Institute of Child Health, University of Birmingham, Birmingham B4 6NH, UK1
Centre for Molecular Microbiology and Immunology, Department of Biosciences, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK2
Author for correspondence: Stuart Knutton. Tel: +44 121 333 8746. Fax: +44 121 333 8701. e-mail: s.knutton{at}bham.ac.uk
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ABSTRACT |
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Keywords: type III secretion, adhesion, cellcell interactions, EPEC
Abbreviations: A/E, attaching and effacing; DAPI, diamidino-2-phenylindole; EPEC, enteropathogenic Escherichia coli; RBC, red blood cell
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INTRODUCTION |
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Tir translocation into host cells requires a chaperone, CesT (Elliott et al., 1999 ), the LEE type III secretion system and type III secreted proteins EspA, EspB and EspD (Frankel et al., 1998
). EspB (Wolff et al., 1998
) and EspD (Wachter et al., 1999
) have been shown to be delivered to the host-cell membrane and, based on homology with pore-forming proteins of Shigella (IpaB/IpaC) and Yersinia (YopB/YopD), are proposed to form a pore complex in the host-cell membrane (Frankel et al., 1998
). EspA, on the other hand, is the major component of a large filamentous structure (Knutton et al., 1998
) proposed to provide a hollow conduit for protein translocation between pores in the bacterial membrane (type III secretion system) and pores in the host cell (EspB/EspD-generated pore) (Frankel et al., 1998
).
Injection of proteins into host cells by Shigella and Yersinia spp. has been correlated with their ability to cause contact-dependent haemolysis of red blood cells (RBCs) in vitro in which bacteria are brought into close contact with the RBC membrane by centrifugation (Clerc et al., 1986 ; Hakansson et al., 1996
); EPEC were also recently shown to exhibit contact-dependent haemolytic activity (Warawa et al., 1999
). However, because long EspA filaments mediate binding of EPEC to RBCs, we showed that close bacterialRBC contact is not a requirement for EPEC-induced haemolysis (Shaw et al., 2001
). Haemolysis was consistent with insertion of a hydrophilic pore into the RBC membrane and, following haemolysis, EspD was associated with the RBC membrane. However, by Western blotting we were unable to detect RBC membrane-associated Tir. As several fundamental questions regarding Tir translocation (e.g. the means by which a soluble bacterial protein becomes an integral membrane protein receptor, the mechanism by which Tir becomes focused beneath attached bacteria and the requirements for Tir modification for intimate Tirintimin interaction) remain unanswered, we have continued to develop this model of type III protein translocation. Here we show that the EPEC type III secretion system can efficiently translocate and correctly insert native Tir into the RBC membrane where it binds intimin and forms an intimate bacterial attachment typical of that seen in A/E lesions.
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METHODS |
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Antibodies and fluorescent stains.
Polyclonal rabbit antibody to the N-terminal region (TirN) or intimin-binding domain of Tir (TirM) (Hartland et al., 1999 ) were used for microscopical studies; a Tir mAb was used for Western blotting studies (Hartland et al., 1999
). Polyclonal rabbit antibody to EspA was used to stain EspA filaments (Knutton et al., 1998
). Polyclonal mouse antibody to the cell-binding domain of intimin (Int280) was used to stain intimin (Hartland et al., 1999
). G410 tyrosine phosphate mAb (Upstate Technology) was used to examine Tir tyrosine phosphorylation. Rhodamine-conjugated wheat germ agglutinin (Sigma) was used to stain the RBC membrane (red). FITC-conjugated phalloidin (Sigma) was used to stain cytoskeletal actin (green). 4,6-Diamidino-2-phenylindole (DAPI; Molecular Probes) was used to stain bacterial DNA (blue). Fluorescent second antibodies used were Alexa488- (green) and Alexa594- (red) conjugated goat anti-rabbit IgG or goat anti-mouse IgG conjugates (Molecular Probes).
Immunofluorescence microscopy.
To stain Tir with the TirN and phosphotyrosine antibodies, and RBC cytoskeletal actin, RBCs were permeabilized with PBS containing 0·1% Triton X-100 for 5 min and washed three times in PBS. All antibody dilutions and immune reactions were carried out in PBS containing 0·2% bovine serum albumin (PBS-BSA). Formalin-fixed, permeabilized and washed RBC monolayers were incubated with antiserum (generally 1:501:100) 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 1:50 in PBS-BSA for 45 min; in most experiments samples were simultaneously stained with rhodamine-conjugated wheat germ agglutinin to visualize the RBC membrane, and with DAPI to stain bacteria. Preparations were washed a further three times in PBS, mounted in glycerol-PBS and examined by incident light fluorescence using a Leica DMR microscope equipped with a DC200 digital camera. In multi-labelling studies individual red (Alexa594, rhodamine), green (Alexa488) and/or blue (DAPI) images were superimposed using Leica IM1000 software. Confocal microscopy was performed using a Leica TCS SP2 Spectral Confocal Microscope.
Electron microscopy.
For immunogold Tir and intimin labelling, RBC monolayers on plastic culture dishes were briefly fixed for 10 min in 0·1% glutaraldehyde, washed three times with PBS, permeabilized in 0·1% Triton X-100 for 5 min and washed a further three times in PBS. Monolayers were incubated with TirN and intimin antisera (1:100) for 4 h at room temperature, washed three times in PBS-BSA and incubated with 5 nm gold-labelled goat anti-rabbit serum and 10 nm gold-labelled goat anti-mouse serum (British Biocell) for 12 h at 4 °C. After further thorough washing, cells were scraped from the dish, pelleted, fixed in 3% buffered glutaraldehyde and processed for thin-section electron microscopy using standard procedures (Knutton, 1995 ). Ultrathin sections were examined in a JEOL 1200EX electron microscope operated at 80 kV.
For scanning electron microscopy EPEC-infected RBC monolayers prepared on glass cover slips were fixed with 3% glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated through graded alcohol solutions and critical-point-dried. For immunogold labelling, monolayers were briefly fixed for 10 min in 0·1% glutaraldehyde, washed, incubated with intimin antiserum (1:100) for 4 h at room temperature, washed again and incubated with 30 nm gold-labelled goat anti-mouse serum (1:100) for 12 h. After further thorough washing, cells were fixed in 3% buffered glutaraldehyde and processed as described above. Mounted specimens were sputter-coated with platinum (Polaron) and examined in a JEOL 1200EX Scanning Transmission Electron Microscope.
RBC membrane preparation.
The method for membrane isolation was adapted from Blocker et al. (1999) . RBCs were sedimented, washed three times in PBS and resuspended in PBS at approximately 5x108 cells ml-1. Washed RBCs (2 ml) were then mixed with 0·4 ml UMD872 and CVD206 in a mixed infection, and 0·4 ml CVD456 as a negative control. PBS (2·4 ml) was added to each infection and the infections incubated at 37 °C for 6 h. Distilled water (0·8 ml) was then added to obtain equal lysis of each infection. The samples were vortexed and spun to pellet any unlysed RBCs. Supernatant (3·4 ml) was then mixed with 6 ml 72% sucrose and transferred to SW40 centrifuge tubes. The mixtures were then overlaid with 2 ml 42% sucrose and 1 ml 25% sucrose, and the sucrose cushions were spun at 15000 g for 16 h. The RBC membranes were collected from the interface between the 42 and 25% sucrose layers and spun at 450000 g for 30 min at 4 °C. The pellets were resuspended in 15 µl SDS gel loading buffer.
E2348/69 whole-cell lysate preparation.
An overnight culture of E2348/69 was diluted 1:100 into DMEM and grown with shaking at 37 °C for 4 h. A sample (1·5 ml) of culture was spun in a microfuge (15000 g) for 5 min to pellet the bacteria. The supernatant was discarded and the bacterial cell pellet was resuspended in 100 µl gel loading buffer.
Immunoblotting.
A sample (15 µl) of the RBC membrane preparation from the UMD872/CVD206 infection, together with 15 µl whole-cell lysate from E2348/69 were separated by SDS-PAGE on a 12% gel run for 1 h at 180 V in a Bio-Rad mini-PROTEAN II cell. Proteins were transferred to a nitrocellulose membrane using a Bio-Rad mini-trans blot apparatus run for 45 min at 100 V. The membrane was blocked with 10% skimmed milk in PBS, 0·05% Tween-20 for 1 h and then washed four times in PBS-Tween. It was then incubated overnight at 4 °C with anti-Tir mAb diluted 1:100 in PBS-Tween (Hartland et al., 1999 ). The blots were washed and detected with anti-mouse alkaline-phosphatase-conjugated antibody as described previously (Knutton et al., 1998
).
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RESULTS |
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To detect Tir in this study we used fluorescence microscopy and an antibody to the N-terminal region of the Tir molecule (TirN antibody), a part of the molecule that is exposed, following translocation, on the cytoplasmic face of the plasma membrane (Hartland et al., 1999 ). Examination of RBC monolayers infected with wild-type EPEC strain E2348/69 for 4 h revealed, in a small number of RBCs, thin bands of fluorescence staining reminiscent of Tir staining seen in typical A/E lesions (Fig. 1a
). Double staining for Tir and intimin confirmed that such fluorescence was indeed Tir that was focused beneath bacteria expressing surface intimin (Fig. 1b
); co-localization of Tir and intimin, and Tir and the RBC membrane demonstrated by confocal microscopy (Fig. 1c
) was consistent with Tir translocation to the RBC membrane and intiminTir intimate attachment. By fluorescence microscopy we were unable to detect unfocused Tir in E2348/69-infected RBC membranes (data not shown). However, a general bacterial stain revealed many adherent bacteria most of which were not associated with focused Tir and, significantly, did not express surface intimin; attachment of these intimin-negative bacteria to the RBCs was mediated, as described previously (Shaw et al., 2001
), by EspA filaments (Fig. 1d
). These results demonstrate that EPEC can translocate Tir into the RBC membrane.
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Optimization of EPEC infection of RBCs
Tir and intimin are believed to be expressed from a single operon (tir) (Mellies et al., 1999 ). Nevertheless, the co-infection studies showed that wild-type E2348/69 could translocate Tir efficiently into the RBC membrane (Fig. 3c
), even in the absence of detectable surface intimin expression (Fig. 1d
). This, therefore, is the most likely explanation for the low level of intimate attachment initially observed (Fig. 1b
). Routinely, we grew bacteria in Luria broth from frozen stock cultures. Since such cultures yielded very low levels of intimate bacterial attachment we investigated other growth conditions to try to modulate intimin expression. Interestingly, serial passaging of E2348/69 in Luria broth for several days resulted in intimin being expressed on the surface of all the RBC-adherent bacteria (Fig. 3d
, inset). Now a 4 h infection of RBCs with E2348/69 that had been serially passaged daily for 4 days yielded levels of intimate intiminTir attachment comparable to those obtained during the co-infection experiments with most RBCs covered with intimately attached bacteria (Fig. 3d
).
Using these optimized infection conditions, examination of defined E2348/69 mutants showed that Tir translocation into the RBC membrane, as for epithelial cell membranes, was dependent on the Tir chaperone, CesT, on a functional type III secretion apparatus and on secreted proteins EspA and EspD; surface intimin expression was required to focus Tir (Table 1).
Translocated Tir does not undergo host-cell modification
In EPEC-infected epithelial cells translocated Tir becomes phosphorylated on tyrosine residues (Rosenshine et al., 1992 ), an event essential for the subsequent interaction with the host-cell cytoskeleton and A/E lesion formation (Kenny, 1999
). In RBCs we were able to detect focused Tir using the TirN antibody and fluorescence staining (Fig. 5a
), but using a tyrosine phosphate antibody we were unable to detect tyrosine phosphorylation of translocated and focused Tir (Fig. 5b
); in contrast, using the same antibody, tyrosine-phosphorylated Tir focused beneath intimately attached bacteria was readily detected in E2348/69-infected epithelial (HEL) cells (Fig. 5c
).
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By Western blotting we were previously unable to detect translocated Tir (Shaw et al., 2001 ). In this study we performed Western blots using a Tir mAb on wild-type EPEC cell lysates and following a 4 h co-culture infection of RBCs with strains CVD206 and UMD872. Tir with an apparent molecular mass of
78 kDa was detected both in bacteria and in fractionated RBC membranes (Fig. 6
); no RBC membrane-associated Tir was detected in a control co-infection using the type III secretion mutant CVD452 and UMD872, whilst Tir detected in infected Hep-2 cell membranes migrated with an apparent molecular mass of 85 kDa, as reported by Kenny (1999)
and Kenny & Finlay (1997)
(data not shown).
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DISCUSSION |
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During A/E lesion formation Tir is translocated to the host-cell membrane by the LEE type III secretion apparatus where it acts as a receptor for intimin; intiminTir binding results in Tir becoming focused beneath intimately attached bacteria (Rosenshine et al., 1996 ). We have proposed a model for the EPEC type III protein translocation apparatus used to translocate Tir which consists of pores in the bacterial and host-cell membranes connected by a hollow EspA filament (Frankel et al., 1998
), thereby providing a continuous channel from the bacterial to the host-cell cytosol. Such a model is consistent with our previously observed interaction of EPEC with RBCs (Shaw et al., 2001
) which demonstrated EspA-filament-mediated interaction of EPEC with the RBC membrane, RBC haemolysis and localization of EspD, a putative pore-forming type III secreted protein, to the RBC membrane. However, our inability to demonstrate membrane-associated Tir suggested that effector protein translocation or membrane insertion might not occur in this RBC model.
Using immunofluorescence and immunoelectron microscopy, this study has now demonstrated that Tir is translocated and correctly inserted into the RBC membrane where it can bind intimin expressed on the bacterial surface to produce an intimate bacterial attachment characteristic of that seen in A/E lesions. Initially we observed very low levels of intimate intiminTir attachment which could have resulted from lack of Tir translocation and/or lack of intimin expression. The co-infection experiments showed that wild-type E2348/69 can translocate Tir efficiently to the RBC membrane, since UMD872 is unable to secrete or translocate Tir (Kenny et al., 1996 ; Knutton et al., 1998
). Single infection experiments using E2348/69 grown from frozen stocks suggested that the initially observed low levels of intimate attachment might be due to a lack of surface intimin expression for Tir to bind to and this was confirmed using serially passaged bacteria; adherent bacteria now expressed surface intimin and were able to efficiently form an intimate intiminTir attachment. In a previous study by Western blotting (Shaw et al., 2001
) and in this study by fluorescence staining we were unable to detect unfocused Tir in the RBC membrane which suggests that, in the absence of intimin binding, translocated Tir is unstable and is either degraded or lost from the membrane. Considering the fact that following natural infections both IgA and IgG Tir antibodies are produced (Jenkins et al., 2000
; Li et al., 2000
), elimination of exposed, unbound Tir from the plasma membrane would be expected to benefit both the bacterium and the host. The reason for the observed difference in intimin expression between fresh and passaged E2348/69 cultures is not clear. Given that Tir and intimin are encoded on the same operon, one would expect both proteins to be co-translated. Either this is not necessarily the case, and there is some evidence that intimin might be expressed from its own promoter in addition to the promoter that transcribes it as part of the tir operon (Gomez-Duarte & Kaper, 1995
), or alternatively, some post-transcriptional regulation of intimin might be involved.
The mechanism of protein translocation has yet to be fully elucidated for any type III secretion system (Hueck, 1998 ). In the case of type III translocated integral membrane proteins such as Tir there are two likely mechanisms for Tir insertion into the host-cell membrane; direct translocation into the host-cell membrane by the type III machinery or translocation into the host-cell cytosol followed by insertion into the cell membrane. Kenny (1999)
has proposed that Tir is first translocated into the host cytosol where it undergoes host-cell modifications followed by insertion into the membrane; these host-cell modifications have now been shown to involve Tir phosphorylation on two serine residues (S434 and S463) by protein kinase A, giving rise to
5 and
2 kDa increases in Tir apparent molecular mass, respectively (Warawa & Kenny, 2001
). It is proposed that the sequential addition of these two phosphate groups may trigger conformational changes in Tir structure that might supply the energy to insert cytosolic Tir into the cell membrane. These studies were carried out following a 4 h infection of HeLa cells. In contrast, Gauthier et al. (2000)
detected Tir in cell membrane fractions after 1·5 h, but only in the cytosolic fraction at very late time points, suggesting that Tir is most likely translocated directly into the host-cell membrane. In the present study it is clear from the fluorescence studies that large amounts of Tir have become translocated to the RBC membrane, and since only Tir, with an apparent molecular mass of
78 kDa, was observed, it can be concluded that Tir detected in the RBC membrane by immunofluorescence and by Western blotting had undergone neither tyrosine nor serine phosphorylation, since there was no Tir staining with a tyrosine kinase antibody and no apparent increase in Tir molecular mass from 78 to 85 kDa. Tir with an apparent molecular mass of
85 kDa, typical of that seen following infection of epithelial cells (Kenny, 1999
; Kenny & Finlay, 1997
), would have been detected in the Western blots had it been present. RBCs, therefore, due to the lack of appropriate serine and tyrosine kinases, appear to provide an infection model that allows Tir translocation to be separated from host-cell Tir modifications. This lack of Tir modification following translocation to the RBC membrane suggests that native unmodified Tir is inserted directly into the host-cell membrane by the type III secretion system and that host-cell Tir modifications occur subsequent to membrane insertion.
Following insertion into the host-cell membrane, EPEC Tir becomes phosphorylated on tyrosine residues, a requirement for the subsequent actin nucleating activity of Tir and A/E lesion formation (Kenny, 1999 ). Tir was recently shown to interact directly with
-actinin which is proposed to provide the linkage between Tir and the actin cytoskeleton (Goosney et al., 2000
). We were unable to demonstrate tyrosine phosphorylation of translocated Tir, and
-actinin is not a component of the RBC cytoskeleton. Thus, although translocated Tir can be focused as a result of intimin binding, it is not surprising that in RBCs there was no actin accretion or pedestal formation.
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ACKNOWLEDGEMENTS |
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Received 16 October 2001;
revised 21 December 2001;
accepted 21 December 2001.