University Department of Paediatric Gastroenterology, Royal Free Hospital, London NW3 2QG, UK1
Centro de Investigaciones Microbiológicas, Benemérita Universidad Autónoma de Puebla, Puebla, México2
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK3
Author for correspondence: Alan D. Phillips. Tel: +44 207 830 2783. Fax: +44 207 830 2146. e-mail: adphill{at}rfhsm.ac.uk
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ABSTRACT |
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Keywords: enteropathogenic Escherichia coli, intimin, cell culture, in vitro organ culture, microvilli
Abbreviations: A/E, attaching and effacing; Bfp, bundle-forming pilus; EAF, EPEC adherence factor; EPEC, enteropathogenic Escherichia coli; Esp, EPEC secreted protein; IVOC, in vitro organ culture; LA, localized adherence; LEE, locus of enterocyte effacement; MLP, microvillus-like processes; SEM, scanning electron microscopy; Tir, translocated intimin receptor
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INTRODUCTION |
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Several genes have been implicated in A/E lesion formation all of these map to a pathogenicity island termed the locus of enterocyte effacement or the LEE region (McDaniel et al., 1995 ). The LEE region encodes a type III secretion system (Jarvis et al., 1995
), with four associated EPEC secreted proteins [EspA, EspB, EspD and a translocated intimin receptor (Tir); Kenny et al., 1997
; Lai et al., 1997
; Donnenberg et al., 1993
; Kenny et al., 1996
; respectively], and an outer-membrane adhesin, intimin (Jerse et al., 1990
). Recently, several significant discoveries concerning EPEC pathogenesis have been reported (reviewed by Frankel et al., 1998
). Kenny et al. (1997)
showed that Tir is in fact an EPEC secreted protein which is encoded by the LEE region and translocated into the host cell from where it is incorporated into the surface membrane of infected host cells, whilst Knutton et al. (1998)
described a surface EPEC organelle, made predominantly from one of the EPEC secreted proteins, EspA, which not only bridges between EPEC and the target host cells but is also required for translocation of proteins, including EspB (Knutton et al., 1998
; Wolff et al., 1998
) and Tir (Kenny et al., 1997
).
The signal(s) and the mechanisms responsible for EPEC-induced cytoskeletal reorganization are currently unknown although Rac-, Rho- and Cdc42-dependent pathways and stimulation of the classical phospholipase C pathway leading to production of IP3 and release of calcium from IP3-sensitive intracellular stores do not appear to be involved (Bain et al., 1998 ; Ben-Ami et al., 1998
). Recent studies have highlighted the possible recruitment and activation of host cell factors involved in actin polymerization, i.e. WiskottAldrich syndrome proteins (WASP) and the Arp2/3 complex, in EPEC-induced pedestal formation (Kalman et al., 1999
). In addition, several other signal transduction pathways appear to be stimulated in epithelial cells following infection with EPEC, including phosphorylation of translocated Tir (Kenny et al., 1997
), intimin-dependent tyrosine phosphorylation of phospholipase C-
1 (Kenny & Finlay, 1995
), tyrosine phosphorylation of several other host cell proteins (Rosenshine et al., 1992
; Rosenshine & Finlay, 1993
), serine/threonine phosphorylation of myosin light chain (Manjarrez-Hernandez et al., 1996
) and dephosphorylation of a 240 kDa host cell protein (Kenny & Finlay, 1997
).
The first gene to be associated with A/E activity was the eae gene encoding the intimate EPEC adhesin, intimin (Jerse et al., 1990 ). We have established that there are four different intimin types (
, ß,
and
) using serological and molecular approaches (Adu-Bobie et al., 1998
). For intimin to mediate adherence to eukaryotic cells it must bind to a cell-surface receptor(s), and studying the intimin family of proteins, we showed that their cell-binding activity is localized to the C-terminal 280 amino acids (Int280) (Frankel et al., 1994
), that purified Int280 can bind to HEp-2 cells on its own (Frankel et al., 1995
, 1996b
), that a specific cysteine residue in EPEC intimin (Cys937) is essential for binding activity (Hicks et al., 1998
; Frankel et al., 1995
), that intimin is required for colonization of the mucosa and A/E lesion formation in a human intestinal organ culture model of infection (Hicks et al., 1998
), and that Int280 can bind to ß1-integrins (Frankel et al., 1996a
).
Recently, the global fold of Int280 in solution was determined by multidimensional NMR (Kelly et al., 1999 ). The structure shows that Int280 is approximately 90
in length and built from three globular domains: domain 1 (residues 191), domain 2 (residues 93181) and domain 3 (residues (183280). The first two domains resemble the type I set of the immunoglobulin super family (IgSF). The IgSF domains in intimin form an articulated linker that most likely extends away from the bacterial surface and confers a highly accessible third domain (D3) for potential interaction. Despite a lack of significant sequence homology, the topology in Int280D3 is reminiscent of the C-type lectins, a family of proteins responsible for cell-surface carbohydrate recognition. Modelling other intimin types (including the enterohaemorrhagic E. coli intimin
derivative) revealed similar structures which define a new family of bacterial adhesion molecules (Kelly et al., 1999
). We also reported that both Int280 and Int280C/S or Int280C/A (biologically inactive forms of Int280 in which Cys937 has been substituted with Ser or Ala, respectively) bind Tir and that Int280 but not Int280C/S binds to cells in the absence of Tir (Hartland et al., 1999
). Together, these data provide strong evidence that intimin interacts not only with Tir but also with a host cell intimin receptor.
The present study, performed on two different prototype EPEC strains (B171 and E2348/69) expressing intimin ß and , respectively, shows that EPEC induce transient proliferation and elongation of microvillus-like processes (MLP) on the eukaryotic cell surface. These structures appeared to enmesh and anchor bacteria to the cell surface. Elongation of microvilli around sites of A/E lesion formation has been described on infection of human intestine in vitro (Hicks et al., 1998
; Knutton et al., 1987b
), and this prompted further investigation of the phenomenon using in vitro cell culture of Caco-2 cells and organ culture of paediatric intestine. Further evidence is provided to show that intimin can bind to non-activated HEp-2 cells: (a) proliferation and elongation of MLP were evident on HEp-2 cells inoculated with Int280-coated plastic beads, and (b) HEp-2 cells responded to infection with UMD864 (intimin-positive, EspB-negative) by increasing the MLP network during early stages; later this developed into cage-like structures surrounding the bacteria. These results show that MLP production is an intimin-dependent event and that intimin can induce significant cytoskeletal reorganization, but not A/E lesion formation, in the absence of Esp-dependent signal transduction and intiminTir interaction.
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METHODS |
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Construction and purification of MBPInt fusion proteins, coating of Covaspheres, HEp-2 adherence assay and actin staining.
MBPInt280 and MBPInt280C/S (MBPInt280 in which Cys937 was replaced with Ser) fusion proteins were constructed, expressed and purified as previously described (Frankel et al., 1994 , 1995
). Polymer micro-spheres (Covaspheres MX; Duke Scientific) were coated with MBPInt280 and MBPInt280C/S fusion proteins according to the manufacturers instructions. For cell-binding assays, 10 µl of the coated Covasphere suspension were added to subconfluent HEp-2 cells in complete medium and the monolayers were incubated for 3 h before washing three times with PBS. Adherence of beads to the cells was visualized by SEM (Frankel et al., 1996b
). Fluorescent actin staining was performed as described by Knutton et al. (1989)
.
Paediatric intestinal in vitro organ culture (IVOC).
This was performed as described previously (Hicks et al., 1998 ). The experiments were maintained for 8 h with a change of tissue culture medium every 2 h and an uninoculated specimen was included with each experimental culture to act as a negative control. Tissue was obtained with fully informed parental consent and ethical approval. All intestinal histology was reported to be normal.
Strains B171 and E2348/69 were each examined in IVOC on three occasions using tissue from different children. The distal duodenum (two male, one female; aged 40, 109 and 120 months) and terminal ileum (one male aged 87 months) was sampled using grasp biopsy forceps during routine endoscopic (Olympus PCF paediatric endoscope) investigation of intestinal disorders. Endoscopically normal ileal resection margin was sampled from two children undergoing formation of an ileostomy (one male aged 190 months and one female aged 172 months). CVD206 (intimin-minus) and UMD864 (EspB-minus) were incubated in IVOC with one duodenal (male aged 24 months) and four ileal (males aged 48, 172 and 190 months, and one female aged 173 months) samples; E2348/69 was used as a positive control in four cases and B171 in one (ileum; aged 172 months). In addition, in two of the cases (males aged 24 and 190 months) CVD206 and UMD864 were co-incubated for the 8 h period. After the culture period specimens were washed thoroughly three times to remove any non-adherent bacteria and then prepared for SEM as described below.
SEM.
After the adherence assay, samples were fixed with 2·5% glutaraldehyde in 0·1 M sodium phosphate buffer, post-fixed in 1% aqueous osmium tetroxide and dehydrated in 2,2- dimethoxypropane. Specimens were transferred to absolute ethanol, critical-point-dried using liquid carbon dioxide in an Emitech K850 apparatus, coated with goldpalladium using a Polaron E5100 sputter coater, and viewed at 30 kV in a JEOL 5300 scanning electron microscope.
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RESULTS |
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Elongation of MLP is intimin-mediated
To study the contribution of individual EPEC virulence factors to the phenomenon of MLP elongation, the interaction of E2348/69 derivatives [strains CVD206 (intimin-minus), UMD864 (EspB-minus) and UMD872 (EspA-minus)] with HEp-2 cells was investigated using SEM (Fig. 3). HEp-2 cells infected with CVD206 for 1 (not shown), 2 (Fig. 3a
), 3 or 6 h (not shown) presented short MLP, similar to control cells, scattered on the cell surface. Small bacterial clusters were seen at 1 h, but larger and more numerous colonies were present at 2 and 3 h. In contrast, strains UMD864 (Fig. 3b
) and UMD872 (data not shown) both induced a profound MLP mobilization and polarization to the area of adherent bacteria. This effect was much more enhanced compared with HEp-2 cells infected with E2348/69. Moreover, the complexity of the MLP network increased in time and by 3 h post-infection the bacteria could be seen trapped within cage-like structures formed by the elongated MLP. These were confined to the area of adherent bacteria (Fig. 3b
). The surrounding cellular membrane presented MLP similar to uninfected control cells. After 6 h incubation, few UMD864 remained attached to HEp-2 cells. Nevertheless, extensive cage-like structures, sometime resembling membrane ruffles, but with few bacteria enmeshed, were present on the cell surface (Fig. 3c
). No debris-like material was observed. When espB was reintroduced into UMD864 on a plasmid, to produce strain UMD864(pMSD3), the phenotype returned towards that seen in the wild-type E2348/69, i.e. elongated MLP were present at early time points (1 and 2 h, data not shown), and were reduced in presence at 3 and 6 h, particularly at sites of A/E lesion formation (Fig. 3d
). UMD864 and UMD872 have been shown to be deficient in transducing Esp-dependent biochemical signals (Kenny et al., 1996
; Lai et al., 1997
) and translocation of Tir (Kenny et al., 1997
), which are required for A/E lesion formation although they still express intimin at a normal level (data not shown). Therefore, these results show that MLP elongation and cage formation is a function dependent upon intimin expression. Supporting this was the finding that incubating HEp-2 cells with Int280-coated, but not Int280C/S-coated, Covaspheres resulted in MLP elongation and mesh formation in association with adhering Covaspheres, in a binding pattern which resembled that of UMD864 (Fig. 4
).
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DISCUSSION |
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In the present investigation we have shown that HEp-2 cells respond to EPEC infection by producing and elongating MLP, which enmesh and appear to anchor the bacteria to the cell surface. In agreement with previous reports (Yamamoto et al., 1992 ; Silva et al., 1989
) we observed that MLP increase in number during initial stages of infection, and then elongate and mobilize around adhering and replicating bacteria. However, by 140180 min post-infection (once a typical bacterial cluster is formed and intimate contact is established), the cell surface neighbouring the LA cluster appears normal, without long MLP, as if they have decreased in numbers and returned to their pre-infected level. The presence of debris implies they degenerate rather than retract.
We demonstrate that, in agreement with previous observations (Knutton et al., 1987b ; Hicks et al., 1998
), microvillus elongation and degeneration is observed on EPEC infection of human intestine in IVOC. This suggests that the phenomena we observed on HEp-2 cells may be important in early events of intestinal colonization by EPEC. The bacterial strains CVD206 (intimin-minus) (Knutton et al., 1987b
, and this paper) and UMD864 (EspB-minus) did not adhere to human IVOC, so we are unable to evaluate their effect on the human intestinal brush border. However, the effects of various EPEC mutants could be evaluated on HEp-2 cells.
We also tested the effect of EPEC E2348/69 and various derived mutants on Caco-2 cells. In contrast to human intestine in IVOC and to HEp-2 cells, there was no evidence of microvillus elongation, either with E2348/69 or with UMD864, although a decrease in microvillus number was observed with CVD206 in a similar manner to E2348/69.
Using defined EPEC mutants which were defective in the production of specific virulence factors required for A/E lesion formation, it was possible to determine the function of these factors during MLP elongation on HEp-2 cells. CVD206, a strain unable to form intimate interactions with host cell membranes due to a deletion mutation in the eae gene, did not induce MLP elongation. In contrast, UMD864 and UMD872, EPEC derivatives deficient in Esp-dependent activation of host cell signal transduction pathways, induced cage-like structures of MLP origin at the site of bacterial adherence. Since UMD864 and UMD872 still express intimin at wild-type levels, they can be considered for practical considerations as intimin-coated particles. Indeed, Int280-coated beads not only induced MLP elongation, but were associated with the MLP in cage-like structures.
An important aspect of this study is the fact that MLP production is induced by intimin (both intimin from strain E2348/69 and intimin ß from strain B171), even in the absence of Esp-mediated cell signalling and translocation of Tir. This result supports observations that Int280 can, on its own, bind to HEp-2 cells (Hartland et al., 1999
; Frankel et al., 1995
, 1996b
), and suggests that intimin can bind to both Tir and to a receptor encoded by the host cell. Our results indicate intimin, by activation of host cell signalling, is the bacterial virulence factor responsible for the alterations in the MLP population during early stages of EPEC interaction with HEp-2 cells, whilst translocation of Tir and/or EspB, possibly in concert with other proteins, induces degeneration of MLP so that they return to pre-infected size and numbers. However, the nature of the signals involved in the MLP production and reduction is at present not known. It is worth noting that when the cell monolayers were infected with wild-type EPEC for 6 h, the adhering bacterial clusters grew to the extent of covering the entire cell surface. In contrast, at 6 h post-inoculation, only a few CVD206 and UMD864 bacteria remained associated with the HEp-2 cell monolayers. These results suggest that the ability to form A/E lesions and to intimately attach to the host cell enhances long-term bacteriahost cell interactions, which is consistent with our recent finding that intimate attachment is required for binding of EPEC to human intestinal cells in IVOC (Hicks et al., 1998
).
Following infection of HEp-2 cells with JPN15(pCVD450) extensive cage-like structures, similar to those induced during infection with UMD864, were observed 1 and 2 h post-infection, although only a few bacteria were seen adhering to the cell surface. In contrast, by 6 h post-infection larger numbers of bacteria forming A/E lesions were observed and no MLP were seen. The pattern of adhesion appeared diffuse and may reflect the absence of Bfp-related three-dimensional colony formation as seen with E2348/69 (Hicks et al., 1998 ). The results indicate that two sequential signal transduction pathways are activated during EPEC infection: intimin-mediated production of MLP, and MLP degeneration and A/E lesion formation.
Evidence is accumulating that intimin binds to more than one receptor (Hartland et al., 1999 ). Previously, we have shown that MBPInt280 binds immobilized ß1 integrins and can mediate integrin-dependent human CD4+ T cell adherence to MBPInt280-coated wells (Frankel et al., 1996a
). Wolff et al. (1998)
have shown that EspB is also translocated into HeLa cells and that intimin is needed for full translocation efficiency, whilst Kenny et al. (1997)
have shown that translocation of Tir is dependent on expression of EspB. The resemblance of the third domain of Int280 to C-type lectins (Kelly et al., 1999
), alternatively indicates that intimin may bind to a carbohydrate moiety. We have also reported that both Int280 and Int280C/S or Int280C/A (biologically inactive forms of Int280 in which Cys937 has been substituted with Ser or Ala, respectively) bind Tir and that Int280, but not Int280C/S, binds to cells in the absence of Tir (Hartland et al., 1999
). Together, these data provide strong evidence that intimin might have two binding activities, i.e. to Tir and to a host-cell receptor; these two binding activities may induce different signalling pathways. The identity of the host-cell intimin receptor is not yet known. Experiments testing the above hypotheses are under way.
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ACKNOWLEDGEMENTS |
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Alan D. Phillips and Jorgé Giròn contributed equally to this work.
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Received 20 December 1999;
revised 21 March 2000;
accepted 24 March 2000.