Institute of Microbiology and Immunology, National Yang-Ming University, Beitou 112, Taipei, Taiwan
Correspondence
Wan-Jr Syu
wjsyu{at}ym.edu.tw
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ABSTRACT |
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INTRODUCTION |
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Most of the LEE genes are organized into five major operons: LEE1, LEE2, LEE3, LEE4 and LEE5. EspA (Kenny et al., 1996), EspB (Donnenberg et al., 1993
) and EspD (Donnenberg et al., 1993
; Lai et al., 1997
) are encoded by LEE4 and secreted by the TTSS; deletion of these three genes abolished the translocation of effector proteins, such as Tir (Kenny et al., 1997
), EspF (Crane et al., 2001
) and MAP (Kenny & Jepson, 2000
). EspA is believed to form a hollow, filamentous structure on the bacterial surface to deliver the effector proteins (Knutton et al., 1998
) and it may also play a role in adherence of enterohaemorrhagic E. coli (EHEC) to the host cell (Cleary et al., 2004
). EspD has been found to target the host-cell membrane (Wachter et al., 1999
), whereas EspB is translocated to both membrane and cytoplasm (Wolff et al., 1998
). Both EspB and EspD are believed to be involved in pore formation on the membranes of the infected cells (Ide et al., 2001
) and have been classified as translocators (Roe et al., 2003
). EspB has also been reported to interact with EspA (Hartland et al., 2000
) and EspD (Ide et al., 2001
), and complexes formed by these three proteins may participate in the initial step of bacterial adherence (Nougayrède et al., 2003
). On the host membrane, the N-terminal region of EspB has been found to bind directly to the intracellular
-catenin and to result in the recruitment of
-catenin underneath the bacterial-adherence site (Kodama et al., 2002
). Recently, it has also been demonstrated that host-cell
1-antitrypsin interacts with EspB and EspD, and this interaction has been implicated to block the formation of the EspB/EspD translocation pore (Knappstein et al., 2004
).
In our previous study on the functional domain of EHEC EspB, one essential region (residues 1118) and two auxiliary segments (residues 118190 and 191282) were defined for EspB secretion (Chiu et al., 2003). When appropriate conditions are supplied, such as the provision of M9 medium for cell cultivation, EspB and other TTSS-secreted proteins can be found in abundance in the medium. In this study, we further dissected the EspB molecule and determined the regions required for its own translocation and its interaction with EspA. As bacteria lacking espB lose adherence ability (Donnenberg et al., 1993
), we also examined whether there is a relationship between the EspB structure and bacterial adherence. We report here the results from a series of deletion analyses.
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METHODS |
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Bacteria were regularly grown in LB medium (Difco). To activate the type III secretion, bacteria were transferred into M9 medium. Media were supplemented with ampicillin (100 µg ml1) or chloramphenicol (34 µg ml1) when necessary. HeLa cells were maintained in Dubelcco's modified Eagle medium (DMEM) supplemented with 10 % (v/v) fetal calf serum and cultured at 37 °C in the presence of 5 % CO2.
General recombinant DNA techniques.
Unless otherwise stated, restriction endonucleases, DNA-modifying enzymes and polymerases were purchased from New England BioLabs. DNA-manipulation procedures were followed either as described by Sambrook & Russell (2001) or as recommended by the manufacturers. DNA was purified from a mini-column (Qiagen) and sequenced automatically by a contract service (Mission Biotech).
Plasmids.
To create plasmid pBP312D, pB312D (Chiu et al., 2003) was amplified with primers PQE60-2R (5'-TGACTGCAGGGTTAATTTCTCCTC-3') and PEspB-9 (5'-TAACTGCAGATGAATACTATTGATAAT-3'). After amplification, the inverted PCR product was digested with PstI and self-ligated by using T4 DNA ligase. To create plasmids pB1312, pB1282, pB1250, pB1220 and pB1190, pBP312D was used as the template and PCR-amplified with primer pairs PB939RS (5'-CGCGGATCCTTACCCAGCTAAGCGACCCG-3')/PEspB-9, PB846RS (5'-CGCGGATCCTTAATCATCCTGCGTTCTGCG-3')/PEspB-9, PB750RS (5'-GCGGGATCCTTAAGTCGATTTGACGGACTC-3')/PEspB-9, PB660RS (5'-GCGGGATCCTTACAGTTTATCTACGGAATTCAA-3')/PEspB-9 and PB570RS (5'-CGCGGATCCTTAAACATCATCTGCAACGCC-3')/PEspB-9, respectively. After amplification, the PCR products were digested with PstI/BamHI and ligated with the vector derived from pBP312D digested with the same enzymes. Plasmids pB
191253, pB
CC, pB
118190 and pB
TM were created by an inverted PCR method (Vandeyar et al., 1988
; Weiner et al., 1993
) using pB312 as template. The primer pairs used were PB570R (5'-TTAAACATCATCTGCAACGCC-3')/PB760 (5'-AATGAACAACGTGCGAAG-3'), PB558R (5'-AACGCCAGATGCACGGCT)/PB649 (5'-GTAGATAAACTGACCAATACC-3'), PEspB-14R (5'-TGCTGCAAAAGAACCTAA-3')/PEspB-19 (5'-GCGAAAGCCACTGAC-3') and PB294R (5'-AGCGGTTGCCGCGGCTTT-3')/PB355 (5'-AACAACGCGGCTAAAGGG-3'), respectively. The PCR products were phosphorylated by T4 DNA kinase and then self-ligated to produce the designated constructs. After so doing, these plasmid-encoded EspB constructs had authentic EspB N termini and carried no tag at the C termini of the molecules.
To create pEspAHis, the entire espA sequence was PCR-amplified from the chromosomal DNA of E. coli 43888; the primers used were PEspA-1 and PEspA-3R. These had the nucleotide sequences 5'-CTAACCATGGATACATCAAATGCA-3' and 5'-CGCAGATCTTTTACCAAGGGATATTGC-3', respectively. After PCR amplification, the obtained DNA fragment was digested with NcoI/BamHI and cloned into NcoI/BamHI-restricted pQE60 (Qiagen); the resulting pEspAHis expressed a recombinant EspA tagged with a hexahistidine epitope at the C terminus (i.e. EspAHisx6).
Fractionation of cell components after bacterial infection.
A previously described method (Gauthier et al., 2000) was followed with a slight modification. In brief, HeLa cells in 100 mm plates cultured to 80 % confluence were washed with PBS. Overnight-grown bacteria were diluted 1 : 100 (v/v) in DMEM and added to the HeLa cells for 6 h. After infection, the cells were washed twice with PBS and scraped off the plates into a cold imidazole buffer (3 mM, pH 7·4) containing 250 mM sucrose, 0·5 mM EDTA, 1 mM sodium orthovanadate, 1 mM sodium fluoride and 1 µM pepstatin. The mixture was passed through a 22-gauge needle eight times and the unbroken cells with the bacteria were pelleted down by centrifugation twice at 3500 g for 15 min. To collect the disrupted membrane fraction of the cells, the recovered supernatant was further centrifuged at 20 000 g for 45 min. Cytosolic proteins in the supernatant were precipitated by adding 1/9 vol. trichloroacetic acid and further incubated at 4 °C for 1 h. Proteins in the precipitates were then collected by centrifugation. Proteins from each sample were dissolved in SDS sample buffer and separated by SDS-PAGE (12 % gel), followed by analysis using Western blotting.
Immunoblotting.
Western blotting analysis of antigens was performed as described previously (Hsu et al., 2000). The anti-EspB antibody has been described previously (Chuang et al., 2001
) and anti-EspA was prepared by immunizing rabbits with gel-purified EspA from the secreted proteins of EHEC. Anti-OmpA was a mouse mAb prepared previously (Yu et al., 2000
). Mouse mAbs against syndecan (Santa Cruz) and actin (Chemicon) were obtained commercially. To detect the primary antibodies bound to the antigens on the nitrocellulose membrane, horseradish peroxidase-conjugated goat secondary antibodies (Sigma) were used. The membranes were finally developed by a chemiluminescence reagent (Hsu et al., 2000
).
Affinity purification with an Ni2+ column.
Ni2+NTA agarose beads (Qiagen) were packed into columns and charged with Ni2+ by passing through five bed volumes of 100 mM NiSO4, and finally equilibrated in Tris-buffered saline (TBS). E. coli lysates derived from bacteria harbouring pEspAHis, pQE60, pB1312, pB1282, pB1190, pB118190 or pB
CC were prepared in TBS and clarified by centrifugation. The lysate containing EspB or its truncated form was mixed with an equal volume of that containing EspAHisx6. The mixture was inverted gently at 4 °C for 1 h and then passed through the TBS-equilibrated Ni2+ column. After washing with TBS containing 150 mM imidazole, the proteins retained in the columns were eluted with 500 mM imidazole in TBS and analysed by Western blotting.
Adherence assay.
Analysis of the adherence of EHEC to cells was modified from the method described by Gansheroff et al. (1999). In brief, 2x105 HeLa cells were plated in 12-well plates. After incubation overnight, the cells were washed with PBS and maintained in DMEM without any additives. Overnight-grown bacteria were diluted 1 : 100 (v/v) and added to the cells, and infection was maintained for 6 h. Thereafter, the cells were harvested and lysed in PBS containing 10 % (w/v) saponin solution (5 mM Tris/HCl, pH 7·4, 0·4 mM NaVO4, 0·1 mM PMSF) at 4 °C for 10 min. The lysates were then diluted serially in LB medium and plated on LB agar plates supplemented with ampicillin (100 µg ml1). After 16 h incubation at 37 °C, c.f.u. were scored and the relative adherence efficiencies were calculated.
Red blood cell (RBC) haemolysis assay.
A previously described method (Warawa et al., 1999) was followed. In brief, human RBCs, type B, were washed with PBS three times and suspended in PBS to a final concentration of 3 % (v/v). The RBCs were then plated on poly-lysine-coated 12-well plates (700 µl per well) for 20 min at 37 °C. After two washes with PBS, the cells were kept in DMEM without phenol red. Overnight-grown bacteria were diluted 1 : 100 (v/v) to the RBC culture. After 6 h incubation at 37 °C in the presence of 5 % CO2, the culture medium was collected and centrifuged. The released haemoglobin was evaluated by measuring OD543 of the supernatant with an ELISA reader (TECAN RainBow). Experiments were carried out in triplicate and the degrees of RBC haemolysis were calculated accordingly (Warawa et al., 1999
).
Immunofluorescence staining.
HeLa cells were infected with bacteria as described above except for cultivation on glass coverslips in six-well plates. After infection, the cells were washed gently with cold PBS twice and fixed with 4 % (v/v) paraformaldehyde for 20 min. The cells were then permeated with 0·5 % Triton X-100 in PBS and blocked with 3 % BSA in PBS (Lin et al., 1999). To stain the bacteria, rabbit anti-O157 antibody (Difco) was used and the bound primary antibodies were in turn detected by FITC-labelled anti-rabbit immunoglobulin (Jackson Laboratory). To stain the actin filaments, cells were treated with tetramethylrhodamine isothiocyanate (TRITC)-labelled phalloidin at 5 µg ml1 (Jackson Laboratory) in PBS containing 1 % (w/v) BSA. The stained cells on coverslips were examined by using a fluorescence microscope (Olympus BX51) and pictures were taken with a CCD camera (Photometrics CoolSNAP).
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RESULTS |
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The products derived from pB1190 and pB118190 were not found in the membrane fractions of the infected cells; nor were they seen in the cytosol (lanes 5 and 8, Fig. 2b, c
). The failure to detect these proteins could not be attributed to a low level of expression, as pB1250, expressed equally well as pB1190 and poorer than pB
118190, was translocated successfully. These facts indicate that the loss of translocation ability with these constructs was because regions critical for translocation had been deleted or altered. Intriguingly, these two constructs are secreted and have been demonstrated repeatedly in the spent medium (Chiu et al., 2003
; Fig. 1c
). Thus, at least for EspB, secretion does not warrant successful translocation and, taken together with the observation that the construct encoded by pB1220 was translocated (lane 4, Fig. 2b and c
), the EspB region spanning residues 118220 must be critical for EspB translocation. Additional products with sizes smaller than expected (Fig. 2a
) were seen with some of the total lysates. They were present in a relatively small amount and may represent minor degradation products.
Interaction of EspB with EspA
EspB has been reported to interact with EspA when secreted into the medium (Hartland et al., 2000) and deletion of espA in EPEC has been found to abolish the translocation of EspB (Knutton et al., 1998
). For these reasons, we investigated whether EspB defective in translocation also fails to interact with EspA. Proteins from pB1190 and pB
118190 were tested, along with three translocatable controls, in an affinity column-retention assay (Fig. 3
). In the assay, EspA tagged with a C-terminal Hisx6 epitope was retained in a nickel-ion column and tested for the co-eluted EspB molecules. The results (Fig. 3b
) indicated that the full-length EspB, encoded by pB1312, and those derived from pB1282 and pB
CC were co-eluted from the column with EspA (Fig. 3c
). In contrast, the products resulting from pB1190 and pB
118190 were not eluted, a fact suggesting that constructs that do not translocate have also lost the ability to interact with EspA.
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EspB-expressing plasmids were transformed into E. coli 43888 B and recovery of the lost bacterial adherence was examined. Fig. 4
shows that E. coli 43888
B, when complemented with the full-length EspB expressed from pB1312, had restored bacterial adherence to a level similar to that of the wild-type strain transformed with a vector control (i.e. pQE60). Constructs with the C-terminal region of EspB deleted, such as those from pB1282, pB1250, pB1220 and pB1190, did not recover bacterial adherence. Furthermore, the construct from pB
191253, with a deletion of residues 191253 of EspB but retaining the C-terminal 59 aa, did not restore the adherence of E. coli 43888
B (Fig. 4
). In contrast, truncation at residues 118190 (in pB
118190), which abolished both translocation and interaction with EspA, apparently did not damage EspB's bacterial-adherence property. Deletion of the hypothetical coiled-coil domain (residues 187216) of EspB or the possible transmembrane domain (residues 99118) alone also did not affect bacterial adherence (Fig. 4
, pB
CC and pB
TM). Therefore, the region of EspB in residues 216312 that locates C-terminally to the hypothetical coiled-coil domain of EspB (see summary in Fig. 7
) must contribute to bacterial adherence, and this region of EspB obviously differs from that required for the functions of translocation and EspA interaction seen above.
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Regions of EspB critical for causing RBC haemolysis
EspB, together with EspD, is thought to form pores at the membranes of the infected cells (Ide et al., 2001; Kenny et al., 1997
; Warawa et al., 1999
). In EPEC, this activity has been assayed by using haemolysis of human RBCs, and deletion of either espA, espD or espB attenuates the haemolytic activity of EPEC (Ide et al., 2001
; Shaw et al., 2001
; Warawa et al., 1999
). To examine the domain of EHEC EspB possibly involved in pore formation, RBCs were incubated with the bacteria used in Fig. 4
, and haemoglobin release was measured after 6 h incubation. Fig. 5
shows that E. coli 43888
B transformed with pQE60 lost the haemolytic activity found with the wild-type EHEC. The constructs of EspB derived from pB1282, pB1250, pB1220, pB1190 and pB
191253 failed to show the complementation activity seen with the full-length EspB. Therefore, truncated EspB that failed in the adherence assay also failed to show haemolysis in RBCs (compare Figs 4 and 5
). Thus, the results with the adherence and haemolysis assays seemed to be in parallel, except for the results with pB
TM, in which deletion of residues 99118 apparently did not affect adherence, but did abolish RBC-haemolysis activity. This observation suggests that residues 99118 of EspB may be involved in pore formation, and this notion is consistent with a transmembrane domain predicted for these residues. Thus, it is proposed that EspB may mediate adherence through a structure that involves the C-terminal region and, after adherence, the transmembrane domain of EspB may further participate in pore formation. In a control with E. coli 43888
T, transformation with pQE60 decreased the haemolysis only to a small degree (Fig. 5
), a result similar to that reported previously (Warawa et al., 1999
).
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DISCUSSION |
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As shown previously, the N-terminal 118 aa region of EspB is critical, but not sufficient, for EspB secretion (Chiu et al., 2003). To serve as a translocator, EspB must retain secretion competence. Therefore, any construct that lacks the N-terminal 118 aa is not secreted at all and could not possibly function for other purposes.
Translocation of EspB into the host cells apparently requires the same region of EspB that is also critical to interact with EspA (Fig. 7a). Thus, these two functions are currently assigned to the same domain. Interaction of EspA and EspB has been reported (Hartland et al., 2000
) and here we have further demonstrated that residues 118190 in the middle region of EspB are needed for this interaction. Less clear is the function of EspB residues 187216, which are predicted to form a coiled-coil structure. In comparison of pB1190 with pB1220 (Fig. 7a
), residues 191216 seem to be crucial for EspB transloction. However, in pB
191253, the deletion includes residues 191216 of EspB, but does not abolish EspB transloction activity. As many factors, such as conformational changes, could account for this inconsistency, we currently prefer a conservative way and assign no function for this region (Fig. 7b
).
Adherence of bacteria to HeLa cells apparently involves EspB, particularly the C-terminal region, and we have narrowed this down to the region spanning from residue 216 to the C terminus (Fig. 7a). This region is also needed for pore formation on the membrane, as measured by the RBC-haemolysis experiment. The latter activity of causing RBC lysis appeared to be associated with an extra domain in residues 99118 that has a high degree of hydrophobicity, as evidenced by the data from pB
TM. Intriguingly, both the C-terminal region and the hydrophobic residues important for RBC haemolysis matched with the regions critical for the formation of the condensed actin structure underneath the sites where bacteria are attached (Fig. 7a
). Actin accumulation has been correlated with successful translocation of Tir, a function that results from the correct formation of membrane pores. Therefore, residues 99118 and 216312 in EspB must play an essential role to function effectively as a translocator.
The functional domains of EspB described above seem to be discernible, and they are grouped accordingly. It is worth noting that, without the EspA-interacting region, the truncated EspB molecule (from pB118190) remains active in helping bacterial adherence (Fig. 7a
). Similarly, EspB losing the pore-forming activity (from pB
TM) did not lose its bacterial-adherence activity. On the other hand, EspB mutants that lost the activity of assisting bacterial adherence all gave a negative result in the pore-formation assay, an observation suggesting that the pore-formation activity of EspB relies on its active function of adherence. It is then proposed that EspB's assistance in bacterial adherence is an action prior to the formation of membrane pores. On the other hand, bacterial adherence and EspB translocation/EspBEspA interaction seem to be independent events, as no clear association could be made (Fig. 7a
).
In our experimental results shown in Fig. 4, we measured the bacteria attached to the cells after 6 h incubation and calculated the relative activity after comparison with the parental strain. To ensure that the measured values did not result from the rate differences of bacterial growth, parallel experiments were performed with individual bacteria, to follow their growth curves. The fact that no apparent differences were found thus excludes this possibility. However, adherence activity normally monitors the ratio of bacteria attached to the number of bacteria inoculated. Our values of the relative adhering activity of different strains should then reflect more than a simple association at the early stage. Away from a direct input of EspB, an indirect effect of EspB on other adherence-assisting molecules, such as EspA (Cleary et al., 2004
) and EspD, may have been measured collectively.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 12 April 2005;
revised 20 June 2005;
accepted 21 July 2005.
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