activity of the yersinia YopE cytotoxin specifically targets the rho pathway: a mechanism for disruption of actin microfilament structure. Mol Microbiol. 2000;36:737–748. - PubMed

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