Yersinia Controls Type III Effector Delivery into Host Cells by Modulating Rho Activity

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Abstract
Yersinia pseudotuberculosis binds to β1 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 β1 integrins, but inefficiently transduces signals through the receptors, was defective for YopE translocation. Interfering with the β1 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 β1 integrin receptors. We propose that Rho activation, mediated by signals triggered by the YopB/YopD translocon and from engagement of β1 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. The type III secretion system (TTSS) is essential for the virulence of a number of Gram-negative human pathogens of enormous clinical significance. The molecular mechanisms by which TTSS effector proteins are translocated into the host cell are not well understood. The work presented here proposes a new model in which the enteropathogen Yersinia pseudotuberculosis manipulates the host cell machinery to control effector translocation. This involves activation of the host cell Rho GTPase by the cooperative action of adhesin-mediated high affinity binding to specific cell receptor molecules known as β1 integrins, and interaction of components of the TTSS with the host cell membrane. This molecular mechanism of controlling TTSS may not be restricted to Y. pseudotuberculosis and might take place during infection of host cells with other pathogens that encode homologues of Yersinia TTSS proteins. Our findings provide a good starting point to study the molecular nature of the complex interaction between bacterial pathogens bearing TTSSs and the host cell. Importantly, components that act by modulating the TTSS are potential targets for novel antimicrobials.