1 Institute of Child Health, University of Birmingham, Birmingham, UK
2 Division of Infectious Diseases, University of Maryland, Baltimore, MD, USA
3 Centre for Molecular Microbiology and Infection, Department of Biosciences, Imperial College, London, UK
Correspondence
Stuart Knutton
s.knutton{at}bham.ac.uk
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ABSTRACT |
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INTRODUCTION |
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BFP, encoded on a large 80 kb EPEC adherence factor plasmid (EAF) (Nataro et al., 1987b
) present in typical EPEC strains (Kaper, 1996
), have been shown to be important for EPEC pathogenicity (Bieber et al., 1998
), to be responsible for bacteriabacteria interaction and microcolony formation (Giron et al., 1991
) and also for dispersal of bacteria from microcolonies (Bieber et al., 1998
; Knutton et al., 1999
). Several studies have also implicated BFP in initial binding of EPEC to host epithelial cells (Donnenberg et al., 1992
; Giron et al., 1991
; Tobe & Sasakawa, 2001
, 2002
) although other studies which used human intestinal organ culture suggested that BFP were not involved in initial EPEC adherence but only at a later stage to promote microcolony formation (Hicks et al., 1998
).
Genes encoding A/E lesion formation are localized to the locus of enterocyte effacement pathogenicity island (LEE PAI) (McDaniel et al., 1995). The LEE encodes a TTSS, several secreted translocator and effector proteins and proteins involved in intimate bacterial attachment (Frankel et al., 1998
). One secreted translocator protein, EspA, forms a long filamentous extension to the TTSS needle complex (Daniell et al., 2001
; Sekiya et al., 2001
), whereas two other translocator proteins, EspB and EspD, are thought to provide a translocation pore in the host cell membrane, thereby allowing translocation of LEE effector proteins into the host cell cytosol (Frankel et al., 1998
). Since EspA filaments form a direct interaction with the host cell during early stages of A/E lesion formation (Knutton et al., 1998
), they could function as an initial EPEC attachment factor, particularly in the case of atypical EPEC which lacks EAF plasmids (Kaper, 1996
).
Intimin is the well characterized EPEC adhesin that promotes intimate attachment to host cells. However, to form an intimate attachment and A/E lesion formation, the LEE-encoded effector protein Tir (translocated intimin receptor) has to be translocated and inserted into the host cell membrane by the TTSS (Kenny et al., 1997); the intiminTir interaction triggers the assembly of actin into pedestals beneath intimately attached bacteria (Campellone & Leong, 2003
). Whilst Tir is a well characterized intimin receptor, additional uncharacterized host cell receptors have been implicated in intimin-mediated EPEC adhesion (Frankel et al., 2001
) and recently a specific host cell protein, nucleolin, was shown to bind a specific intimin subtype, intimin
, expressed by some EPEC and enterohaemorrhagic E. coli strains (Sinclair & O'Brien, 2002
).
The difficulty in assessing an adhesive role of BFP, EspA filaments and intimin when they are being expressed simultaneously in wild-type EPEC prompted us to generate a set of single, double and triple mutants in bfpA, espA and eae and to examine the role of these putative adhesins individually and in pairs. Human intestinal brush border cells provide a better model of EPEC infection than do undifferentiated epithelial cells of non-intestinal origin. Hence, in this study, we examined EPEC adhesion to Caco-2 brush border cells. The study supports a role for BFP and EspA filaments in initial brush border attachment of EPEC and intiminTir interaction in intimate EPEC adhesion and A/E lesion formation. The study found no evidence for other adhesins able to promote EPEC adhesion.
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METHODS |
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Infection of Caco-2 cells.
Caco-2 intestinal cells were grown in DMEM/20 % fetal calf serum on 24 mm diameter Transwell permeable culture supports for 12 days to ensure differentiation of the brush border. EPEC infections were performed in HEPES-buffered Modified Eagle's Medium (MEM)/2 % fetal calf serum. Cell monolayers were infected with an overnight LB culture of EPEC [10 µl broth culture (ml culture medium)-1] for up to 6 h at 37 °C; for long infections, the medium was changed after 3 h. In co-culture experiments, 5 µl broth culture (ml medium)-1 of each strain was used. At the end of the infection period, cells were washed three times in PBS for 1 min on an orbital shaker and fixed in either 4 % formalin (for immunofluorescence studies) or 3 % buffered glutaraldehyde [for scanning electron microscopy (SEM)]. In some experiments, a gentle washing procedure was used. In this case cells were washed three times for 20 s with gentle rocking by hand. Quantitative assessment of adhesion was made from two separate infections by counting numbers of bacteria in three representative microscope fields taken with a 63x objective lens. Adhesion (mean number of bacteria per field±SD) is expressed relative to that of E2348/69 (100 %).
Infection of human red blood cell (RBC) monolayers.
RBC monolayers were produced as described previously (Knutton et al., 2002). EPEC infections were performed in HEPES-buffered DMEM and cell monolayers infected with an overnight LB culture of EPEC [10 µl broth culture (ml culture medium)-1] for 3 h at 37 °C. After three washes in PBS, monolayers were fixed in 4 % formalin.
Infection of human intestinal mucosa.
Normal paediatric duodenal mucosal biopsies taken with informed consent were transported to the laboratory in ice-cold transport medium as described previously (Knutton et al., 1987) and used immediately. Bacteria grown for 3 h in DMEM express LEE genes and are primed ready to produce A/E lesions (Collington et al., 1998
). Primed bacteria were used to infect mucosal biopsies. Biopsies were placed in Bijoux bottles with 1·5 ml primed bacterial culture and 1·5 ml fresh culture medium (DMEM : NCTC135/10 % FCS) and the bottles placed on a rotary mixer inside an incubator for 1·5 h at 37 °C. Biopsies were washed three times in warm PBS on the rotary mixer and fixed in 3 % glutaraldehyde.
Antibodies and fluorescent stains.
Polyclonal rabbit or mouse antibodies to BFP (Knutton et al., 1999), EspA (Knutton et al., 1998
), intimin (Batchelor et al., 1999
), Tir (Hartland et al., 1999
) and H6 flagellin (Yona-Nadler et al., 2003
) were used for immunofluorescence staining in conjunction with Alexa488 (green) and Alexa594 (red) goat anti-rabbit IgG or goat anti-mouse IgG secondary antibody conjugates (Molecular Probes). In addition, bacteria were stained with propidium iodide (red) or DAPI (blue) (Molecular Probes), cell actin with fluorescein (green) or rhodamine (red) conjugated phalloidin (Sigma) and RBCs and the brush border membrane with rhodamine-conjugated wheat germ agglutinin (WGA) (Sigma).
Immunofluorescence microscopy.
To stain Tir and cytoskeletal actin, Caco-2 cells were permeabilized with PBS containing 0·1 % Triton X-100 for 5 min and washed three times in PBS prior to immunostaining. All antibody dilutions and immune reactions were carried out in PBS containing 0·2 % bovine serum albumin (PBS/BSA). Formalin-fixed, permeabilized and washed cells were incubated with antiserum (1 : 100) in PBS/BSA for 45 min at room temperature. After three 5 min washes in PBS, samples were stained with either Alexa488- or Alexa594-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Molecular Probes) diluted 1 : 100 in PBS/BSA for 45 min. Where appropriate, samples were simultaneously stained with propidium iodide to stain bacteria and/or phalloidin to stain cytoskeletal actin- or rhodamine-conjugated WGA to stain the brush border membrane. RBC samples were stained with rhodamine-conjugated WGA to stain the RBC membrane and with DAPI to stain bacteria. Preparations were washed a further three times in PBS and mounted in glycerol/PBS. HEp-2 and Caco-2 cell preparations were examined by confocal microscopy using a Leica TCS SP2 Spectral Confocal Microscope, equipped with an Argon (488 nm) and two Helium/Neon (543 nm, 633 nm) lasers and 40x or 63x PL APO objectives. Confocal illustrations show either image sections or image projections through the brush border region of the cell. In triple-stained specimens using Alexa488, Alexa594 and propidium iodide, the propidium iodide emission was artificially coloured blue to distinguish it from Alexa594 (red). RBC samples were examined by conventional epifluorescence microscopy using a Leica DMR microscope equipped with a 63x PL APO objective and a Leica DC200 digital camera.
SEM.
Glutaraldehyde-fixed Caco-2 cell monolayers and intestinal mucosal biopsy tissue were post-fixed in 1 % osmium tetroxide, dehydrated through graded alcohol solutions and critical-point-dried. Mounted specimens were sputter-coated with platinum (Polaron) and examined in a Philips XL30 scanning electron microscope (Knutton, 1995).
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RESULTS |
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Wild-type EPEC adhere to Caco-2 cells and form A/E lesions
Caco-2 cells grown on permeable filters for 12 days produce a polarized sheet of columnar epithelial cells with a well developed apical brush border surface that can be visualized by confocal microscopy after staining the microvillous membrane with WGA or the microvillous actin cytoskeleton with phalloidin (Fig. 1a, b) or visualized by SEM (Fig. 1c
). To examine initial EPEC attachment to Caco-2 cells, we examined cell monolayers at early time points (10 min) when single bacteria were first seen bound to the brush border surface (Fig. 2
a). At this stage, fluorescence staining of E2348/69 revealed expression of BFP fibrils and surface expression of intimin; EspA filaments were expressed by some but not all bacteria at this time point and they were frequently shorter than mature EspA filaments seen at later time points (Knutton et al., 1998
). The longer and more numerous BFP fibrils appeared to mediate bacterial attachment to the brush border surface (Fig. 2b
). Actin accretion, indicative of A/E lesion formation (Knutton et al., 1989
), was not observed at this stage. Adhesion of individual bacteria to Caco-2 cells was followed by bacterial aggregation giving rise first to small aggregates (Fig. 2c
) and later after 3 h to larger microcolonies (Fig. 2d
) typical of localized adherence seen with HEp-2 cells (Table 1
). All bacteria within microcolonies expressed BFP and BFP appeared to mediate bacteriabacteria aggregation (Fig. 2d, e
); actin accretion beneath bacteria in contact with the apical cell surface indicated that they had formed A/E lesions (Fig. 2f, g
). By 6 h only bacteria in contact with the cell surface that had produced A/E lesions were observed, indicating that the three-dimensional bacterial microcolonies had dispersed (Fig. 2g, h
), a phenomenon we demonstrated previously in E2348/69-infected HEp-2 cells (Knutton et al., 1999
). Since BFP, EspA filaments and intimin were expressed when EPEC first adhered to Caco-2 cells, we examined double deletion mutant strains UMD880, UMD886 and UMD883 (Table 1
) to assess the role in adhesion of BFP, EspA filaments and intimin independently.
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Intimin is known to bind Tir following its translocation and insertion into the host cell membrane; strain UMD883 lacks EspA filaments and thus a functional TTSS and so is unable to translocate Tir. To assess the adhesive properties of UMD883 to cells possessing translocated Tir, we performed co-culture infections with strain CVD206 or UMD886, which lack the intimin gene but which possess a functional TTSS and can translocate Tir. In co-culture with CVD206 (Fig. 5) or UMD886, strain UMD883, identified by surface intimin staining, was able to adhere intimately to Caco-2 cells and produced typical A/E lesions. However, the extent of UMD883 adhesion and A/E lesion formation was much more pronounced when co-cultured with CVD206 compared to UMD886, presumably due to the much more efficient adhesion of CVD206 (Table 1
). Focused Tir translocated by CVD206 (Fig. 5c
) or UMD886 was also detected beneath intimately attached UMD883 bacteria.
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Flagella expression was also examined during cell adhesion. Virtually all E2348/69 bacteria attaching initially to Caco-2 cells expressed flagella (Fig. 7a) but as bacteria formed microcolonies and produced A/E lesions (3 h) the percentage of flagellated bacteria was greatly reduced (Fig. 7b
); after 6 h few flagella were observed (Fig. 7c
). A similar decrease in the number of flagellate bacteria following initial attachment was seen with the other adherent strains (UMD880, UMD872, UMD901, CVD206). Strains UMD883 and UMD888 (Fig. 7d
) were non-adherent to Caco-2 cells but the presence of flagellate bacteria was confirmed by immunofluorescence of bacteria present in the culture medium (Fig. 7d
, inset).
EPEC adhesion to RBC monolayers
In recent studies we demonstrated EPEC adhesion to RBC monolayers, initially by EspA filaments and subsequently with strains able to translocate Tir, by intiminTir intimate interaction (Shaw et al., 2001, 2002
). In this study we also screened the collection of mutants in bfpA, espA and eae for their ability to adhere to RBC monolayers. As reported previously, wild-type E2348/69 adhered to RBC monolayers (Fig. 8
a) and induced a high level (
80 %) of haemolysis (Shaw et al., 2001
). Similar levels of RBC adhesion (and haemolysis) were produced by strains UMD886 (Fig. 8d
), UMD901 (Fig. 8f
) and CVD206 (Fig. 8h
), whereas strains UMD880 (Fig. 8b
), UMD883 (Fig. 8c
), UMD888 (Fig. 8e
) and UMD872 (Fig. 8g
) did not adhere to RBC monolayers and did not induce haemolysis. Thus, adhesion to RBCs was independent of BFP expression but correlated with expression of EspA filaments; under the assay conditions used, adhesion of the four positive strains was shown to be mediated by EspA filaments (data not shown).
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DISCUSSION |
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Several studies have implicated BFP in initial binding of EPEC to host cells. The initial studies of Giron et al. (1991) demonstrated a significant reduction in localized adherence of EPEC in the presence of BFP antibodies; residual adherence can now be explained by EspA filament and intiminTir adherence. Tobe & Sasakawa (2001)
implicated BFP in host cell attachment by demonstrating preferential binding of BFP, producing EPEC to the Caco-2 cell surface rather than to existing EPEC microcolonies formed during an earlier infection. These authors also concluded that differential EPEC adherence to cells of different species origin was due to BFP (Tobe & Sasakawa, 2002
). This present study, which examined EPEC strains at an early stage when they first adhere to cells, also supports such a cell adhesive role for BFP. Early rapid adhesion of individual bacteria correlated with the expression of BFP and, by immunofluorescence, BFP fibrils appeared to connect bacteria to the brush border surface. Subsequently, individual bacteria aggregated to produce small and then larger microcolonies with BFP appearing to link bacteria in the aggregates. Thus, these morphological data also support a direct role for BFP in initial cell attachment in addition to their recognized role in microcolony formation and dispersion. An adhesive role for BFP implies a specific host cell BFP receptor. Numerous candidate BFP receptors have been proposed, including a variety of oligosaccharides and, most recently, the phospholipid phosphatidylethanolamine (PE) (Nougayrede et al., 2003
). This study also confirmed our previous conclusion that EPEC adhesion to RBCs is independent of BFP (Shaw et al., 2001
) and indicates a BFP receptor that is lacking on the RBC surface. A PE receptor would be consistent with these RBC data because in the erythrocyte membrane PE is predominantly localized on the cytosolic side of the membrane.
A study by Hicks et al. (1998) employing in vitro intestinal organ culture to examine EPEC adhesion to human small intestinal mucosa concluded that BFP was not involved in initial EPEC adherence but only at a later stage to promote microcolony formation. This conclusion was based on the fact that strain JPN15, which lacks the large plasmid encoding BFP but expresses EspA and intimin, adhered to cultured paediatric small intestinal mucosa in two-dimensional microcolonies and produced A/E lesions whereas strain CVD206 (bfpA+ espA+ eae-) did not adhere. We also demonstrated previously A/E adhesion of a plasmid-cured derivative of E2348/69 to human intestinal mucosa and noted that the extent of adhesion was highly attenuated compared to that of the wild-type (Knutton et al., 1987
). This could now be explained by a lack of BFP-mediated initial attachment with initial attachment of the plasmid-cured strain being promoted by the much less efficient EspA filaments. In this study we examined early stages of EPEC adhesion to human intestinal mucosa and, in contrast to Hicks et al. (1998)
, we did observe adhesion of strain CVD206. CVD206, unlike E2348/69, lacks intimin and so cannot form an intiminTir intimate attachment and A/E lesions but it does express BFP and EspA filaments which could be promoting non-intimate adhesion. Although the CVD206 adhesins involved were not identified, the observation that CVD206 did adhere does cast doubt on the conclusions of Hicks et al. that BFP does not promote initial mucosal attachment of EPEC. Failure to detect mucosal adhesion of CVD206 by Hicks et al. may have been due to differences in the adhesion assay protocols used and/or to poor BFP expression by the CVD206 strain used. It was particularly noticeable in the study by Hicks et al. that in a 3 h HEp-2 cell adhesion assay they only demonstrated adhesion of individual CVD206 bacteria to the HEp-2 cell surface, whereas others had observed adhesion of large bacterial microcolonies by 3 h (Donnenberg & Kaper, 1991
; Knutton et al., 1999
).
EspA filaments are a component of the EPEC TTSS whose primary function is the delivery of virulence proteins into host cells (Daniell et al., 2001). However, since EspA filaments interact with host cells during the early stages of A/E lesion formation, it has been proposed that they may also function as adhesins. We showed previously that EspA filaments promoted attachment of strains lacking BFP to epithelial cells and to RBCs (Knutton et al., 1998
; Shaw et al., 2001
) and similar results supporting a role of EspA filaments as initial attachment factors were demonstrated with Shiga toxin-producing E. coli (Ebel et al., 1998
) which also lacks BFP. This present study also supports an adhesive role for EspA filaments in EPEC adhesion to brush border cells since adhesion correlated with EspA filament expression and, by immunofluorescence, EspA filaments appeared to connect bacteria to the brush border surface. However, adhesion was much less efficient than with strains expressing BFP. The extent of EspA-filament-mediated adhesion was dependent on the washing procedure used, suggesting that EspA-filament-mediated adhesion is weak compared to BFP and intiminTir-mediated adhesion where the washing procedure had no effect on levels of adhesion. Compared to BFP, the less efficient EspA-filament-mediated adhesion probably reflects the small number of EspA filaments produced (
12 EspA filaments per bacterium) (Daniell et al., 2001
) and the nature of their interaction with host cells which is currently unknown. EPEC have been divided into typical EPEC which possess EAF plasmids and BFP and atypical EPEC which lack EAF plasmids and BFP (Kaper, 1996
). The use of EspA filaments as initial attachment factors could explain why atypical EPEC is still able to colonize the gut and produce A/E lesions but, due to a lack of BFP, is a much less efficient colonizer than typical EPEC (Levine et al., 1985
).
Intimin expressed on the bacterial surface binds Tir following its translocation and insertion into the host cell membrane; intiminTir interaction produces the characteristic intimate EPEC attachment and triggers subsequent A/E lesion formation (Campellone & Leong, 2003; Frankel et al., 1998
). Strain UMD883 expressing only intimin did not adhere to Caco-2 cells although it was able to bind and form an intimate attachment and A/E lesions following translocation of Tir into the Caco-2 cell membrane. The adhesive role of intimin binding to translocated Tir is well established (Kenny et al., 1997
). However, there is also a considerable body of evidence for a host cell intimin receptor, not least of which is the binding of the cell-binding domain of intimin to epithelial cells in the absence of Tir (Frankel et al., 2001
) and the recent demonstration, in the case of E. coli O157 : H7, of a host cell surface protein, nucleolin, which specifically binds intimin
expressed by this serotype (Sinclair & O'Brien, 2002
). Although available evidence supports the presence of host cell intimin receptors, the inability of strain UMD883 to adhere to Caco-2 and HEp-2 cells in the absence of Tir clearly indicates the lack of a receptor able to support bacterial adhesion.
The present study examined three particular EPEC adhesive factors. However, other putative EPEC adhesive factors have been proposed, including other fimbrial antigens (Giron et al., 1993), Efa1/LifA (Badea et al., 2003
) and flagella (Giron et al., 2002
). Other than type 1 fimbriae, which have been demonstrated not to be involved in epithelial cell adhesion, strain E2348/69 has not been reported to produce other recognized fimbriae (Elliott & Kaper, 1997
). Efa1/LifA, a protein unique to EPEC (Klapproth et al., 2000
) and other A/E pathogens, appears to have cell-binding activity and might contribute to adhesion in some manner. However, the lack of adhesion of UMD888 (bfpA- espA- eae-) implies the absence of other adhesive factors sufficient to promote adhesion of EPEC to intestinal brush border cells. A direct role for flagella in EPEC adhesion was suggested recently (Giron et al., 2002
). We thought previously that adherent EPEC did not express flagella because we did not see flagella by SEM. However, it is now clear from the immunofluorescence staining performed as part of this study that adherent EPEC do possess flagella which could therefore play a role in adhesion. Nevertheless, this study found no direct evidence for an adhesive role for flagella since strains UMD883 (bfpA- espA- eae+) and UMD888 (bfpA- espA- eae-) produced flagella and yet were totally non-adherent. However, it is clear that EPEC flagella can interact with host cells and stimulate host immune responses (Zhou et al., 2003
) and thus a contribution to adhesion cannot be ruled out although this study indicates that flagella, in the absence of other adhesins, cannot by themselves support bacterial adhesion.
In summary, the results of this study, using defined EPEC E2348/69 mutants and Caco-2 brush border cells, are consistent with data obtained previously using undifferentiated epithelial cells of non-intestinal origin and indicate that BFP, EspA filaments and, in a Tir-dependent manner, intimin can each support bacterial adhesion to intestinal epithelial cells; no evidence was found for other adhesins which could support EPEC adhesion. BFP appeared to be the predominant initial attachment factor but, in the absence of BFP, EspA filaments were able to promote a less efficient initial bacterial attachment. Intimin functioned as an adhesin in the presence of translocated Tir but we found no evidence for an independent host cell intimin receptor capable of promoting EPEC adhesion.
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ACKNOWLEDGEMENTS |
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Received 28 August 2003;
revised 18 November 2003;
accepted 25 November 2003.
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