CesAB is an enteropathogenic Escherichia coli chaperone for the type-III translocator proteins EspA and EspB

Elizabeth A. Creasey1, Devorah Friedberg2, Robert K. Shaw3, Tatiana Umanski2, Stuart Knutton3, Ilan Rosenshine2 and Gad Frankel1

1 Centre for Molecular Microbiology and Infection, Department of Biological Sciences, Imperial College, London SW7 2AZ
2 Departments of Molecular Genetics and Biotechnology, The Hebrew University, Faculty of Medicine, POB 12272, Jerusalem 91120, Israel
3 Institute of Child Health, University of Birmingham, Birmingham B4 6NH, UK

Correspondence
Gadi Frankel
g.frankel{at}imperial.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Enteropathogenic Escherichia coli (EPEC) are extracellular pathogens that colonize mucosal surfaces of the intestine via formation of attaching and effacing (A/E) lesions. The genes responsible for induction of the A/E lesions are located on a pathogenicity island, termed the locus of enterocyte effacement (LEE), which encodes the adhesin intimin and the type III secretion system needle complex, translocator and effector proteins. One of the major EPEC translocator proteins, EspA, forms a filamentous conduit along which secreted proteins travel before they arrive at the translocation pore in the plasma membrane of the host cell, which is composed of EspB and EspD. Prior to secretion, many type III proteins, including translocators, are maintained in the bacterial cytoplasm by association with a specific chaperone. In EPEC, chaperones have been identified for the effector proteins Tir, Map and EspF, and the translocator proteins EspD and EspB. In this study, CesAB (Orf3 of the LEE) was identified as a chaperone for EspA and EspB. Specific CesAB–EspA and CesAB–EspB protein interactions are demonstrated. CesAB was essential for stability of EspA within the bacterial cell prior to secretion. Furthermore, a cesAB mutant failed to secrete EspA, as well as EspB, to assemble EspA filaments, to induce A/E lesion following infection of HEp-2 cells and to adhere to, or cause haemolysis of, erythrocytes.


Abbreviations: A/E, attaching and effacing; EPEC, enteropathogenic Escherichia coli; FAS, fluorescence actin staining; LEE, locus of enterocyte effacement; RBC, red blood cells; TTSS, type III secretion system


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Enteropathogenic Escherichia coli (EPEC) is the archetype of a group of pathogens that adhere tightly to host enterocytes and cause extensive host cell cytoskeletal rearrangements resulting in the formation of a pedestal beneath the adherent bacteria and the localized destruction of microvilli, a phenomenon known as the attaching and effacing (A/E) lesion (reviewed by Frankel et al., 1998). The genes encoding the A/E phenotype are located in the locus of enterocyte effacement (LEE), a pathogenicity island present in the chromosome of all A/E pathogens (McDaniel et al., 1995). Characterization of this locus revealed that it encodes a type III secretion system (TTSS; Jarvis et al., 1995), translocator and effector proteins, their chaperones and the adhesin intimin (Elliott et al., 1998).

TTSSs are utilized by many Gram-negative pathogens to deliver virulence factors or effectors, directly into host cells (reviewed by Hueck, 1998). Components of the delivery apparatus are highly conserved among TTSSs and secretion complexes have very similar organization (Kubori et al., 1998; Tamano et al., 2000; Blocker et al., 2001; Sekiya et al., 2001; Daniell et al., 2001). The secretion apparatus spans both bacterial membranes and extends from the bacterial cell via a needle-like projection. In order to translocate proteins across the host cell's membrane, a pore is formed by type III secreted proteins known as translocators (reviewed by Buttner & Bonas, 2002). In addition to these common structures, the LEE-encoded TTSS possesses a long filamentous structure that connects the distal end of the needle structure to the host cell membrane (Knutton et al., 1998; Daniell et al., 2001; Sekiya et al., 2001; Wilson et al., 2001). The filament is composed of many copies of EspA forming a helical bundle with a central pore through which secreted proteins may pass (Daniell et al., 2003).

Secretion of many type III substrates requires the presence of cytoplasmic chaperones. There is little or no sequence similarity between TTSS chaperones; however, they have some common characteristics, i.e. low molecular mass (<15 kDa), an acidic pI and a C-terminal amphipathic helix near their C-terminal end (Wattiau et al., 1994). The identified TTSS chaperones have been broadly categorized into three groups (reviewed by Page & Parsot, 2002). The largest group acts as a chaperone for a single effector protein and includes SycE and SycT of Yersinia, IpgE of Shigella, SicP and SigE of Salmonella, and SpcU and Orf1 of Pseudomonas aeruginosa. These chaperones function as a dimer and bind a discrete region in the N-terminal part of their cognate effectors; they have been described as stabilization factors (Frithz-Lindsten et al., 1995; Day & Plano, 1998; Fu & Galan, 1998; Niebuhr et al., 2000), secretion pilots (Woestyn et al., 1996; Day & Plano, 1998; Cambronne et al., 2000) and anti-association factors (Hartland & Robins-Browne, 1998; Niebuhr et al., 2000). A second group chaperone the translocator proteins and include SycD of Yersinia, IpgC of Shigella, SicA of Salmonella and CesD of EPEC. These chaperones do not bind to a single discrete region of the translocators (Neyt & Cornelis, 1999; Francis et al., 2000) and may interact independently or as a complex, with two or more translocator proteins (Neyt & Cornelis, 1999; Anderson et al., 2002); they have been described as anti-association factors preventing premature interactions that would target the proteins for degradation (Neyt & Cornelis, 1999; Tucker & Galan, 2000). A third group that chaperones more than one effector protein has been proposed; as yet only two chaperones have been ascribed to this group – Spa15 of Shigella (Page et al., 2002) and CesT of EPEC (Creasey et al., 2003a); in other respects CesT displays all the characteristics of the SycE family of chaperones, it is not known if the same is true of Spa15. Recent research has also demonstrated that chaperones play an important role in regulation, both at the level of transcription and at the level of secretion (Boyd et al., 2000; Cambronne et al., 2000; Darwin & Miller, 2001; Mavris et al., 2002; Wulff-Strobel et al., 2002).

In LEE-encoded TTSSs, chaperones have been identified for the translocators EspD (CesD – Wainwright & Kaper, 1998; and CesD2 – Neves et al., 2003) and EspB (CesD – Wainwright & Kaper, 1998) and the effector proteins Tir (CesT – Abe et al., 1999; Elliott et al., 1999), Map (CesT – Creasey et al., 2003a) and EspF (CesF – Elliott et al., 2002); however, no chaperone has been identified for EspA, the EspA filament subunit. This is surprising given the tendency of EspA to aggregate (B. C. Neves & G. Frankel, unpublished data), which would be deleterious within the bacterial cell. In a recent survey of interactions between LEE-encoded proteins using the yeast two-hybrid system (Creasey et al., 2003b) we identified an interaction between EspA and the uncharacterized protein encoded by orf3; Orf3 also bound EspB, leading us to speculate that Orf3 performs a chaperone-like function for these translocator proteins. In this study we tested this hypothesis experimentally.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and media.
Bacterial strains and plasmids used in this study are listed in Table 1. Bacteria were grown at 37 °C in Luria broth or on agar supplemented with ampicillin (100 µg ml-1), chloramphenicol (34 µg ml-1) or kanamycin (30 µg ml-1) as required. EPEC cytosolic or secreted proteins were identified from whole-cell lysates or supernatants of strains grown in Dulbecco's modified Eagle's medium (DMEM).


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Table 1. Strains and plasmids used in this study

 
Generation of CesAB and EspA plasmids.
To create a complementing plasmid for EPEC cesAB, the cesAB gene was amplified by PCR (primer pair 5'-GGAATTCATGAGTATTGTGAGCCAAAC-3'/5'-AACTGCAGGTCATACTATTTTTCTATTATTTC-3'). Following digestion with EcoRI/PstI, the product was cloned into pSA10, a modified pKK177-3 that contains the lacIq gene (Schlosser-Silverman et al., 2000) to construct pDF1388.

To create a plasmid allowing overexpression of EspA in an EPEC background, the espA gene was amplified by PCR (primer pair 5'-AAGAATTCATGGATACATCAACTACAGC-3'/5'-AAACTGCAGTTATTTACCAAGGGATATTCC-3'). Following digestion with EcoRI/PstI, the product was cloned into pSA10 to construct pDF1614.

Co-purification of CesAB with EspA and EspB in EPEC.
To create a construct for expression of CesAB with a C-terminal hexahistidine tag within EPEC, the cesAB gene was amplified by PCR (plasmid pair 5'-AAGAATTCATGAGTATTGTGAGCCAAAC-3'/5'-AAACTGCAGTCAATGATGATGATGATGATGTACTATTTTTCTATTATTTCTATT-3'). Following digestion with EcoRI and PstI, the product was cloned into pSA10 to create pICC287.

Plasmid pICC287 was transformed into EPEC escF, ICC171, which produces wild-type levels of Esps but is deficient in secretion (Wilson et al., 2001). ICC171(pICC287) was inoculated 1 : 50 into DMEM, protein expression was induced by the addition of 1 mM IPTG, and cells were grown at 37 °C for 6 h. Cells were disrupted by sonication and cleared lysates were loaded onto a nickel affinity column. Following washing, retained proteins were eluted and analysed by Western blotting using anti-His antibodies (Santa Cruz Biotechnology) and polyclonal antisera raised against EspA (Knutton et al., 1998) and EspB (Knutton et al., 1998). ICC171 without plasmid pICC287 was used as a control.

Construction of cesAB mutant strain.
We applied the one-step inactivation method of chromosomal gene disruption that utilizes the Lambda Red recombinase (Datsenko & Wanner, 2000) to construct an EPEC cesA : : kan mutant. A DNA fragment containing the kan gene was amplified from plasmid pKD4 (Datsenko & Wanner, 2000) using primers 82 F cesAB-mut (5'-GGACTATAGAGATAATGCGAATCAGGATTAATAATAAATAGAGGATGAGTGTGTAGGCTGGAGCTGCTTC-3') and 83 R cesAB-mut (5'-CTATTTTATTAAAAATTGTCATACTATTTTTCTATTATTTCTATTCCGTTGCATATGAATATCCTCCTTAG-3'). This fragment was used to replace wild-type cesAB in E2348/69 by homologous recombination, constructing EPEC cesAB : : kan (DF1358) as described by Datsenko & Wanner (2000).

Haemolysis assay.
Quantitative haemolysis assays were performed using monolayers of human red blood cells (RBCs) infected with EPEC for 3 h at 37 °C as described previously (Knutton et al., 2002). RBC monolayers were also examined for lysis and bacterial adhesion by phase-contrast microscopy following the removal of non-adherent bacteria by washing with PBS.

Microscopy
Fluorescence actin staining (FAS).
This was performed using fluorescein conjugated phalloidin (Sigma) as described previously, following a 3 h infection of Hep-2 cell monolayers with EPEC strains (Knutton et al., 1989).

Immunofluorescence.
EspA immunofluorescence was performed as described previously using rabbit EspAEHECO157 : H7 polyclonal antiserum and a goat anti-rabbit Alexa 488 fluorescence conjugate (Molecular Probes) (Knutton et al., 1998).

Preparation of secreted proteins for Western blots.
EPEC strains were inoculated 1 : 1000 into DMEM to induce type III protein secretion. Cultures were incubated aerobically at 37 °C for 16 h (OD600 approx. 0·7). Cells were pelleted by centrifugation, resuspended in 2xSDS sample buffer and the supernatants concentrated 50-fold using a centrifugal filter device (Millipore); an equal volume of 2xSDS sample buffer was added to the concentrated supernatants. Supernatants and cell fractions were analysed by Western blotting using polyclonal antisera against EspA (Knutton et al., 1998), EspB (Knutton et al., 1998) and Tir (Hartland et al., 1999) and mAbs against EspD (Ebel et al., 1998) and EspF (Umanski et al., 2002), all at a 1 : 500 dilution.

Measurement of gene expression by flow cytometry.
Gene expression of bacterial strains containing gfp transcriptional fusions was monitored by flow cytometry as described previously (Umanski et al., 2002). Cultures grown to an OD600 of 0·3–0·35 were centrifuged and suspended in filtered PBS to an OD600 of 0·3. Fluorescence intensity of 104 bacterial-sized particles was acquired and the mean values were evaluated. Fluorescence measurements were performed using a FACScan cytometer (Becton Dickinson). Data analysis was performed using the CELLQUEST program. Experiments were performed in triplicate and standard deviations were calculated.

Analysis of EspA stability.
EPEC wild-type and cesAB strains harbouring pDF1614 were grown at 37 °C to an OD600 of 0·35, then 1 mM IPTG was added to induce EspA production. After 30 min, chloramphenicol was added to a final concentration of 200 µg ml-1, in order to immediately block protein synthesis and cell growth. Samples (1·5 ml) were taken at 10 min intervals from time 0 to 80 min, cells were pelleted by centrifugation (14 000 g, 4 °C, 2 min) and suspended in 40 µl lysis buffer containing 50 mM Tris pH 7·5 and 1 % SDS. Samples were heated to 100 °C for 5 min and analysed by Western blotting using {alpha}-EspA mAbs (Hartland et al., 2000) followed by {alpha}-mouse IgG–horseradish peroxidase conjugate and ECL detection reagents (Amersham). Membranes were developed simultaneously.


   RESULTS
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Orf3–EspA and Orf3–EspB protein interactions
We recently conducted a systematic yeast two-hybrid screen for interactions between LEE-encoded proteins (Creasey et al., 2003b). Among the protein interactions identified were Orf3–EspA (10-fold increase in {beta}-galactosidase units above single plasmid yeast transformants) and Orf3–EspB (fivefold increase in {beta}-galactosidase units above single plasmid yeast transformants). To assess whether the interactions of Orf3 with EspA and EspB in the yeast are physiological, we constructed a carboxy terminal His-tagged Orf3 (pICC287) (Table 1) and expressed it in the EPEC escF mutant (ICC171), which produces wild-type levels of secreted proteins but is defective in protein secretion (Wilson et al., 2001). ICC171(pICC287) was grown in DMEM for 16 h, in the presence of IPTG. The Orf3–His protein was purified from cell lysates on a nickel affinity column and bound proteins were analysed by Western blotting. We found that both EspA and EspB specifically co-purify with Orf3–His, indicating an interaction between these proteins within the bacterial cytoplasm (Fig. 1).



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Fig. 1. EspA and EspB specifically co-purify with Orf3–His (pICC287) expressed in an EPEC escF strain (ICC171). ICC171(pICC287) (lanes 1 and 2) and ICC171 (lanes 3 and 4) were grown in DMEM to induce LEE expression. Cleared lysates were loaded onto nickel columns and retained proteins were eluted. Lysates (lanes 1 and 3) and eluted proteins (lanes 2 and 4) were subjected to immunoblotting. EspA (a) and EspB (b) were found to specifically co-purify with Orf3–His (lane 2).

 
orf3 is essential for EspA filament formation, haemolysis and A/E lesion formation
In order to define a role for Orf3, we disrupted its gene in the prototype EPEC strain E2348/69 and subjected the mutant (DF1358) to a variety of phenotypic characterizations.

(i) EspA filaments are expressed when bacteria are grown in tissue culture medium (DMEM) for 3 h. Under these conditions DF1358 was unable to produce EspA filaments; this ability was restored when the mutant was complemented in trans by a cloned orf3 (pDF1388) (Fig. 2).



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Fig. 2. Immunofluorescence staining of EspA filaments (a–c) and FAS (d–f) following a 3 h infection of HEp-2 cells with wild-type EPEC strain E2348/69 (a, d), an orf3 mutant (DF1358) (b, e) and orf3 complemented strain DF1358(pDF1388) (c, f). E2348/69 and DF1358(pDF1388) produced EspA filaments and a positive FAS assay indicative of A/E lesion formation; DF1358 did not produce EspA filaments and was FAS negative, i.e. it did not produce A/E lesions. Bar, 1 µm.

 
(ii) Haemolysis of RBCs is an in vitro measure of EPEC interaction with host cells and insertion of a TTSS translocation pore into the host cell plasma membrane, which results in osmotic swelling and lysis (Shaw et al., 2001). DF1358 was unable to adhere to the RBC membrane or cause haemolysis (Fig. 3). These abilities were restored to wild-type levels when the mutant was complemented by pDF1388 (Fig. 3).



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Fig. 3. (a) Haemolytic activity of wild-type EPEC strain E2348/69, an orf3 mutant (DF1358) and an orf3 complemented strain, DF1358(pDF1388). (b) Phase-contrast micrographs showing an uninfected RBC monolayer (i) and RBC monolayers infected for 4 h with E2348/69 (ii), DF1358 (iii) and DF1358(pDF1388) (iv). E2348/69 and DF1358(pDF1388) adhered to RBCs and were highly haemolytic, whereas DF1358 was non-adherent and non-haemolytic. Bar, 5 µm.

 
(iii) Induction of actin accretion beneath bacteria is an in vitro measure of A/E lesion formation (Knutton et al., 1989). Rearrangement of filamentous actin was examined using the FAS test 3 h after infection of HEp-2 cell monolayers. DF1358 adhered to HEp-2 cells in small microcolonies but was unable to induce actin accretion beneath adherent bacteria, i.e. it was unable to induce A/E lesions in vitro. A/E lesions comparable to the wild-type were produced when DF1358 was complemented in trans with pDF1388 (Fig. 2).

These results show that orf3 is required for EspA filament formation and therefore, indirectly, for haemolysis of RBCs and formation of A/E lesions.

Orf3 is required for type III protein secretion
The inability of the orf3 mutant to assemble EspA filaments indicates a secretion defect. In order to test this experimentally, EPEC strains E2348/69, DF1358 and DF1358(pDF1388) were grown overnight in conditions known to stimulate type III secretion (Kenny et al., 1997). Supernatants from these cultures were concentrated 50-fold and evaluated by Western blotting using antisera raised against EPEC secreted proteins. These experiments revealed that DF1358 could not secrete EspA or EspB (Fig. 4); in contrast, the strain secreted wild-type levels of EspD, EspF (Fig. 4) and Tir (data not shown). Complementation of DF1358 with pDF1388 restored secretion of EspA and EspB to near wild-type levels (Fig. 4).



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Fig. 4. orf3 is required for the secretion of EspA and EspB but not of EspD or EspF. Secreted proteins from wild-type (lane 1, E2348/69), orf3 (lane 2) and orf3 complemented (lane 3) EPEC backgrounds were analysed by Western blotting. The orf3 strain failed to secrete EspA and EspB (lane 2); secretion could be recovered by complementing the strain with a plasmid encoding orf3 (lane 3).

 
Taken together, the data presented so far indicate that Orf3 has characteristics typical of a type III chaperone. Therefore, orf3 has been renamed cesAB (chaperone for E. coli secreted proteins EspA and EspB) in accordance with the accepted nomenclature.

CesAB is required for EspA stability in the cytoplasm
Several type III chaperones have been shown to be required for stability of their targets within the bacterial cell (Frithz-Lindsten et al., 1995; Abe et al., 1999; Elliott et al., 1999, 2002; Tucker & Galan, 2000; Creasey et al., 2003a). To address the role of CesAB in the stability of EspA and EspB, we analysed cell pellets of EPEC strains E2348/69, DF1358 and DF1358(pDF1388), which had been grown overnight in TTSS-inducing conditions. Low levels of EspA were detected in the cell pellet of the cesAB strain; complementation restored the level of EspA to near wild-type levels (Fig. 5a). In contrast, EspB was detected in the cell pellet of the cesAB strain at levels significantly higher than wild-type, indicating that EspB accumulates in the cytoplasm when it is not secreted (Fig. 5a).



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Fig. 5. CesAB is required for the stability of EspA in the bacterial cytoplasm. (a) Greatly reduced levels of EspA are detected in the cell pellet of cesAB mutant cells; in contrast, EspB is present at elevated levels. Lanes: 1, cesAB; 2, cesAB complemented; 3, E2348/69. (b) EspA stability was measured in wild-type and cesAB backgrounds after protein expression was blocked by the addition of chloramphenicol. EspA levels remained constant in the wild-type strain, whereas the protein was rapidly degraded from 30 min after the addition of chloramphenicol to the cesAB mutant strain.

 
To analyse further the contribution of CesAB to the stability of EspA, we overexpressed EspA from an inducible promoter (pDF1614) in wild-type and cesAB backgrounds. After growth for 30 min in the presence of IPTG to induce EspA expression, protein synthesis was stopped using chloramphenicol, samples were taken every 10 min and the cells were analysed by Western blotting using {alpha}-EspA mAbs (Fig. 5b). In the wild-type strain, the level of EspA remains approximately constant; however, in the cesAB mutant strain, EspA is rapidly degraded from 30 min after the addition of chloramphenicol, with none detectable after a further 50 min.

CesAB is not required for transcription of the LEE4 operon
The effect on secretion and stability of EspA could be due to a defect in expression; however, the espA gene along with espB and espD are encoded by the LEE4 operon and because there was only a slight reduction in espD secretion from DF1358 we hypothesized that the defect in EspA and EspB secretion from strain DF1358 is not a result of decreased transcription of the LEE4 operon. To confirm this hypothesis experimentally, we utilized a fusion between the LEE4 promoter region and gfp. We found no significant difference in fluorescence intensity in CesAB+ or CesAB- backgrounds (Table 2). Moreover, transcription of the other LEE operons was also unaffected by the absence of cesAB (Table 2). Therefore, we concluded that lack of CesAB does not have a negative effect on transcription and that the defects in stability and secretion of EspA seen in DF1358 are due to a post-transcriptional mechanism involving CesAB.


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Table 2. Expression of LEE genes fused to gfp in wild-type and cesAB EPEC backgrounds

Fluorescence intensity was measured as described in Methods. Numbers in parentheses are standard deviation values.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
EspA is an EPEC translocator that forms a long filamentous extension to the needle complex of the TTSS apparatus connecting the bacterium to the host cell (Knutton et al., 1998; Daniell et al., 2001; Sekiya et al., 2001). Type III secreted proteins, including translocators, often require the presence of cytosolic chaperones for storage in the bacterial cytoplasm and efficient secretion. Despite the fact that EspA has been observed to form large aggregates when expressed in E. coli (B. C. Neves & G. Frankel, unpublished data), no chaperone that might keep it soluble prior to secretion had yet been identified. Here we report the identification of CesAB, the product of LEE orf3, a previously uncharacterized ORF, as a chaperone of EspA and EspB.

CesAB shares some characteristics with type III chaperones; it is small (12·3 kDa) and mainly {alpha}-helical; however, it has an alkaline pI, which, although not without precedent (Jackson et al., 1998; Ruiz-Albert et al., 2003; Zurawski & Stein, 2003), is unusual. Here we show that CesAB is specifically required for secretion and stability prior to secretion of EspA; conversely, CesAB is required for secretion of EspB but does not play a role in intracellular stability of EspB. Importantly, efficient secretion of EspB is also dependent on the EspD chaperone CesD, although a direct interaction between CesD and EspB has not been demonstrated (Wainwright & Kaper, 1998). Because we demonstrated a specific interaction between CesAB and EspB, we propose that CesAB is the primary chaperone for EspB and that CesD plays an auxiliary role. It appears that both pore-forming translocators of EPEC are chaperoned by two chaperones as EspD also has two reported chaperones (Wainwright & Kaper, 1998; Neves et al., 2003). This hypothesis is further supported by recent reports showing that the Salmonella SseA is a basic Salmonella Pathogenicity Island 2 (SPI2)-encoded type III chaperone that binds to, and is required for stability and secretion of, SseB (EspA homologue) and SseD (EspB homologue) (Ruiz-Albert et al., 2003; Zurawski & Stein, 2003). However, there is no significant sequence homology between CesAB and SseA and the C-terminal coiled-coil domain within SseA is not present in CesAB. It is possible that in EPEC, CesAB, CesD and CesD2 play a role in the order by which the translocator proteins are targeted to the secretion machinery. Indeed, we suggested that EspB is the last of the translocators to be secreted following EspA filament assembly and EspD targeting to the plasma membrane (Frankel et al., 1998). It is also clear that there is some interplay between the EPEC translocators with regard to secretion: in the absence of espA, there is increased secretion of EspB (Kenny et al., 1996; Lai et al., 1997); conversely, in the absence of espB, there is a slight reduction in secretion of EspA, although this does not have an effect on EspA filament formation (Knutton et al., 1998). The absence of the third translocator protein, EspD, results in reduced EspA and EspB secretion (Lai et al., 1997).

The LEE pathogenicity islands of several pathogens have been sequenced (Elliott et al., 1998; Deng et al., 2001; Zhu et al., 2001; Tauschek et al., 2002); cesAB is present in all strains, although in the rabbit pathogens REPEC and RDEC-1 the cesAB gene lacks a standard ATG start codon. Considering the essential role CesAB plays in EspA secretion and EspA filament assembly it is likely that in these strains translation starts from the alternative start codon GTG (Val), located three codons downstream from the position of the start codon of EPEC and EHEC cesAB. Indeed, we have recently shown that REPEC strain E22 elaborates EspA filaments on its surface (S. Knutton, E. A. Creasey, R. K. Shaw & G. Frankel, unpublished data); this is the first direct observation of EspA filaments produced by an A/E pathogen of rabbits.

Three families of type III chaperones have been described (reviewed by Page & Parsot, 2002). The characteristics of CesAB suggest that it belongs to the family of translocator chaperones; this family is associated with secretion of one or more translocator proteins and reported roles include the protection of their secreted targets from premature degradation by preventing premature interactions (Menard et al., 1994; Neyt & Cornelis, 1999; Tucker & Galan, 2000). It is likely that the CesAB–EspA interaction averts polymerization of EspA within the bacterial cell. Indeed, we have shown that in the absence of CesAB, EspA rapidly degrades; furthermore, only monomeric EspA co-purifies with CesAB. The mechanism by which CesAB prevents EspA aggregation is unknown but it is possible that interacting domains of EspA are sequestered in the CesAB–EspA complex. Disruption of the coiled-coil region, known to be essential for multimerization of EspA (Delahay et al., 1999), is not sufficient to allow secretion of EspA from the cesAB strain (data not shown) indicating that masking this region is not the only role of CesAB. EspA is also known to bind EspB (Hartland et al., 2000); however, we have not investigated whether CesAB can prevent this interaction.

We have demonstrated, using the yeast two-hybrid system, that CesAB can bind EspA and EspB independently of each other, although the data presented in this study do not rule out the possibility of the existence of a ternary complex between CesAB, EspA and EspB. However, yeast three-hybrid experiments indicated that this is not the case (data not shown).

CesAB is a basic type III chaperone; the association of basic chaperones with their cognate proteins remains uncharacterized. Crystallization of the CesAB–EspA complex should provide insights into the pairing mechanism, but this remains a challenge for future experimentation.


   ACKNOWLEDGEMENTS
 
This work was supported by the Wellcome Trust, the EC (grant QLK2-2000-0060) and BSF (grant 2001108).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 27 August 2003; revised 13 October 2003; accepted 15 October 2003.