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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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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). One of the pathogenic mechanisms used by the bacterium is to cause attaching and effacing (A/E) lesions on the intestine, a histopathological change that results from bacteria attaching to enterocytes, destroying the host microvilli and causing the formation of a pedestal-like structure (Frankel et al., 1998). The major virulence factors involved in A/E lesions reside on a locus of enterocyte effacement (LEE), a pathogenicity island on the chromosome that has also been found in human enteropathogenic E. coli (EPEC), rabbit EPEC and a mouse pathogen called Citrobacter rodentium. Among these LEEs, there are many genes in common (McDaniel et al., 1995) and the sequences of the various LEEs show a high degree of homology and gene similarity (Deng et al., 2001).
The LEE in EHEC consists of 41 ORFs; these encode the type III secretion (TTS) apparatus (Esc and Sep proteins) (Pallen et al., 2005), effectors (EspF, EspG, EspH, EspI, MAP and Tir) (Crane et al., 2001; Elliott et al., 2001, 2002; Lai et al., 1997; Mundy et al., 2004), translocators (EspA, EspB and EspD) (Chiu et al., 2003; Kenny et al., 1996; Kresse et al., 1999), chaperones (e.g. CesAB, CesD, CesD2, CesF and CesT) (Creasey et al., 2003a, c; Wainwright & Kaper, 1998), regulators (Ler, GrlR, GrlA and Mpc) (Deng et al., 2004; Mellies et al., 1999; Tsai et al., 2006) and adhesin (intimin) (Donnenberg et al., 1993; Jerse & Kaper, 1991; Kenny et al., 1997b). While some of them have been thoroughly studied, others remain less well characterized, and gene l0017 of EHEC is one of the latter.
The TTS system secretes translocators and effectors across the inner and outer membranes of the bacteria after appropriate induction. To achieve this function, the system consists of a basal apparatus with proteins distributed over the inner membrane, periplasm and outer membrane (Roe et al., 2003). While the effectors and the translocators are all secreted by the TTS system, subtle differences have been found in their secretion. Overall, 19 LEE genes, when individually deleted, affect secretion of both translocators and effectors, while four others preferentially affect the secretion of translocators (Deng et al., 2004). The counterpart of l0017 in C. rodentium, orf29, is among the 19 genes required for all TTS. This ORF encodes a protein of 92 aa of which 86 % are identical between C. rodentium and EHEC (Deng et al., 2001). In a yeast two-hybrid screening, the product of orf29 has a positive interaction with that of orf2 (l0053 in EHEC). Furthermore, orf2 is homologous to Pseudomonas aeruginosa pscE and Yersinia pestis yscE, and the latter binds to YscG (Pallen et al., 2005). By correlation, orf29 is speculated to play the YscG-like role. YscG shares 47 % sequence identity with PscG and has a chaperone-like activity (Day et al., 2000; Quinaud et al., 2005). However, no homology has been found between YscG and the orf29 product (Pallen et al., 2005). In this study, we have experimentally confirmed the effect of l0017 by deletion and determined the location and biochemical properties of L0017. Intriguingly, L0017 was found to have a chaperone-like function that interacts with and stabilizes EspA.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Generation and verification of the deletion mutants.
To delete l0017 from the EHEC chromosome, a previously used method of homologous recombination without inserting a selectable marker was employed (Link et al., 1997; Tsai et al., 2006). First, a 5' fragment flanking l0017 was amplified from the chromosomal DNA of the parental WT strain (WT) using primers L17-33909F (5'-GCTGAAGATCTTGCAGAC-3') and L17-34978R(XbaI) (5'-TGCTCTAGACCGCCCACACCAGTATCTTATT-3'). The PCR product was ligated into the pGEM-T Easy vector (Promega) to create pGEMT-1/2-L17. A plasmid, pGEMT-3/4-L17, containing an l0017-3' flanking fragment, was similarly generated using primers L17-35188F(XbaI) (5'-TGCTCTAGAGGTAGTGGCTGGGTACGAGGATTT-3') and L17-36194R(SalI) (5'-GTCGACGACTTTTAAGCTCTGTGCGC-3'). From the above plasmids, the two l0017-flanking fragments were cut and ligated to take advantage of the engineered XbaI and SalI sites within the PCR primers; by so doing, pGEMT-A/B-L17 was obtained. This plasmid was subsequently digested with NotI and SalI and the fragment encompassing the two l0017-flanking segments was gel-purified and ligated with NotI/SalI-restricted pKO3 (Link et al., 1997), to generate pKO3-d17. Thereafter, pKO3-d17 was transformed into the EHEC WT strain to create an l0017-deleted mutant strain, which was named 
L17. To confirm the construction of the l0017-deleted strain, PCR was performed using two primer pairs (Fig. 1). Amplification using the primers L19-34637F(XbaI) (5'-TGCTCTAGACGGAATTTGGTTCGT-3') and L19-35660R(SalI) (5'-GTCGACGCGGGCTTAAAACCTAAAGC-3') should give a fragment of 1035 bp from the WT and of 800 bp from L17. A second primer pair L17 F (5'-ATGGTTAATGATATTTCTGC-3') and C1R (5'-CCACTCGAGTTAAAATCCTCGTACCCAGCC-3') was also used that should amplify a DNA fragment of 280 bp from the WT and a 50 bp fragment from L17. In both cases the putative deletion strain L17 produced the latter fragment sizes, confirming deletion of l0017. Generation of the l0036 deletion mutant (L36) has been described previously (Tsai et al., 2006).
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); it was renamed pQ-EspB for this paper.
Transcriptional fusion and translation fusion reporter constructs.
The EspA-LacZ translational fusion construct pKMespAL and the transcriptional fusion construct pKMespAX were generated using pKM005 that contains a promoterless lacZ reporter gene (Masui et al., 1983; Lee & Cerami, 1987). The espA fragment was PCR-amplified from pQ-EspA using the primers XT5 (5'-GCTCTAGACCCGAAAAGTGCCACCTG-3') and ESPARB (5'-TTGGATCCTTACCAAGGGATATTGC-3'). The PCR product was restricted with XbaI/BamHI and ligated into pKM005 that had been digested with the same enzymes; the result was pKMespAL. Similarly, espA was PCR-amplified using the primers XT5 and ESPARX (5'-GCTCTAGATTATTTACCAAGGGATATTGC-3'), and the product was treated with XbaI and then ligated into XbaI-treated, calf intestinal phosphatase-digested pKM005 to generate pKMespAX.
pEspA-His-L0017 was constructed by amplification of espA from pQ-EspA using primers XhoI-EspA-F (5'-CCGCTCGAGAAATCATAAAAAAT-3') and XhoI-EspA-R (5'-CCGCTCGAGTTTACCAAGGG-3'). The PCR product was inserted into XhoI-treated pQE-30 after digestion with XhoI. The resulting plasmid, pT5-EspA, was next restricted with BamHI and KpnI, and ligated with a BamHI–KpnI fragment obtained from pQH-L17 that encodes RGS-Hisx6-tagged L0017. The plasmid thus obtained simultaneously encodes tag-free EspA and RGS-Hisx6-tagged L0017. By making the above construction, expression of each gene in either transcriptional fusion or translational fusion is driven by an exogenous T5 promoter.
Measurement of β-galactosidase activity.
The method (Miller, 1972) used ONPG as the substrate. Basically, bacterial transformants were cultivated at 30 °C, and three representative colonies were picked and measured in triplicate for enzyme activity. Each value was individually calibrated with respect to bacterial growth density (Miller, 1972).
Adherence activity of bacteria to HeLa cells.
A previously described method (Chiu & Syu, 2005) was followed with a slight modification. HeLa cells were seeded in 6-well plates (2x105 cells per well) in DMEM supplemented with 10 % fetal calf serum (FCS) and ampicillin (100 µg ml–1) for 40 h. Prior to infection, the wells were washed with DMEM without FCS. The monolayer of cells was infected with the various EHEC strains, which had been grown overnight and then diluted 1 : 100. The infections were then allowed to proceed for 6 h. After discarding the supernatants, the wells were washed with phosphate-buffered saline (PBS) five times at 4 °C. Then the HeLa cells were scraped into new microcentrifuge tubes and pelleted down by centrifugation at 1800 g for 5 min at 4 °C. Finally, the HeLa cells were lysed and the total number of bacteria associated with the cells in a well was counted on LB-ampicillin agar plates.
Actin fluorescence staining of HeLa cells.
Immunofluorescence microscopy analysis was carried out as described by Kresse et al. (1999).
Immunoblotting.
EHEC-secreted proteins and bacterial total protein lysates were prepared and analysed as described previously (Chiu & Syu, 2005; Kenny et al., 1997a). Anti-L0017 was prepared by immunizing mice with purified recombinant proteins from E. coli JM109. Anti-EspA, anti-EspB and anti-Tir have been described previously (Tsai et al., 2006). Immunoblots were developed with Renaissance Western Blot Chemiluminescence Reagent Plus (NEN) and the images were captured using X-ray film (Fuji).
Bacterial cell fractionation.
To separate bacterial proteins into fractions, the method of Neves et al. (2003) was slightly modified. EHEC was inoculated into M9 medium (200 ml) and grown for 6 h at 37 °C in the presence of 5 % CO2. The bacteria were then pelleted by centrifugation at 4 °C. After removing the supernatants, the pellets were weighed and resuspended by adding 50 ml osmotic shock buffer A (20 % sucrose, 20 mM Tris, pH 8.0) g–1. EDTA, at a final concentration of 1 mM, was subsequently added and this was followed by gentle shaking at 4 °C for 10 min. The cells were then centrifuged at 8000 g for 20 min at 4 °C and then the supernatants were removed. The pellets were resuspended by adding 50 ml osmotic shock buffer B (5 mM MgSO4) g–1, which was followed by gentle shaking at 4 °C for 10 min. The supernatants were collected by centrifugation as described above. The resulting supernatants were combined for individual cultures and the periplasmic proteins thus obtained were concentrated using a Centricon (Millipore). The bacterial pellets were resuspended in 16 ml lysis buffer (100 mM Tris/HCl, pH 7.5, 1.0 mM PMSF, 0.5 µl aprotinin ml–1) and the bacteria were disrupted by five passages through a French pressure press. After centrifugation at 14 000 g for 20 min at 4 °C, the collected supernatants were spun in an ultracentrifuge at 80 000 g for 30 min at 4 °C. The resulting supernatants were then concentrated using a Centricon and saved as the cytosolic fraction. The pellets with the membrane fractions were further processed into outer- and inner-membrane fractions by washing once with lysis buffer and resuspension in 1.6 ml sarkosyl buffer (10 mM Tris/HCl, pH 8, containing 100 mM NaCl, 1.0 mM PMSF, 0.5 µl aprotinin ml–1 and 0.5 % N-lauroylsarcosine). The suspensions were incubated at 4 °C for 4 h to obtain suitable dissolution of the inner-membrane proteins. After another ultracentrifugation step, the supernatants containing the inner-membrane proteins were obtained and subsequently concentrated using a Centricon. Finally, the remaining pellets were dissolved in SDS sample buffer and these samples were defined as the outer-membrane fraction. To inspect how well the fractionation was performed, proteins known to be enriched within each compartment were examined by Western blotting. CesD2 has been reported as a chaperone for EspD (Neves et al., 2003) and this protein is found in both the inner-membrane and the cytosolic fraction; the protein was detected with mouse anti-CesD2 polyclonal antibodies (M. S.-W. Su & W.-J. Syu, unpublished data). The outer-membrane protein OmpC was detected by a mouse polyclonal anti-OmpC. The periplasmic maltose-binding protein (MBP) was detected using a mouse mAb (SC1D7) (Hsu et al., 1997).
Co-purification of EspA and L0017.
Affinity binding of a Hisx6-tagged protein to a Ni2+-NTA agarose column (Qiagen) was carried out as described previously (Chiu & Syu, 2005) except for the buffers. In this case, the buffer used for equilibration and lysis was 50 mM NaH2PO4, pH 8.0, containing 300 mM NaCl, 10 mM imidazole and 1 mM PMSF, and the washing buffer consisted of 50 mM NaH2PO4, pH 8.0, 300 mM NaCl and 50 mM imidazole. Finally, the elution buffer was identical to that used for washing except that imidazole was added at 250 mM.
Bacterial two-hybrid assay.
The system (Stratagene) was used as described previously (Tsai et al., 2006). In essence, pBT, which encodes the 
cI protein, was used to produce a bait protein fused with cI, and pTRG was used to generate a target protein fused to the C terminus of an N-terminal domain of the RNA polymerase 
subunit. Interactions of the bait and target proteins in the system would then yield a high level of β-galactosidase expression, the activity of which was measured as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The resultant mutant strain (L17) was confirmed by PCR using two sets of primer pairs that gave amplified PCR fragments of the expected sizes. Direct sequencing of an 800 bp PCR fragment, amplified using primers L19-34637F(XbaI) and L19-35660R(SalI), indicated a precise excision at the intended junction (Fig. 1). This mutant was then tested for adherence activity with HeLa cells. L17 appeared to have lost most of its adherence activity, retaining only 4 % residual adherence when compared to the parental strain transformed with the control vector pQE-30. After complementation with L0017 expression from pQH-L17, this mutant's adherence activity was restored to 94 % of the control. When L17 was used to infect HeLa cells that were subsequently examined by immunofluorescence staining, no cytoskeleton rearrangement and actin aggregation were observed (data not shown).
Localization of L0017 in the inner-membrane fraction
To elucidate the potential function of l0017, it is of value to establish the location to which L0017 moves after ribosomal synthesis. To do so, WT bacteria were activated to express LEE proteins in M9 medium, and total bacterial lysate was fractionated. No fraction gave a positive L0017 signal when analysed by Western blotting using a specific antibody raised against recombinant L0017. We reasoned that the intrinsically expressed L0017 might be at such a low level that it was beyond the sensitivity of the Western blot (Fig. 2a). To circumvent this difficulty, plasmid pQH-L17 was transformed into the EHEC WT strain and protein fractionation was carried out again. Proteins from the individual fractions were likewise analysed by Western blotting, except that an additional antibody, anti-Hisx6 tag, was used (compare Fig. 2a and b). Fig. 2(b) shows that L0017 was concentrated in the inner-membrane fraction (lane IM). The identification of L0017 in the inner-membrane fraction is supported by positive detection using both anti-L0017 and anti-Hisx6. The fractionation was itself successful as shown by the correct detection of the fraction marker proteins OmpC, MBP and CesD2.
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). To further explore the effect of deleting this gene, the synthesis and secretion of representative LEE proteins were examined. Like the C. rodentium orf29 strain (Deng et al., 2004), no Tir, EspA and EspB were detected in the spent media produced by L17 (Fig. 3b, lane 2), unless L17 was further transformed with pQH-L17 (Fig. 3b, lane 3). It is worth noting that overexpressed L0017 was not found in the concentrated media and was only found in the bacterial lysate (Fig. 3a). In the bacterial lysate, it was also notable that the deletion of l0017 affected the intracellular level of EspA, but not that of Tir or EspB (Fig. 3a, lane 2). Again, the decrease in the level of EspA in L17 could be restored by complementation by L0017 expression ectopically (Fig. 3a, lane 3).
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, lanes 3–6). While comparable levels of EspB were detected in the WT and L17 strains (lanes 5 and 6), the levels of EspA in the two strains differed greatly (lanes 3 and 4). Therefore, the absence of L0017 indisputably affects the intracellular level of EspA, regardless of their associated promoters. To carry out controls, we examined expression in two additional mutants of EHEC. In C. rodentium, it has been observed that deletion of orf16 (equivalent to EHEC l0032) affects the secretion of translocators, but favours that of effectors. Therefore, we created an l0032 deletion mutant (L32) (J. C.-W. Lio & W.-J. Syu, unpublished results), and repeated the transformation and analyses for EspA and EspB as described above. The results of Fig. 4(b) show that there is no apparent difference in the intracellular levels of these proteins between the WT and L32 strains (EspA, lanes 1 and 2; EspB, lanes 3 and 4). The observed protein levels in the second mutant, L36 (Fig. 4c), were similar to those observed in the WT and L32 strains. Therefore, the low expression level of EspA in L17 is a unique property. It is worth noting that L36 (orf12 in C. rodentium) differs from L32 by a complete loss of secretion in both translocators and effectors, as reported by Deng et al. (2004) and confirmed in Fig. 4(d). It is therefore conceivable that the intracellular level of EspA in a LEE mutant is independent of bacterial TTS capacity.
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. First, a transcriptional fusion was made in pKMespAX where lacZ with intact translational initiation elements was inserted immediately downstream of the espA stop codon (Fig. 5a). By doing so, the T5 promoter controls the transcription of an mRNA encoding both espA and lacZ. Since EspA and β-galactosidase are independently translated but share a common mRNA, any factors from the different host cells that hinder transcription or accelerate the degradation of the mRNA are likely to affect β-galactosidase activity. Second, a fusion of lacZ in-frame with the 3' end of espA before the stop codon was constructed in pKMespAL; in this case β-galactosidase translation directly follows that of EspA (Fig. 5b) because β-galactosidase with an N-terminal EspA fusion is produced when espA-lacZ is transcribed, and this is subsequently translated. Any effect of the host cells (with or without L0017) on the translation process or on the degradation of the fusion protein will be reflected in the level of β-galactosidase activity.
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). The enzyme activity was seen to show a 6.8- and 8.1-fold increase in the WT and L17 strains, respectively, when the transformants containing pKMespAX were compared to those carrying the control plasmid (pKM005). On the other hand, when the β-galactosidase activities were compared with those from the pKMespAL transformants, an intriguing difference was observed. There was a 9.6-fold increase in enzyme activity from the pKMespAL-transformed WT strain compared to that from the pK005-transformed WT, whereas there was an opposite effect (a 22-fold reduction) in the case of pKMespAL-transformed L17 compared to L17 carrying pKM005. The 1.2-fold difference found between the transcriptional fusion data obtained with the WT and L17 strains is likely to be due to experimental variation. In contrast, the 210-fold difference seen between the two translational fusion (pKMespAL) results strongly suggests that L0017 regulates at the protein level after the transcription.
There are a number of possible reasons for the post-transcriptional effects of the L0017 protein. These include the stability of the translated protein, which is the easiest scenario to test. To do this, pQ-EspA was transformed separately into the WT and L17 strains. After inducing LEE expression for 5 h, ribosomal translation was blocked with chloramphenicol (at a final concentration of 200 µg ml–1). The existing intracellular EspA was then detected over a period of time by Western blotting. Fig. 6(a) shows that the level of EspA in L17 declined quickly within the first hour. By the second hour, EspA is barely detectable. On the other hand, in the WT strain, EspA was relatively stable during the first 2 h, and substantial amounts of EspA remained detectable after inoculation for 16 h (Fig. 6b). To exclude the possibility that the decreased stability of EspA in L17 is simply due to degradation of EspA arising from the loss of bacterial secretion capability, we performed a similar experiment in the control L36 mutant. Fig. 6(c) shows that EspA was comparably detected in L36 over the same period of time as seen in the WT strain (Fig. 6d). Therefore, it would seem that the presence of L0017 stabilizes intracellular EspA in EHEC.
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shows that EspA was abundantly detected in the eluted fractions when the bacterial proteins loaded into the column were prepared from bacteria harbouring pEspA-His-L0017. Remarkably, it was seen that EspA was most prominently detected in fractions 2–4 with intensities directly proportional to the amount of Hisx6-L0017 detected, a fact that unequivocally reveals an interaction between EspA and L0017. In contrast, in a control where EspA alone was expressed from pT5-EspA, EspA was detected only in the pass-through and washing fractions, but not in the eluant. Therefore, these biochemical data support that the interaction between EspA and L0017 does occur when they are expressed in the same bacterium. To corroborate this interaction, a bacterial two-hybrid assay was performed (Fig. 7b, inset). When L0017 was cloned as bait, a positive signal was found after pairing with the target of EspA. The interaction seemed to be as strong as L0017 paired with L0053, a previously reported binding protein in the yeast two-hybrid system (Creasey et al., 2003b).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Secretion of translocaters (exemplified by EspB) and effectors (exemplified by Tir) were disrupted and the bacterial activities of cell adherence and actin accumulation disappeared. In addition, we located L0017 in the inner-membrane fraction of EHEC, a biochemical property that is consistent with the proposed role of the protein as a TTS component (Deng et al., 2004).
In examining whether L0017 has any additional functions, we have found for the first time that a lack of an inner-membrane component from EHEC resulted in a decrease in the intra-bacterial level of EspA, but not of EspB or Tir. Mechanistically, we have ruled out the possibility that L0017 regulates the EspA level at the corresponding promoter. The first line of evidence was obtained by placing espA under the control of an exogenous promoter, such as the T5 promoter, and we found that L17 produced a low level of intracellular EspA when compared with its congenic counterpart (Fig. 4). Second, by using a reporter gene transcriptionally fused downstream to espA, the reporter activity of the two strains demonstrated no apparent difference (Fig. 5c). Therefore, it would seem that L0017 regulates EspA expression at a level other than transcription.
When a translational fusion assay was carried out to monitor the EspA-β-galactosidase fusion protein (Fig. 5b), a 210-fold difference was found between L17 and its parental strain (Fig. 5c). This difference was consistent with the direct detection of the EspA protein seen in Fig. 4. Therefore, these results indicate that for EHEC to keep a constant and high level of EspA in the cytoplasm, the presence of L0017 is required, perhaps to retain an effective TTS system. The amount of L0017 needed is far less than that of EspA, as revealed by the fact that the level of intrinsic L0017 was very hard to detect in the WT strain. However, when L0017 is missing, newly synthesized EspA is degraded quickly and the intracellular level of EspA drops aberrantly.
EspA constitutes the major part of the filamentous surface appendages of pathogenic E. coli (Ebel et al., 1998) and it alone is sufficient to form filamentous structures in the absence of other LEE proteins in vitro (Delahay et al., 1999; Yip et al., 2005). How this protein, which has a strong tendency to polymerize, reaches the extracellular surface, remains a puzzle. In a previous model proposed by Yip et al. (2005), the newly synthesized EspA is maintained partially unfolded, with two extensive coiled coils preserved after binding to its intracellular chaperone, CesAB. Since it is known that the CesAB chaperone is not secreted and thus can only act to maintain the monomeric status of EspA monomeric inside the bacterium (Yip et al., 2005), a component of the TTS system must have an adaptor that allows consecutive transit of EspA. From our data above, it can be seen that once the TTS is handicapped by a lack of L0017, intracellular EspA destabilizes. A bridging protein on the membrane apparently is needed to prevent EspA degradation. A study with a yeast two-hybrid system did not detect EspA interacting with a membrane component of the TTS system, except for the known CesAB chaperone (Creasey et al., 2003b). However, such an interaction within the bacterium cannot be completely ruled out by a genetic system that functions in yeast. A prominent undetected example is the interaction of CesD2 and EspD (Creasey et al., 2003b; Neves et al., 2003). Inner-membrane association and the ability to stabilize EspA have made L0017 a compelling candidate for handing over the unfolded EspA. This is supported by the co-expression experiment, and we have demonstrated that EspA and L0017 indeed do interact with each other.
Two chaperones (CesD in the cytosol and CesD2 in the inner-membrane fraction) have been found with EspD, which has a high tendency to aggregate (Daniell et al., 2001). However, so far, CesAB is the only known chaperone reported to both stabilize and directly interact with EspA. The characteristic whereby L0017 interacts with EspA, stabilizes EspA expression and is localized on the inner membrane are analogous to those seen with CesD2 and EspD (Neves et al., 2003). Therefore, L0017 should probably be categorized as one of the TTS chaperones and named CesA2, as the second EspA chaperone. This uniqueness makes CesA2 distinct from the inner-membrane-associated SepD-SepL that may form a molecular switch to ensure the secretion of translocators prior to effectors, but not to affect their stability (Deng et al., 2005; O'Connell et al., 2004). A speculation is that EspA chaperoned by CesAB and CesA2 may transit outwards via some kind of contact with the SepD-SepL complex. If so, it remains to be explored whether and how EspA interacts with these proteins, including those indirect binders such as L0053, to result in transient complexes.
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ACKNOWLEDGEMENTS |
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Edited by: B. Kenny
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REFERENCES |
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Received 10 October 2007;
revised 6 January 2008;
accepted 8 January 2008.
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