Báo cáo khoa học: Cytoskeleton-modulating effectors of enteropathogenic and enterohemorrhagicEscherichia coli: role of EspL2 in adherence and an alternative pathway for modulating cytoskeleton through Annexin A2 function pot

MINIREVIEW Cytoskeleton-modulating effectors of enteropathogenic and enterohemorrhagic Escherichia coli: role of EspL2 in adherence and an alternative pathway for modulating cytoskeleton through Annexin A2 function Toru Tobe Department of Microbiology and Immunology, Graduate School of Medicine, Osaka University, Japan Introduction Adherence to the surface of host epithelia is a first step in infection by enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic Escherichia coli (EPEC). EHEC and EPEC are capable of intimate adherence to the mucosal surface via a specific mechanism which is mediated by the interaction between host-surface- oriented translocated intimin receptor (Tir) and a bac- terial outer membrane protein intimin [1]. Tir is one of the effectors injected into host cells through a type III secretion system (T3SS) and modulates cellular func- tion [2]. Injected Tir is localized in the host-cell membrane and forms a receptor for intimin. Binding of intimin to the extracellular domain of Tir induces the cytoplasmic domain of Tir to recruit the cellular factors needed for actin polymerization [3]. By changing the F- actin-based cytoskeleton, the morphology of bacteria- adherent host cells is dramatically modified, leading to disruption of the microvilli and the formation of a pedestal-like structure beneath the adherent bacteria, which is called an attaching ⁄ effacing (A⁄ E) lesion [4]. In addition, the host cell’s cytoskeleton is targeted by other virulence factors, such as mitochondrial Keywords actin dynamics; A ⁄ E pathogen; colonization; lipid raft; membrane morphology; prophage; pseudopod-like structure; transcriptional regulation; type III secretion; virulence factor Correspondence T. Tobe, Department of Microbiology and Immunology, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan Fax: +81 6 6879 3309 Tel: +81 6 6879 3301 E-mail: torutobe@bact.med.osaka-u.ac.jp (Received 10 November 2009, revised 14 January 2010, accepted 3 February 2010) doi:10.1111/j.1742-4658.2010.07654.x Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic Escheri- chia coli (EPEC) are attaching ⁄ effacing pathogens that possess a type III secretion system and deliver a variety of effectors into host cells for suc- cessful infection. EHEC produces at least 20 effector families with various functions. Reorganization of the cellular cytoskeleton at the adherent site is a hallmark of these pathogens. EspL2 of EHEC is a novel effector class that can modulate the cellular cytoskeleton. By interacting with and activa- ting Annexin A2, EspL2 contributes to the formation of a condensed microcolony and may adhere to host cells in a translocated intimin recep- tor-independent manner. The interaction of EspL2 with Annexin A2 increases F-actin bundling activity and strengthens the membrane–cytoskel- eton linkage, resulting in the condensation of actin fibers and the induction of a pseudopod-like structure. Membrane microdomains, namely the lipid raft, which is rich in Annexin A2, may be a platform by which EHEC ⁄ EPEC infection modulates cellular signaling and the cytoskeleton. Abbreviations A ⁄ E, attaching ⁄ effacing; EHEC, enterohemorrhagic Escherichia coli; EPEC, enteropathogenic Escherichia coli; LEE, locus of enterocyte effacement; Map, mitochondrial associated protein; SpLE3, Sakai prophage-like element 3; T3SS, type III secretion system; Tir, translocated intimin receptor. FEBS Journal 277 (2010) 2403–2408 ª 2010 The Author Journal compilation ª 2010 FEBS 2403 associated protein (Map), EspH and EspB [5]. EHEC O157:H7 possesses > 20 types of effectors, the func- tions of many of which are yet to be fully determined. Among these, EspL2 is encoded by the espL2 gene on Sakai prophage-like element 3 (SpLE3) which also car- ries two other effector genes, nleB1 and nleE [6,7]. Interaction of EspL2 with Annexin A2 After the initial infection of epithelial cells, EHEC delivers bacterial proteins, effector proteins, into cells through the T3SS. Although EHEC O157:H7 Sakai possesses > 60 genes that show similarity with the amino acid sequences of known effectors, only 39 encode proteins that are secreted and translocated into host cells through T3SS; the others are either pseudog- enes or unexpressed genes [7]. EHEC O157:H7 Sakai harbors two genes encoding EspL family proteins (espL1 and espL2). Of these, EspL2 is secreted and delivered into host cells in a T3SS-dependent manner, whereas espL1 seems to be a dead gene because it does not produce any detectable product or transcript (T. Tobe, unpublished). Using FLAG-tagged EspL2 protein, T3SS-delivered EspL2 has been shown to localize to the cytosolic side of the plasma membrane. Adherence of EHEC induces an F-actin-rich structure beneath the sites of bacterial adherence which extends from the plasma membrane to the cytoplasm. EspL2 colocalizes with the F-actin structures although only at the membrane proximal part. Isolation of host proteins bound to EspL2 using affinity chromatography revealed that Annexin A2 (also known as annexin II) may be the target of EspL2 in host cells. Coprecipita- tion of Annexin A2 with EspL2 from a purified mix- ture of the two in vivo supported the interaction of both proteins and, furthermore, indicated a direct interaction between EspL2 and Annexin A2 [8]. Ann- exin A2 binds to the cytosolic surface of the cellular membrane and this has been linked to the formation or stabilization of actin assembly sites [9]. Localization of EspL2 at regions close to the membrane suggests that EspL2 interacts directly with Annexin A2 on the cellular membranes of infected cells. Morphological changes in the microcolony and surface of infected host cells Infection by an EHEC mutant strain lacking the espL2 gene showed little difference in terms of adherent capac- ity and the morphology of host cells compared with a wild-type strain. However, a microcolony of EHEC har- boring multiple copies of the espL2 gene has a highly condensed 3D structure and strongly induces extension of the host-cell membrane, forming pseudopod-like structures at the bacterial attachment sites [8]. Beneath the bacterial adherence site, intimate attachment of bacteria induces the formation of actin polymers and a pedestal-like structure sometimes forms. Even bacteria in the upper layer of the 3D colony are associated with F-actin and the plasma membrane, indicating that upward expansion of the bacterial colony is achieved by the extension of actin polymers. By contrast, a microcol- ony of EHEC espL2 mutant on epithelial cells is flat, spreading two-dimensioally on the cell surface, and bacterial density is lower than those formed by EHEC harboring multiple copies of the espL2 gene with spaces between bacteria (Fig. 1). Meanwhile, the wild-type EHEC strain forms a microcolony of intermediate phe- notype. In the microcolony of EHEC harboring multiple copies of the of espL2 gene, the host-cell membrane is extended to form filopodia or pseudopod-like structures among the bacteria in the microcolony. However, adher- ence of the EHEC espL2 mutant causes only a slight EHEC Tir Annexin A2 F-actin EspL2 EHEC Δ espL2 EHEC wild type Nucleus Fig. 1. Modulation of actin dynamism and membrane morphology by the interaction of EspL2 and Annexin A2. Attachment of EHEC induces actin reorganization beneath adherent sites via Tir-induced actin polymerization. Formation of a membrane microdomain, the lipid raft, is induced by EHEC microcolony formation, and lipid raft- associated protein Annexin A2 also accumulates at the adherent site. The interaction of EspL2 with Annexin A2 induces the conden- sation of F-actin, leading to the formation of a condensed bacterial colony and a pseudopod-like structure (right cell). By contrast, with- out EspL2, attachment of EHEC induces the formation of F-actin pedestals, but each pedestal is separated from others (left cell). EspL2 effector modulates cytoskeleton T. Tobe 2404 FEBS Journal 277 (2010) 2403–2408 ª 2010 The Author Journal compilation ª 2010 FEBS morphological change in the cell surface. The induction of a pseudopod-like structure is observed even with infection of an EHEC tir mutant harboring multiple copies of the espL2 gene, which does not form intimate attachment via Tir–intimin interaction and can not induce actin polymerization at the site of adherence. This clearly indicates that morphological changes in the cell membrane, possibly with changes in the cytoskele- ton, are induced by EspL2 without Tir-mediated actin polymerization. Other effectors have been shown to induce the forma- tion of filopodia or a pseudopod-like structure, but the induction is only transient before the formation of inti- mate attachment via the Tir–intimin interaction [10,11]. Filopodia are formed at an early stage of infection and later become dissipated when Tir is phosphorylated to begin recruiting the cellular factors involved in actin polymerization. Map is necessary for transient filopodia formation and EspH is involved in supporting Tir-med- iated actin polymerization [12,13]. Overexpression of Map in EPEC not only increases the number of micro- colonies with filopodia, but also prolongs the duration of filopodia [12]. Deletion of the espH gene results in enhanced filopodia formation [13]. These effectors seem to act only at the early stages of adherence. Therefore, it is unlikely that Map and ⁄ or EspH are necessary for the formation of a condensed microcolony with F-actin pedestals and induction of the morphological changes in the cell surface caused by EspL2. Moreover, pheno- types caused by EspL2, such as the formation of a pseu- dopod-like structure into a microcolony, were similarly observed with adherence of the tir mutant or the wild- type strain [8], suggesting that EspL2-induced pseudo- pod-like structure formation is distinct from Map ⁄ EspH-regulated formation of filopodia. Modification of activity of Annexin A2 by EspL2 Annexins are a family of proteins that bind to mem- brane phospholipids in a Ca 2+ -dependent manner. This property links Annexins to many cellular events related to the membrane, such as membrane–cytoskele- ton linkage, cell signaling, the assembly of certain membrane domains, endocytosis and ion fluxes across the membrane [14]. Annexin A2 binds directly and spe- cifically to phophatidylinositol (4,5)-bisphosphate, and this binding is responsible for recruiting the Annexin A2–p11 complex to the submembraneous actin-assem- bly site of EPEC-infected cells or Arf6-activated cells [15]. In EPEC-infected cells, Annexin A2 accumulates at sites of EPEC attachment where formation of actin- rich pedestals is induced [16]. It has been suggested that Annexin A2 at the cytoplasmic membrane surface beneath the EPEC adherent site plays a role in reorga- nization of the membrane ⁄ cytoskeleton following EHEC infection [16]. Notably, recruitment of Annex- in A2 to EPEC adherent sites is independent of Tir-induced actin polymerization, suggesting that Annexin A2 is not linked directly to actin pedestal formation [16]. In addition, Annexin A2 has been shown to interact directly with and bundle polymerized actin [17,18]. Therefore, it is most likely that Annex- in A2 at the EPEC adherent site links F-actin, most of which is polyerimzed by Tir-activated cellular factors, to the cytoplasmic membrane, affecting the morphology of the membrane and cytoskeleton. Morphological changes in the bacterial microcolony and cellular membrane at the bacterial adherence site of cells infected with EHEC harboring multiple copies of the espL2 gene or espL2 mutant are may be the result of membrane-associated cytoskeleton reorganization. F-actin pedestals beneath the microcolony of an EHEC strain with multiple copies of the espL2 gene also con- densed along with adherent bacteria. In addition, pseu- dopod-like structures are induced in infected cells. These observations are explained by activation of Annexin A2: strengthening the linkage between polymerized actins and reorganization of the cytoskeleton–membrane inter- action may result in the aggregation of actin pedestals and the induction of membrane protrusions (Fig. 1). An in vitro assay for the actin bundling activity of Annex- in A2 clearly indicated enhancement of Annexin A2 activity in the presence of EspL2 protein [8]. Further- more, depletion of Annexin A2 in EHEC-infected cells reduced the EspL2-associated phenotypes and resulted in the formation of a microcolony similar to that formed by the espL2 mutant, even with EHEC harboring multi- ple copies of the espL2 gene [8]. Consequently, it is possible that injected EspL2 interacts with Annexin A2, which is bound to the cytosolic plasma membrane, and enhances the activity of bundling actin filament and linking of the membrane to the cytoskeleton. Role of the membrane microdomain, the lipid raft, in EHEC ⁄ EPEC infection Adherence of EPEC ⁄ EHEC to epithelial cells is closely associated with the formation of a membrane microdo- main, called a lipid raft. Lipid rafts are enriched in cho- lesterol, sphingolipids and specific proteins that mediate a variety of cellular functions, such as cell sig- naling, cell adhesion, membrane trafficking, mem- brane–actin interactions and membrane-domain formation [19,20]. At EPEC-adherent loci, cholesterol and glycosyl phosphatydilinositol-anchored proteins T. Tobe EspL2 effector modulates cytoskeleton FEBS Journal 277 (2010) 2403–2408 ª 2010 The Author Journal compilation ª 2010 FEBS 2405 accumulate in a Tir-independent manner [16]. Annex- in A2 is a member of a group of proteins clustered at the lipid raft, and is accumulated at EPEC- and EHEC-adherent sites. Clustering of Annexin A2 at the EPEC-adherent site is independent of actin pedestal formation, indicating that actin reorganization induced by the Tir–intimin interaction is not necessary for Annexin A2 accumulation. Also, accumulation of Annexin A2 at EHEC-adherent sites has been shown to be independent of the espL2 gene [8]. Although Annexin A2 binds directly to phophatidylinositol (4,5)-bisphosphate, it is not clear whether Annexin A2 triggers the segregation of certain lipids or whether for- mation of the lipid raft induces Annexin A2 clustering. Although formation of a lipid raft is necessary to estab- lish intimate adherence by bundle-forming pili-deficient EPEC, a wild-type EPEC strain expressing bundle- forming pili or EHEC can form intimate adherence with actin pedestals even on cells treated with methyl- b-cyclodextrin, an inhibitor of lipid raft formation [21]. Moreover, actin pedestal formation by EHEC is observed in Annexin A2-depleted COS7 cells [8]. These results suggest that formation of a membrane microdo- main is enhanced by the adherence of EPEC ⁄ EHEC, but EPEC ⁄ EHEC are not required for the intimate adherence and reorganization of the actin cytoskeleton by Tir. Formation of a lipid raft micordomain may contribute to EPEC ⁄ EHEC adherence and assist in microcolony formation by creating a microenvironment that is preferable for reorganization of the cytoskeleton at the adherent site or by modulating cell signaling. Regulation and distribution of the espL gene family in EHEC ⁄ EPEC Orthologs of the EHEC O157:H7 Sakai espL2 gene have been found in all EHEC and EPEC strains exam- ined by hybridization or sequencing [22,23]. Further- more, nucleotide sequences surrounding the espL2 genes of many EHEC and EPEC strains, including EHEC O26:H-, EHEC O103:H2, rabbit EPEC O15:H- and EPEC O127:H6, are highly conserved (99–100% identity) and other two effector genes, nleB and nleE, are found downstream of the espL2 gene in these strains (NCBI database). EspL2 must be an essential type III effector for the pathogenicity of EHEC and EPEC. In addition, because the nucleotide sequences of espL2 orthologs in sequenced strains of EHEC and EPEC showed 99–100% similarity, it is likely that the espL2 gene was recently acquired by EHEC ⁄ EPEC or that the EspL2 protein is highly conserved among EHEC ⁄ EPEC strains. Part of the EspL2 amino acid sequence shows similarity with OspD3 ⁄ ShET ⁄ SenA of Shigella spp. OspD3 has been shown to be an effector protein secreted through the T3SS of Shigella flexneri [24], but the role of OspD3 in Shigella infection remains unknown. Although OspD3 ⁄ ShET ⁄ SenA was reported to be an enterotoxin that was secreted by E. coli K12 harboring the gene [25], EspL2 of EHEC does not show any cytotoxic activity (A. Miyahara & T. Tobe, unpublished). It is likely that EspL2 and OspD3 have the same origin and have evolved as pro- teins with different functions. espL2 nleB1 nleE nutrient starvation butyrate Environmental stimuli espJ tccP LEE Sp14 SpLE3 Pch Ler H-NS tir eae (intimin) map espH espB ler pchA Sp4 etc Fig. 2. Coordinated regulation of the espL2 gene on SpLE3, and LEE genes in EHEC O157:H7 Sakai. LEE genes are positively regulated by Ler, which is encoded in LEE. Transcription of the LEE1 operon, including the ler gene, is positively regulated by Pch, which is encoded by pchA on another prophage-like element Sp4. Effector genes located outside LEE are regulated by Pch (e.g. espL2, nleB1 and nleE on SpLE3) or by Ler with Pch (e.g. espJ and tccP on Sp14). All the genes in the Pch–Ler regulon are repressed by H-NS, but once expression of pchA and ler is stimulated by environmental signals, such as nutrient starvation or butyrate [27,28], transcription of the genes in the regulon are activated coordinately. EspL2 effector modulates cytoskeleton T. Tobe 2406 FEBS Journal 277 (2010) 2403–2408 ª 2010 The Author Journal compilation ª 2010 FEBS The espL2 gene is on prophage-like element SpLE3 in EHEC O157:H7 Sakai, and two other effector genes, nleB1 and nleE, are downstream of it on the same element. Expression of these three genes is coor- dinately regulated by locus of enterocyte effacement (LEE) genes through the action of Pch [26] (Fig. 2). LEE genes are composed of five operons and several cistrons. Transcription of LEE operons and genes is positively regulated by Ler, which is encoded by the LEE1 operon, transcription of which is positively reg- ulated by Pch regulators encoded on prophage-like elements. The espL2 gene belongs to a subgroup of the Pch–Ler regulon, expression of which is depen- dent on Pch but less so on Ler [26]. Indeed, a chro- matin immunoprecipitation assay showed that Pch, but not Ler, binds to the chromosomal loci around the espL2 gene [26]. Interestingly, the region contain- ing the espL2–nleB1–nleE genes is often found next to the LEE region. Consequently, espL2, together with nleB1 and nleE, may be closely associated with LEE genes in regulation and have roles in pathogenicity. Conclusions Modulation of the cellular cytoskeleton seems to be essential for establishing the tight adherence of EHEC and EPEC. A main contributor must be the Tir-medi- ated polymerization of actin beneath the adherent bacteria. In addition, EHEC ⁄ EPEC translocate several effectors that modulate organization of the actin fiber and cytoskeleton. EspL2 is one effector that can mod- ify membrane morphology and F-actin organization. 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Coprecipita- tion of Annexin A2 with EspL2 from a purified mix- ture of the two in vivo supported the interaction of both proteins and, furthermore, indicated a direct interaction between EspL2 and Annexin A2. condensed microcolony and may adhere to host cells in a translocated intimin recep- tor-independent manner. The interaction of EspL2 with Annexin A2 increases F-actin bundling activity and strengthens the membrane–cytoskel- eton