EspG of enteropathogenic and enterohemorrhagic E. coli binds the Golgi matrix protein GM130 and disrupts the Golgi structure and function Abigail Clements, 1 Katherine Smollett, 1 Sau Fung Lee, 2 Elizabeth L. Hartland, 2 Martin Lowe 3 and Gad Frankel 1 * 1 Centre for Molecular Microbiology and Infection, Imperial College, London SW7 2AZ, UK. 2 Department of Microbiology and Immunology, University of Melbourne, Australia. 3 Faculty of Life Sciences, University of Manchester, UK. Summary The enteric pathogens enteropathogenic Escheri- chia coli (EPEC), enterohaemorrhagic E. coli (EHEC) and Shigella flexneri all translocate at least one effector protein of the EspG protein family into host cells via a type III secretion system (T3SS). The EspG family comprises EspG, EspG2 and VirA. From a Y2H screen, we identified the Golgi matrix protein GM130 as a potential binding partner of EspG. We confirmed EspG:GM130 protein interaction by affinity co-purification. In co-immunoprecipitation experi- ments EspG was co-precipitated with GM130 while both GM130 and tubulins were co- precipitated with EspG. When expressed ectopi- cally in HeLa cells, the EspG protein family all localized to the Golgi and induced fragmentation of the Golgi apparatus. All EspG family proteins were also able to disrupt protein secretion to a greater extent than the T3SS effector NleA/EspI, which has previously been shown to localize to the Golgi and interact with SEC24 to disrupt COPII vesicle formation. We hypothesize that EspG:GM130 interaction disrupts protein secre- tion either through direct disruption of GM130 function or through recruitment of other EspG interacting proteins to the Golgi. Introduction Many important bacterial pathogens rely on secretion systems to target bacterial proteins for delivery into the host cell [reviewed in (Hayes et al., 2010)]. Examples include the gastrointestinal pathogens enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli (EHEC) [reviewed in (Croxen and Finlay, 2010)] and the closely related Shigella spp. [reviewed in (Ray et al., 2009)], which all utilize a type III secretion system (T3SS) to translocate bacterial ‘effector’ proteins into the host cell (Hayes et al., 2010). Once inside the host cell, effector proteins manipulate host cell processes in order to create a favourable environment to replicate and survive. Genome sequencing of these pathogens has enabled their effector repertoires to be predicted and then confirmed experimentally. The prototype EPEC strain E2348/69 has 21 predicted effector proteins (Iguchi et al., 2009), EHEC O157:H7 has 49 predicted effector proteins (Tobe et al., 2006) and Shigella has approximately 25 T3SS effectors (Parsot, 2009). Within this large repertoire of proteins a single effector protein can manipulate a number of different cellular processes, for example the EPEC effector NleH can inhibit apoptotic pathways (Hem- rajani et al., 2010) or NF-kB signalling (Gao et al., 2009) depending on the host cell partner protein they bind. It also appears that more than one effector can manipulate the same host process, for example in EPEC, EspF and Map both induce apoptosis by permeabilizing the outer mitochondrial membrane (Kenny and Jepson, 2000; Nou- gayrede and Donnenberg, 2004) and NleE and NleC inhibit activation of NF-kB (Newton et al., 2010; Pearson et al., 2010; Vossenkamper et al., 2010; Yen et al., 2010). Therefore, within an infected cell there is a highly compli- cated and regulated reorganization of host processes that occurs because of the presence of the bacterial effector proteins. One effector group shared among EPEC, EHEC and Shigella is the EspG protein family, which consists of EspG, EspG2 and VirA (Elliott et al., 2001). EPEC EspG and EHEC EspG are 98% identical. Some EPEC strains also encode a second EspG protein called EspG2 (Elliott et al., 2001; Smollett et al., 2006), which shares 43% Received 10 February, 2011; revised 13 May, 2011; accepted 17 May, 2011. *For correspondence. E-mail g.frankel@imperial.ac.uk; Tel. (+44) 20 7594 5253; Fax (+44) 20 7594 3069. Cellular Microbiology (2011) 13(9), 1429–1439 doi:10.1111/j.1462-5822.2011.01631.x First published online 11 July 2011 © 2011 Blackwell Publishing Ltd cellular microbiology