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A novel EspA-associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells
1998; Springer Nature; Volume: 17; Issue: 8 Linguagem: Inglês
10.1093/emboj/17.8.2166
ISSN1460-2075
AutoresStuart Knutton, Ilan Rosenshine, Mark J. Pallen, Israel Nisan, Bianca C. Neves, Christopher Bain, Carmel Wolff, Gordon Dougan, Gad Frankel,
Tópico(s)Enterobacteriaceae and Cronobacter Research
ResumoArticle15 April 1998free access A novel EspA-associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells Stuart Knutton Corresponding Author Stuart Knutton Institute of Child Health, University of Birmingham, Birmingham, B16 8ET UK Search for more papers by this author Ilan Rosenshine Ilan Rosenshine Departments of Molecular Genetics and Biotechnology, The Hebrew University, Faculty of Medicine, POB 12272, Jerusalem, 9112 Israel Search for more papers by this author Mark J. Pallen Mark J. Pallen Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Present address: Microbial Pathogenesis Research Group, Department of Medical Microbiology, St. Bartholomew's and the Royal London Schools of Medicine and Dentistry, London, EC1A 7BE UK Search for more papers by this author Israel Nisan Israel Nisan Departments of Molecular Genetics and Biotechnology, The Hebrew University, Faculty of Medicine, POB 12272, Jerusalem, 9112 Israel Department of Clinical Microbiology, The Hebrew University, Faculty of Medicine, POB 12272, Jerusalem, 9112 Israel Search for more papers by this author Bianca C. Neves Bianca C. Neves Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Present address: Departamento de Microbiologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo Av. Prof. Lineu Prestes, 1374, Sao Paulo-SP-Cep, 05508-900 Brazil Search for more papers by this author Christopher Bain Christopher Bain Institute of Child Health, University of Birmingham, Birmingham, B16 8ET UK Search for more papers by this author Carmel Wolff Carmel Wolff Departments of Molecular Genetics and Biotechnology, The Hebrew University, Faculty of Medicine, POB 12272, Jerusalem, 9112 Israel Search for more papers by this author Gordon Dougan Gordon Dougan Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Search for more papers by this author Gad Frankel Gad Frankel Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Search for more papers by this author Stuart Knutton Corresponding Author Stuart Knutton Institute of Child Health, University of Birmingham, Birmingham, B16 8ET UK Search for more papers by this author Ilan Rosenshine Ilan Rosenshine Departments of Molecular Genetics and Biotechnology, The Hebrew University, Faculty of Medicine, POB 12272, Jerusalem, 9112 Israel Search for more papers by this author Mark J. Pallen Mark J. Pallen Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Present address: Microbial Pathogenesis Research Group, Department of Medical Microbiology, St. Bartholomew's and the Royal London Schools of Medicine and Dentistry, London, EC1A 7BE UK Search for more papers by this author Israel Nisan Israel Nisan Departments of Molecular Genetics and Biotechnology, The Hebrew University, Faculty of Medicine, POB 12272, Jerusalem, 9112 Israel Department of Clinical Microbiology, The Hebrew University, Faculty of Medicine, POB 12272, Jerusalem, 9112 Israel Search for more papers by this author Bianca C. Neves Bianca C. Neves Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Present address: Departamento de Microbiologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo Av. Prof. Lineu Prestes, 1374, Sao Paulo-SP-Cep, 05508-900 Brazil Search for more papers by this author Christopher Bain Christopher Bain Institute of Child Health, University of Birmingham, Birmingham, B16 8ET UK Search for more papers by this author Carmel Wolff Carmel Wolff Departments of Molecular Genetics and Biotechnology, The Hebrew University, Faculty of Medicine, POB 12272, Jerusalem, 9112 Israel Search for more papers by this author Gordon Dougan Gordon Dougan Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Search for more papers by this author Gad Frankel Gad Frankel Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK Search for more papers by this author Author Information Stuart Knutton 1, Ilan Rosenshine2, Mark J. Pallen3,4, Israel Nisan2,5, Bianca C. Neves3,6, Christopher Bain1, Carmel Wolff2, Gordon Dougan3 and Gad Frankel3 1Institute of Child Health, University of Birmingham, Birmingham, B16 8ET UK 2Departments of Molecular Genetics and Biotechnology, The Hebrew University, Faculty of Medicine, POB 12272, Jerusalem, 9112 Israel 3Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AZ UK 4Present address: Microbial Pathogenesis Research Group, Department of Medical Microbiology, St. Bartholomew's and the Royal London Schools of Medicine and Dentistry, London, EC1A 7BE UK 5Department of Clinical Microbiology, The Hebrew University, Faculty of Medicine, POB 12272, Jerusalem, 9112 Israel 6Present address: Departamento de Microbiologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo Av. Prof. Lineu Prestes, 1374, Sao Paulo-SP-Cep, 05508-900 Brazil *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:2166-2176https://doi.org/10.1093/emboj/17.8.2166 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, employ a type III secretion system to deliver effector proteins across the bacterial cell. In EPEC, four proteins are known to be exported by a type III secretion system—EspA, EspB and EspD required for subversion of host cell signal transduction pathways and a translocated intimin receptor (Tir) protein (formerly Hp90) which is tyrosine-phosphorylated following transfer to the host cell to become a receptor for intimin-mediated intimate attachment and 'attaching and effacing' (A/E) lesion formation. The structural basis for protein translocation has yet to be fully elucidated for any type III secretion system. Here, we describe a novel EspA-containing filamentous organelle that is present on the bacterial surface during the early stage of A/E lesion formation, forms a physical bridge between the bacterium and the infected eukaryotic cell surface and is required for the translocation of EspB into infected epithelial cells. Introduction Enteropathogenic Escherichia coli (EPEC), an important cause of severe infantile diarrhoeal disease in many parts of the developing world, colonizes the small intestinal mucosa and, by subverting intestinal epithelial cell function, produces a characteristic histopathological feature known as the 'attaching and effacing' (A/E) lesion (Moon et al., 1983) and diarrhoea. The A/E lesion is characterized by localized destruction (effacement) of brush border microvilli, intimate attachment of the bacillus to the host cell membrane and the formation of an underlying pedestal-like structure in the host cell consisting of polymerized actin, α-actinin, ezrin, talin and myosin (Ulshen and Rollo, 1980; Knutton et al., 1987, 1989; Finlay et al., 1992); similar A/E lesions are produced by EPEC in a variety of tissue culture cell lines (Knutton et al., 1989). In vitro studies employing cultured epithelial cells and defined EPEC mutants support a three-stage model of A/E lesion formation: (i) initial non-intimate attachment; (ii) signal transduction and cytoskeletal rearrangements in host cells and (iii) intimate bacterial adhesion, actin accumulation and pedestal formation (Donnenberg et al., 1997). An emerging theme in the pathogenesis of bacterial infections is subversion by bacterial pathogens of host cell functions including signal transduction pathways and cytoskeletal organization (Cornelis and Wolf-Watz, 1997; Donnenberg et al., 1997; Finlay and Cossart, 1997; Finlay and Falkow, 1997). Like many other Gram-negative pathogens of animals (e.g. Yersinia, Salmonella and Shigella) and plants (e.g. Pseudomonas syringae, Erwinia amylovora, Ralstonia solanaceum and Xanthomonas campestris), EPEC also relies for its exploitation of host cell machinery on a complex specialized secretion system—a type III secretion system (Jarvis et al., 1995; Lee, 1997). The EPEC type III secretion system is part of a chromosomal pathogenicity island, designated the LEE (for locus for enterocyte effacement) which contains all the genes required to produce A/E lesions (McDaniel et al., 1995, 1997). The LEE also encodes intimin, an outer membrane protein adhesin, required for the intimate attachment of EPEC to host cells (Jerse et al., 1990; Frankel et al., 1994) and for the organization of polymerized actin into a cup-like pedestal beneath each attached bacterium (Donnenberg et al., 1997). Type III secretion systems have been shown to deliver effector proteins across the bacterial cell envelope and eukaryotic cell membrane to the host cell cytosol (Mecsas and Strauss, 1996; Lee, 1997). The components responsible for the secretion of proteins across the bacterial cell envelope are broadly conserved in all type III secretion systems, so that one type III secretion system can export proteins usually secreted by another type III system (Rosqvist et al., 1995). However, the proteins secreted across the bacterial cell wall vary from system to system: in Yersinia they are termed Yops (Yersinia outer-membrane proteins; Cornelis and Wolf-Watz, 1997), in Shigella flexneri Ipas (Invasion plasmid antigens) (Menard et al., 1996) and in Salmonella Sips and Sops (Salmonella inner- and outer- membrane proteins, respectively; Wood et al., 1996; Collazo and Galan, 1997). In EPEC, four proteins are known to be exported by a type III secretion system. Three EPEC-secreted proteins (or Esps), EspA, EspB and EspD (Donnenberg et al., 1993; Kenny et al., 1996; Lai et al., 1997), all of which are required for signal transduction in host cells, and a translocated intimin receptor (Tir) (formerly Hp90) which is tyrosine-phosphorylated following transfer to the host cell membrane to become the receptor for intimin-mediated intimate attachment and A/E lesion formation (Kenny et al., 1997). In this paper, we now show that one of the Esps, EspA, is a major component of a large extracellular filamentous appendage which appears on the bacterial surface prior to intimate EPEC attachment, forms a direct link between the bacterium and the host cell surface and is required for the translocation of another Esp, EspB, into infected host cells. Results Anti-EspA labels EPEC surface filamentous structures We used the His-tag system to produce recombinant EspA. The purified protein was used to raise a rabbit polyclonal EspA antiserum which was then used in immunofluorescence studies to stain the EPEC wild-type strain E2348/69 and its derivatives (Table I). We examined bacteria grown in broth- and tissue-culture media since expression of EPEC-associated virulence factors has been shown to be highest in mid-log growth phase in Dulbecco's modified Eagle's medium (DMEM) (Gomez-Duarte and Kaper, 1995; Tobe et al., 1996; Knutton et al., 1997). No labelling with the anti-EspA antibody was seen in overnight bacterial cultures grown in L-broth from any of the strains (data not shown). However, when the antiserum was applied to bacterial cells of the wild-type strain grown to mid-log phase in DMEM, numerous filamentous structures, ∼50 nm in diameter and up to 2μm long, were seen on the bacterial surface (Figure 1a). These structures were also produced by plasmid-cured E2348/69 strain JPN15 (Figure 1b), by an espB− strain UMD864 (Figure 1c) and by a tir− strain CVD463 (data not shown) but they were not detected on cells of the espA− strain UMD872 (Figure 1d). They were seen, however, when espA was re-introduced into UMD872 on a plasmid, to produce strain UMD872(pMSD2) (data not shown). The filamentous structures were also absent from the sepB− strain CVD452 (Figure 1f), which is deficient in the secretion of Esps (Jarvis et al., 1995). Although of uniform diameter, there was some variability in the length of filaments present on individual bacteria and among different strains. Compared with wild-type E2348/69, plasmid-cured JPN15 and espB− strain UMD864, an espD− strain, UMD870, produced structures that were very short (Figure 1e) whereas strain UMD870(pLCL123), where the espD gene is supplied in trans, produced filaments longer than those of the wild-type (data not shown). When observed in suspension culture by immunofluorescence, the filaments appeared to be rigid, but hinged at the bacterial surface, and were seen to wave about in the medium, thus making it difficult to obtain sharp fluorescence micrographs of these structures (Figure 1). Figure 1.Combined fluorescence/phase contrast micrographs showing EPEC strains stained with the EspA antiserum. The antiserum stained filamentous surface structures on wild-type strain E2348/69 (a), plasmid-cured strain, JPN15 (b) and the espB− strain, UMD864 (c), and very short filaments on the espD− strain, UMD870 (e). There was no staining of the espA− strain, UMD872 (d) or the sepB− strain, CVD452 (f). (Scale bar, 5 μm). Corresponding Western blots (g) showed similar levels of EspA in culture supernatants of strains E2348/69 (lane 1), JPN15 (lane 2), UMD864 (lane 3), reduced levels of EspA in culture supernatants of strain UMD870 (lane 5) and no EspA in culture supernatants of strain UMD872 (lane 4). Molecular weight markers are shown in thousands on the left. Download figure Download PowerPoint Table 1. List of strains and plasmids Description Reference Strain E2348/69 Wild-type Levine et al. (1985) JPN15 EAF-plasmid cured Jerse et al. (1990) UMD872 espA− Kenny et al. (1996) UMD870 espD− Lai et al. (1997) UMD864 espB− Donnenberg et al. (1993) CVD452 sepB− Jarvis et al. (1995) CVD463 tir− S.Elliott and J.B.Kaper (unpublished) Plasmid pMSD2 cloned espA Kenny et al. (1996) pMSD3 cloned espB Donnenberg et al. (1993) pLCL123 cloned espD Lai et al. (1997) The presence of EspA filaments correlated with levels of EspA detected in supernatants of mid-log phase culture from various EPEC-derived strains using the polyclonal antiserum and Western blots (Figure 1g). Levels of EspA similar to those produced by the wild-type were detected in supernatants from the plasmid-cured derivative JPN15, espB− strain, UMD864 and from the mutant strains complemented in trans with the relevant genes on recombinant plasmids (Table I); no EspA was detected in the supernatant from the espA− strain UMD872, and only low levels of EspA were secreted by the espD− strain, UMD870, which is in accordance with a previous report (Lai et al., 1997). Ultrastructure of EspA filaments To investigate the structure of the EspA filaments we examined bacteria grown to the mid-log phase in DMEM using negative staining and immunogold labelling electron microscopy. Anti-EspA-labelled filaments were seen when wild-type E2348/69 and plasmid-cured derivative JPN15 were examined by immunogold labelling (Figure 2a,b) but no such structures were seen on the espA− strain UMD872 (Figure 2c). Somewhat high background levels of gold labelling were seen routinely with strains secreting EspA but not with strains lacking EspA, suggesting that the antiserum is also staining unpolymerized EspA present in the bacterial suspension. In formalin-fixed preparations (Figure 2b) the filaments appeared as rigid cylindrical rods similar to those seen by immunofluorescence, whereas in unfixed preparations the filaments appeared somewhat collapsed and less rigid (Figure 2a). When combined with negative staining, the EspA filaments appeared to have a substructure and to be composed of smaller structures. This was most clearly seen in filaments present in culture supernatants (Figure 2d and e). The EspA antiserum stained aggregates of smaller, ∼7–8 nm diameter, pilus-like structures; filaments with three pilus structures typical of those illustrated in Figure 2d and e were the most common. EspA-associated filaments were not seen when cells of wild-type E2348/69 or other strains were examined by negative staining without immunolabelling, suggesting that these structures are stabilized by the antiserum. Figure 2.Electron micrographs showing EPEC strains immunogold labelled with the EspA antiserum. The antiserum stained filamentous surface structures on strains JPN15 (a) and E2348/69 (b) but there was no staining of the espA− strain, UMD872 (c). Negative staining revealed a substructure in these filaments (b) and, at higher magnification, filaments present in culture supernatants were seen to be composed of small bundles of thinner ∼7–8 nm diameter pilus-like structures (d and e). Many bacteria produced other ∼7 nm diameter fimbriae which did not stain with the antiserum (b, arrow). (Scale bars, 200 nm). Download figure Download PowerPoint Negative staining electron microscopy also revealed the structurally and antigenically distinct bundle-forming pili (bfp) produced by wild-type strain E2348/69 (not shown) and other rod-like fimbriae, ∼7 nm in diameter, produced by some E2348/69 and JPN15 bacteria that also did not stain with the EspA antiserum (Figure 2b, arrow); mannose-sensitive haemagglutination of bacterial cultures suggests that these are probably somatic type 1 fimbriae. EspA filaments interact with target cells To avoid complications due to bfp expression by wild-type E2348/69 and the formation of large three-dimensional bacterial microcolonies which obscure interaction of bacteria with the cell surface, we used the plasmid-cured derivative, JPN15, to investigate the possible interaction of the EspA-associated organelles with target cells. In tissue-culture cell-adhesion assays we routinely examined EPEC adhesion after 3 and 6 h incubation periods. Since the plasmid-cured derivative, JPN15, adheres less efficiently than the wild-type, we initially examined HEL cell adhesion assays after 6 h by immunofluorescence using the EspA antiserum. At this stage, we observed that most bacteria had formed A/E lesions (assessed by fluorescence actin staining; Knutton et al., 1989) but showed no or scant staining with the antibody. Since bacteria grown in tissue-culture medium express EspA filaments, we speculated that, in the absence of adhesins such as bfp, initial attachment of strain JPN15 might be weak, and that weakly adherent bacteria might be removed in the vigorous washing procedure of the assay. We therefore modified our adhesion assay to include a much gentler washing procedure. Immunofluorescence studies now revealed a large population of cell-adherent bacteria that had not formed A/E lesions, and these bacteria were covered with EspA filaments (Figure 3a); at higher magnification the filaments were seen to form a direct link between the bacterium and the HEL cell surface (Figure 3b). Similarly, when Hep-2 cell preparations infected with JPN15 were pre-incubated with the EspA antiserum and then examined by transmission electron microscopy, anti-EspA labelled filaments were seen forming a bridge between the bacteria and the eukaryotic cell surface (Figure 4a). Structures morphologically similar to EspA filaments produced by JPN15 were also seen by scanning electron microscopy, and in cell adhesion assays such structures also formed a bridge between bacteria and the eukaryotic cell surface (Figure 5a); no such structures were seen by scanning electron microscopy with the EspA deletion mutant (data not shown). Figure 3.Fluorescence micrographs showing adhesion of EPEC strain, JPN15, to cultured HEL cells. Bacteria were double stained for EspA (green) and for cellular actin (red). After a 5 h incubation and gentle washing, some bacteria had formed A/E lesions indicated by actin accumulation (a, arrowheads); the remainder of the cell surface was covered with bacteria that had not formed A/E lesions but were covered with EspA staining filaments. At higher magnification EspA staining filaments could be seen to bridge bacteria and the cell surface (b, arrows). (Scale bar, 5 μm). Download figure Download PowerPoint Figure 4.Transmission electron micrographs showing JPN15 immunogold-labelled with the EspA antiserum after a 3 h incubation with Hep-2 cells. Prior to A/E lesion formation, labelled filaments were seen to bridge bacteria and the Hep-2 cell surface (a, arrow). EspA staining filaments were eliminated from the site of intimate bacterial attachment and reduced EspA staining was seen following A/E lesion formation (b). (Scale bar, 200 nm). Download figure Download PowerPoint Figure 5.Scanning electron micrographs showing strain JPN15 adhering to HEL cells. Prior to A/E lesion formation, filamentous surface structures similar to those stained by the EspA antiserum can be seen on the bacterial surface and forming a bridge between bacteria and the HEL cell surface (a). Note that filaments which interact with the cell surface appear very rigid and to be embedded in the HEL cell membrane (a, arrows). Filaments can also be seen on the exposed surface of bacteria that have formed A/E lesions (b, arrow). (Scale bar, 0.25 μm). Download figure Download PowerPoint In a similar manner, using short (<1 h) incubation times, EspA filaments linking bacteria and the HEL cell surface were also demonstrated for wild-type strain E2348/69 (data not shown). EspA filament expression during A/E lesion formation After a 6 h incubation of cells with plasmid-cured strain JPN15 and thorough washing, the lack of EspA staining indicated that the EspA filaments were absent from mature A/E lesions. To characterize EspA filament expression by wild-type EPEC strain E2348/69 and its plasmid-cured derivative, JPN15, during A/E lesion formation we performed adhesion assays at 1 h intervals for 6 h and stained cells for EspA and for cellular actin (to assess A/E lesion formation). Several stages of EspA filament expression were identified although these were difficult to quantitate because of the dynamic nature of the adhesion process. Stage 1. EspA filaments were first expressed (in the absence of cells) when bacteria were grown in tissue culture medium (Figure 1a and b). Stage 2. Prior to A/E lesion formation, bacteria formed a non-intimate cell attachment, with EspA filaments forming a bridge between the bacterium and eukaryotic cell surface (Figures 3b, 4a, 5a and 6a). Figure 6.Fluorescence micrographs showing different stages of EspA filament expression during adhesion of strain JPN15 to cultured HEL cells. Bacteria were double stained for EspA (green) and for cellular actin (red). Initially bacteria formed a non-intimate attachment with EspA filaments covering the bacterial surface and bridging bacteria and the cell surface (a). When bacteria formed A/E lesions, EspA filaments were excluded from the region of intimate contact but were still present on the remainder of the bacterial surface (b, arrow). Staining of EspA filaments reduced gradually (c, arrow) until none was detectable in mature A/E lesions (d). (Scale bar, 5μm). Download figure Download PowerPoint Stage 3. Most wild-type E2348/69 and some plasmid-cured JPN15 bacteria progressed to form intimate cell attachment with associated actin accumulation. At this stage, EspA filaments were excluded from the site of intimate attachment but were present on the remainder of the bacterial surface (Figures 4b, 5b and 6b). Stage 4. Progressive loss of the remainder of the EspA filaments from the bacterial surface (Figure 6c) produced fully developed A/E lesions (often with extended pedestal structures) devoid of EspA filaments (Figure 6d). EspA is required for translocation of EspB into host cells Previous studies showed that both espA and espB mutants share a common phenotype, i.e. deficiency in subverting host cell signal transduction pathways and A/E lesion formation (Donnenberg et al., 1993; Kenny et al., 1996). Moreover, Lai et al. (1997) showed that secretion of EspB is not affected by an espA mutation. In this study, we have shown that EspA filaments did not stain with an EspB antiserum (data not shown). However, building upon our observation that the EspA filaments form a bridge between the incoming EPEC and the infected eukaryotic cells and a report by Wolff et al. (1998) that EspB is translocated into the host cell, we hypothesized that EspA filaments play a direct role in the translocation process. In order to test this, HeLa cells were infected with wild-type E2348/69, with the espA− strain UMD872, and with strain UMD872(pMSD2) where the espA gene is supplied in trans, and translocation of EspB was tested by immunofluorescence microscopy using the EspB antiserum. Zones of accumulated EspB were detected following permeabilization of E2348/69-infected HeLa cells (Figure 7C and D) but were not detected in non-permeabilized cells (Figure 7A and B); no EspB staining could be detected in the espA− strain UMD872-infected HeLa cells with or without permeabilization (Figure 7E and F) but staining was restored to permeabilized cells when espA was supplied in trans [strain UMD872(pMSD2)] (Figure 7G and H). Therefore, EspA filaments not only connect EPEC with the eukaryotic cell membrane, but are also required for translocation of EspB. Figure 7.EspA is required for EspB translocation. HeLa cells were infected for 3.5 h with E2348/69 and UMD872(pMSD2) or for 5 h with UMD872. The infected cells were fixed, permeabilized or not permeabilized and stained with the EspB antiserum. Zones of EspB accumulation were not detected in non-permeabilized HeLa cells infected with wildtype E2348/69 (A and B) but are evident in permeabilised HeLa cells infected with E2348/69 (C and D, arrows); zones of EspB accumulation were not detected in HeLa cells infected with the espA− strain UMD872 (E and F) but they were evident when espA was supplied in trans [strain UMD872(pMSD2)] (G and H) (×2000). Download figure Download PowerPoint Wolff et al. (1998) showed that translocated EspB can be found in association with the eukaryotic cell membrane and in the cytosolic fraction. In this study, we tested whether EspB could be detected in either fraction following infection with espA− strain, UMD872. Cytosolic and membrane (Triton X-100 soluble) fractions of E2348/69, as a control, and UMD872-infected HeLa cells were analysed for the presence of EspB by immunoblotting using the EspB antiserum. In addition, these fractions were examined for the presence of tyrosine-phosphorylated Tir using anti-phosphotyrosine antibody. Tyrosine-phosphorylated Tir is membrane-associated (Rosenshine et al., 1996a; Kenny et al., 1997), and thus served as an internal control for assessing the efficiency of the fractionation procedures. As reported by Wolff et al. (1998), in HeLa cells infected with E2348/69, Tir was found only in the membrane fraction, whereas EspB was found in both membrane and cytosolic fractions (Figure 8A). In contrast, neither Tir nor EspB were detected in either fraction of HeLa cells infected with espA− strain UMD872 (Figure 8A). Figure 8.EspA is required for EspB translocation. (A) HeLa cells were infected for 3.5 h with either E2348/69 or UMD872. The infected cells were fractionated into membrane and cytosolic fractions, and the fractions analysed by immunoblotting with the EspB antiserum or with anti-phosphotyrosine antibody. With E2348/69, EspB was localized to both membrane and cytosolic fractions, but EspB was not detected in either fraction of cells infected with the espA− strain UMD872; tyrosine phosphorylated Tir was localized solely in the membrane fraction. (B) Lack of translocation of EspB by UMD872 was not due to lack of secretion since comparable levels of EspB were found both in the medium and in free infecting bacteria. Download figure Download PowerPoint In order to quantify the relative levels of translocated EspB, Wolff et al. (1998) used the adenylate cyclase (AC) reporter system (Sory and Cornelis, 1994) and constructed a fusion between the espB gene and a DNA fragment encoding the catalytic domain of CyaA (EspB–CyaA fusion protein encoded by plasmid pEspB–CyaA). To further determine the importance of EspA filaments in the translocation of EspB, we compared the ability of 'activated' E2348/69 and espA− strain UMD872 to translocate the EspB–CyaA fusion protein into infected HeLa cells by measuring the level of intracellular [3H]cAMP. Compared with HeLa cells infected with E2348/69 as a control, infection for 1.5 h with E2348/69(pEspB–CyaA) led to a ∼143-fold increase in intracellular [3H]cAMP. In contrast, no increase in the level of [3H]cAMP was detected even after prolonged infection (3 h) of HeLa cells with espA− strain UMD872(pEspB–CyaA) (Figure 9A). In order to rule out the possibility that the absence of intracellular EspB is not due to reduced secretion of the fusion protein in the espA− strain background, we compared levels of extracellular EspB and EspB–CyaA produced by E2348/69 and UMD872. In agreement with a previous report (Lai et al., 1997), we found, using immunoblotting, similar levels of EspB between the two strains both in the growth medium and in free bacteria (Figure 8B). Secretion of EspB–CyaA by espA− strain UMD872 was reduced 7.6-fold compared with that of E2348/69(pEspB–CyaA), as determined by measuring AC enzymatic activity in the medium (Figure
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