Cellular Microbiology (2006) 8(9), 1504–1515 doi:10.1111/j.1462-5822.2006.00729.x First published online 2 May 2006 © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd Blackwell Publishing LtdOxford, UKCMICellular Microbiology 1462-5814© 2006 The Authors; Journal compilation © 2006 Blackwell Publishing Ltd ? 20068915041515Original ArticleRole of YopE and YopT during Yersinia infectionG. I. Viboud, E. Mejía and J. B. Bliska Received 11 October, 2005; revised 3 March, 2006; accepted 7 March, 2006. *For correspondence. E-mail gviboud@ms.cc.sunysb.edu; Tel. (+1) 631 632 4175; Fax (+1) 631 632 9797. Comparison of YopE and YopT activities in counteracting host signalling responses to Yersinia pseudotuberculosis infection Gloria I. Viboud,* Edison Mejía and James B. Bliska Department of Molecular Genetics and Microbiology, Center for Infectious Diseases, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-5222, USA. Summary Pathogenic Yersinia species share a type III secretion system that translocates Yop effector proteins into host cells to counteract signalling responses during infection. Two of these effectors, YopE and YopT, downregulate Rho GTPases by different mechanisms. Here, we investigate whether YopT and YopE are func- tionally redundant by dissecting the contribution of these two effectors to the pathogenesis of Yersinia pseudotuberculosis in a mouse infection and tissue culture model. Four days after oral infection, a YopE + T – strain and a YopE + T + strain colonized spleens of mice at similar levels, suggesting that YopT is not required for virulence. In contrast, spleen coloniza- tion by a YopE – T – strain was significantly reduced. A YopE – T + strain colonized spleen at levels comparable to those of the YopE + T – strain, arguing that YopT can promote virulence in the absence of YopE. Infection of HeLa cells with a YopE – T – H – J – strain expressing either YopE or YopT showed that YopE had a stronger antiphagocytic activity than YopT. Expression of YopE strongly inhibited activation of JNK, ERK and NF k B, and prevented production of IL-8; whereas YopT mod- erately inhibited these responses. On the other hand, pore formation was inhibited equally by YopE or YopT. In conclusion, YopE is a potent inhibitor of infection- induced signalling cascades, and YopT can only par- tially compensate for the loss of YopE. Introduction The type III secretion system (TTSS) is a virulence mech- anism required for the pathogenesis of several Gram- negative human pathogens, including Yersinia pestis , Yersinia pseudotuberculosis and Yersinia enterocolitica (Cornelis, 2002a; Ramamurthi and Schneewind, 2002; Viboud and Bliska, 2005). This secretion machinery is composed of structural proteins that form an internal basal body and a needle-like appendage, translocator proteins involved in the delivery of toxins into the host cell, and the toxin or effector proteins (Cornelis, 2002b). In the patho- genic Yersinia species, six effectors target different signal- ling molecules in the host cell to disarm the innate and adaptive immune system (Cornelis, 2002c; Juris et al ., 2002; Aepfelbacher, 2004; Viboud and Bliska, 2005). YopE, YopH, YopO and YopT target various signalling proteins to provide antiphagocytic or anti-inflammatory functions. YopJ thwarts the inflammatory response by inactivating the mitogen-activated protein kinase (MAPK) and NF κ B signalling pathways. This activity prevents production of cytokines and induces apoptosis in macrophages. YopM has been found in the nucleus (Skrzypek et al ., 1998; Benabdillah et al ., 2004) or in complex with two protein kinases in the cytoplasm (McDonald et al ., 2003). Its molecular pathogenic function remains unclear. The YopB, YopD and LcrV proteins are required for efficient delivery of effector Yops into the host cell. It is thought that YopB and YopD insert in the plasma mem- brane forming a channel through which the Yops are translocated (Rosqvist et al ., 1994; Sory and Cornelis, 1994; Persson et al ., 1995). Purified YopB and YopD have been shown to have pore-forming properties. They can insert into liposomes, and lipid-bound Yops can form ion- conducting channels in planar lipid membranes (Tardy et al ., 1999). Infection of cultured cells with Yersinia mutants lacking multiple effectors Yops (multi-Yop mutants) results in the formation of plasma membrane pores of an estimated size of 1.2–3.5 nm (Håkansson et al ., 1996). Infection of cultured cells with wild-type Yersinia strains, on the other hand, does not induce pore formation during the translocation process. It has been suggested that effectors prevent pore formation under these infection conditions either by filling the transloca- tion channels (Håkansson et al ., 1996; Marenne et al ., 2003) or by counteracting signals within the host cell that are required for pore formation (Viboud and Bliska, 2001).