Characterization of Enteropathogenic Escherichia coli Mutants That Fail To Disrupt Host Cell Spreading and Attachment to Substratum
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感染与免疫杂志 2006年第2期
Department of Molecular Genetics and Biotechnology, The Hebrew University, Faculty of Medicine, POB 12272, Jerusalem 91120, Israel
ABSTRACT
Upon infection of host cells, enteropathogenic Escherichia coli (EPEC) delivers a set of effector proteins into the host cell cytoplasm via the type III secretion system (TTSS). The effectors subvert various host cell functions. We found that EPEC interferes with the spreading and ultimately with the attachment of suspended fibroblasts or epithelial cells, and we isolated mini-Tn10kan insertion mutants that failed to similarly affect host cells. In most mutants, the insertion sites were mapped to genes encoding TTSS components, including cesD, escC, escJ, escV, espD, sepL, espB, and escF. Other mutants contained insertions in micC or upstream of bfpP, yehL, or ydeP. The insertion upstream of ydeP was associated with a reduction in TTSS protein production and was studied further. To determine whether the apparent repression was due to constitutive expression of the downstream encoded genes, ydeP and ydeO expression vectors were constructed. Expression of recombinant YdeP, YdeO, or EvgA, a positive regulator of both ydeP and ydeO, repressed TTSS protein production. Our results suggest that upon activation of the EvgAS two-component system, EvgA (the response regulator) activates both ydeP and ydeO expression and that YdeP and YdeO act conjointly, directly or indirectly repressing expression of the TTSS genes.
INTRODUCTION
Enteropathogenic Escherichia coli (EPEC) causes infantile diarrhea, which results in the death of several hundred thousand children each year in developing countries (10). EPEC infection is characterized by intimate binding to intestinal enterocytes, localized effacement of absorptive microvilli, transient filopodium formation, and accumulation of polymerized actin beneath the bacteria (26, 46, 54). This histopathology is termed the attaching and effacing lesion (18, 28). The genes responsible for attaching and effacing lesion formation are clustered in the locus of enterocyte effacement (LEE) (37). The LEE consists of 41 genes encoding a type III secretion system (TTSS), as well as several effector proteins. The LEE genes are organized in five major operons, LEE1 to LEE5 (16, 17). Operons LEE2 to LEE5 are positively regulated by Ler, which is encoded by the LEE1 operon (19, 38). Regulation of the LEE1 operon is complex and involves many factors, including H-NS (3, 56), integration host factor (19), Fis (21), PerC (3, 38), BipA (23), GadX (48), GrlA, and GrlR (9), as well as quorum sensing (27, 49). In the closely related organism enterohemorrhagic E. coli (EHEC), the LEE operons are regulated by EtrA, EivF, RpoS, and ClpX (25, 63). For efficient attachment, EPEC uses the plasmid-encoded bundle-forming pilus (BFP) (18, 46). The BFP is encoded by two operons, perABC (bfpTVW) and bfpA-L, which contains 13 genes encoding the pilus structural genes (22, 53).
E. coli K-12 has 32 two-component systems (39), one of which is the EvgAS system, whose physiological role is obscure. EvgS is the sensor histidine kinase, and EvgA is the corresponding response regulator (40, 43, 44). The transcriptome of an E. coli K-12 evgAS mutant has been compared with that of the wild type, using DNA array analysis (42). In addition, the attempts to identify the evgAS mutant phenotype have included a high-throughput phenotype microarray analysis (64), in which nearly 2,000 different phenotypes were examined. However, neither study showed that there was a significant difference between the evgAS mutant and the wild-type strain. Additionally, an in vitro examination revealed low levels of EvgS autophosphorylation and EvgA transphosphorylation (59). The results of these studies may imply that the EvgAS two-component system does not function in E. coli or that the input signal for evgAS activation has not been found.
Here we show that EPEC prevents spreading and ultimately interferes with the attachment of suspended fibroblasts, or epithelial cells, to the substratum. Furthermore, we isolated mini-Tn10kan insertion mutants deficient in inhibition of host cell attachment. These mutants contain insertions in LEE genes, including cesD, escC, escJ, escV, espD, sepL, espB, and escF, in non-LEE genes, including micC, and in the regions upstream of bfpP, yehL, and ydeP. Further characterization of the insertion upstream of ydeP indicated that it is associated with a reduction in TTSS protein production, probably as a result of constitutive expression of ydeP and ydeO. Our results suggest that upon EvgA activation, ydeP and ydeO are expressed and possibly act jointly, directly or indirectly, to repress the expression of the TTSS genes.
MATERIALS AND METHODS
Bacterial strains, plasmids, and oligonucleotides. The strains and plasmids used in this study are listed in Table 1, and the oligonucleotides used are listed in Table 2. Bacteria were grown in either LB broth, M9 minimal medium (35), or Dulbecco's modified Eagle's medium (DMEM) (Sigma). When indicated below, the medium was supplemented with either streptomycin (100 μg/ml), tetracycline (10 μg/ml), ampicillin (50 μg/ml), kanamycin (50 μg/ml), or chloramphenicol (25 μg/ml).
Analysis of inhibition of host cell spreading and attachment. Bacterial cultures that were grown overnight at 37°C in LB broth without shaking were diluted 1:50 in DMEM and grown without shaking under conditions known to stimulate TTSS expression for 3.5 h in the presence of 5% CO2 at 37°C to the mid-logarithmic growth phase (optical density at 600 nm, 0.3), which created preactivated cultures (19). Prior to infection, DU17 fibroblasts (47) were trypsinized, washed with phosphate-buffered saline (PBS), and resuspended in DMEM supplemented with 10% fetal calf serum. A 500-μl suspension of DU17 cells (105 fibroblasts) was infected with 500 μl of a culture of preactivated bacteria (5 x 107 bacteria) at a multiplicity of infection (MOI) of 1:500. The infected cells were seeded onto coverslips in wells of 24-well plates. After 15, 60, or 180 min of infection, cells were fixed for 10 min using 3.7% paraformaldehyde in PBS, washed, permeabilized for 2 min with 0.1% Triton X-100, rewashed, and actin stained using phalloidin-rhodamine (Sigma). Slides were prepared, and micrographs were obtained by fluorescence microscopy.
Mutagenesis and isolation of mutants deficient in blocking host cell attachment. EPEC strain E2348/69 was mutated using a mini-Tn10kan transposon that was delivered into EPEC strain E2348/69 (Str) by mating with E. coli SM10pir (Kanr) containing pLOF/Km (Ampr Kanr), as previously described (8). Transconjugate EPEC colonies were selected on LB medium plates supplemented with kanamycin and streptomycin and tested for ampicillin sensitivity to confirm the lack of plasmid integration into the EPEC chromosome. Each mutated colony was inoculated into LB broth in 96-well tissue plates (master plates) and grown overnight at 37°C. The cultures were then preactivated, maintaining the 96-well format. Concurrently, DU17 fibroblast cultures were trypsinized, washed, and resuspended in DMEM supplemented with 10% fetal calf serum. Using a 96-well plate, 50 μl of suspended DU17 cells (3.4 x 104 fibroblasts) was inoculated with 50-μl portions of different mutants (5 x 106 bacteria) (MOI, 1:66). DU17 was infected with each mutant in duplicate. Infection with wild-type EPEC and infection with the escN mutant served as positive and negative controls, respectively. After 1 h of infection at 37°C, the wells were washed three times with PBS, stained with 100 μl Giemsa stain (Sigma) for 10 min, and then washed with PBS to remove the excess Giemsa stain. The wells were then examined. An inability to block cell attachment resulted in blue staining of a well. Mutants that were unable to block host cell attachment were recovered from the master plates.
Quantitative attachment assay. Using 24-well tissue culture plates, 500 μl of suspended DU17 cells (2.5 x 105 fibroblasts) was infected with 500 μl of preactivated cells of different mutants (5 x 107 bacteria) (MOI, 1:50). Specific wells in each plate in which the cells were infected with wild-type EPEC and with the escN mutant served as positive and negative controls, respectively. After 1 h of infection at 37°C, the wells were washed three times with DMEM and incubated for 20 min with 200 μl lysis buffer (0.1% Triton X-100, 20 mM MgCl2), which specifically lysed the DU17 cells. Next, 180 μl of the lysate was cleared by 5 min of centrifugation (174 x g). The protein concentration of the cleared lysate was directly correlated with the number of attached DU17 cells (data not shown). The protein concentration was measured using a BCA protein assay kit (Sigma). Assays were carried out in triplicate, and standard errors were calculated. The effect of each mutant was compared with the effects of the controls; the effect of wild-type EPEC was defined as 0%, and the effect of the escN mutant was defined as 100%.
Determination of the mini-Tn10kan insertion sites. All mutants were analyzed by PCR using primers for various LEE genes (6) and for the mini-Tn10kan cassette (C1_Tn10_out_R and C4_Tn10_out_F), followed by sequencing (Table 2). DNA fragments containing insertions in non-LEE mutants were cloned. To this end, the total DNA of each mutant was digested with either EcoRI, PstI, MfeI, or SalI and subjected to Southern analysis. As a probe, we used a digoxigenin-labeled kanamycin cassette generated by PCR, using primers C2_Tn10_in_F and C3_Tn10_in_R (Table 2) and a DIG kit (Roche). Digested fragments smaller than 5 kbp were purified from the agarose gel and ligated into pUC18, using either EcoRI, PstI, MfeI, or SalI depending on the enzyme used for genomic DNA fragmentation. Clones containing mini-Tn10kan were selected using LB agar plates supplemented with kanamycin. Sequencing using primers M13_F and M13_R revealed the exact location of the mini-Tn10kan insertion.
Construction of mutants and plasmids. Specific genes were deleted with the aid of the red system, using the DY378 strain or the pKD46 plasmid, as previously described (7, 62). We used pKD3, pKD4 (7), and the chromosomal DNA of Salmonella containing Tn10d-Tet (15) as chloramphenicol, kanamycin, and tetracycline DNA template-resistant cassettes, respectively. The primers used for mutant analysis are shown in Table 2. In some cases, mutations were first generated in DY378 and then transferred into wild-type EPEC harboring pKD46. To generate the expression plasmids, genes were amplified with specific primers (Table 2) and cloned into the pSA10 cloning vector (45) as previously described (2). The plasmid sequence was verified by sequencing, using analysis primers pSA10_F and pSA10_R (Table 2).
Analysis of protein production and secretion under conditions stimulating TTSS and BFP production. One milliliter of preactivated bacteria was centrifuged, and supernatants containing the secreted proteins, as well as pellets containing total proteins, were recovered. Where indicated below, isopropyl--D-thiogalactopyranoside (IPTG) was added 1 h after dilution in DMEM. The protein levels in the different samples were adjusted according to the cell density, and the samples were used for immunoblot analysis. Membranes were incubated with different primary polyclonal rabbit antibodies (anti-EspB, -Tir, -intimin, -Ler, -EscJ, -BfpA, or -SSB) diluted in Tris-buffered saline (150 mM NaCl, 20 mM Tris-HCl; pH 7.5) containing 1% bovine serum albumin. Binding of secondary anti-rabbit immunoglobulin G conjugated to alkaline phosphatase (Sigma) was detected using BCIP (5-bromo-4-chloro-3-indolylphosphate)-nitroblue tetrazolium (Promega).
Conditions used for comparison of the evgA mutant with wild-type EPEC. To create TTSS-repressing conditions, we compared EspB production in overnight cultures grown without shaking in LB broth, either at 37°C or at 27°C, or in M9 minimal medium. To create bvgAS-repressing conditions (31), 5 mM nicotinic acid or 20 mM MgSO4 was added to the LB broth.
Qualitative and quantitative analysis of actin pedestal formation in infected HeLa cells. HeLa cells were infected, fixed, stained with phalloidin-rhodamine, and analyzed by fluorescence microscopy as previously described (61). To quantify pedestal formation efficacy, the number of HeLa cells with more than four actin pedestals in several randomly chosen fields was divided by the total number of HeLa cells, which yielded the percentage of HeLa cells with actin pedestals. About 100 cells were examined for each strain.
RESULTS
EPEC inhibits cell spreading and ultimately interferes with cell attachment by a TTSS-dependent mechanism. EPEC induces infected host cells to detach from the substratum, and the detachment is particularly enhanced in DU17 fibroblasts (1, 47). We tested whether EPEC also prevents the attachment of suspended host cells to the substratum. Adherent DU17 fibroblasts were trypsinized and infected with activated wild-type EPEC or the EPEC escN mutant or not infected. Immediately upon infection, infected cells were seeded, and at different times the unattached cells were washed off. The remaining cells were fixed, actin stained, and examined microscopically (Fig. 1). At 15 min after seeding, infected and uninfected cells were found to be similarly attached. At 60 min after seeding, uninfected cells and cells infected with the escN mutant began to spread. Cells infected with wild-type EPEC failed to spread and later (180 min) detached and were washed away (Fig. 1). In contrast, uninfected cells and cells infected with the escN mutant remained attached and spread. These findings indicate that wild-type EPEC allows initial host cell attachment to the substratum. However, EPEC ultimately interferes with host cell attachment and spreading in a TTSS-dependent manner. We called this sequence of events "attachment inhibition."
Isolation of mutants deficient in inhibiting cell attachment. Using Giemsa staining in a 96-well format, cell attachment was visible to the naked eye as bluish turbidity at the bottom of a well, whereas a lack of attachment resulted in clear wells (Fig. 2B). We used this assay to screen 4,000 mini-Tn10kan mutants of EPEC for a deficiency in preventing fibroblast attachment (Fig. 2). Mutants that repeatedly failed to prevent host cell attachment were analyzed further by (i) mapping the insertion site, (ii) quantifying the inhibition of cell attachment, (iii) testing EspB secretion, and (iv) microscopically observing the infected HeLa cells. The latter procedure was used to analyze the formation of actin pedestals in infected cells and to determine the pattern of bacterial attachment. We identified 26 different mutants; 21 of these mutants contained insertions located within the LEE, and 5 mutants contained insertions located outside the LEE. The 21 LEE mutants had insertions in cesD, escC, escJ, escV, espD, sepL, espB, and escF (Table 3).
In most cases, insertion within the LEE genes resulted in mutants deficient in EspB secretion and in inducing actin pedestal formation. These characteristics were also observed for five of the escV::mini-Tn10kan mutants but not for SN59, which secreted EspB and only partially inhibited cell attachment (26%) (Table 3). The insertion in SN59 was mapped 9 bp upstream of that of SN50, flanking the sequence CGTTATGCG (the boldface C and G are mini-Tn10kan insertion sites in SN59 and SN50, respectively, and the underlined methionine codon represents M79). We speculate that in SN59, the mini-Tn10kan sequence incidentally forms a ribosomal binding site, which allows translation initiation from M79. This may lead to synthesis of truncated EscV that is sufficient for assembly of a partially active TTSS, which may explain the differences in the escV mutant phenotypes.
Another interesting observation was an observation made with the SN67 mutant, which despite its active TTSS, suggested by its EspB secretion and its actin pedestal induction, allowed only partial cell attachment (50%) (Table 3). This may suggest a role for a possible effector; however, the SN67 insertion has been localized to escD, which is known to code for a structural protein that together with EscJ forms a ring-like structure in the TTSS inner membrane (30, 50, 51). The insertion in SN67 is located 12 bp upstream of the M14 codon, which may allow translation initiation from M14, as described for SN59. The putative truncated EscD may support the formation of a partially active TTSS. This suggests that high translocation efficiency may be required to block host cell attachment.
Similar phenotypes were observed in SN6 and SN51 mutants, both of which contained insertions in cesD encoding a TTSS chaperone for EspD and EspB. These mutants exhibited attenuated TTSS activity and allowed cell attachment, like an escN mutant, again suggesting that high translocation efficiency is required to block host cell attachment. In conclusion, aside from mutants with insertions in cesD and escD all LEE mutants exhibited similar phenotypes, which supports the notion that ultimately EPEC inhibition of host cell spreading and attachment is TTSS dependent. In addition, the results validate our screening methodology.
Five non-LEE mutants were isolated, SN61, SN63, SN64, SN66, and SN116 (Table 4). Strain SN66, in which the insertion was in the bfpF-P intergenic region, failed to form microcolonies on infected HeLa cells compared to the wild type (data not shown). Mutants SN61 and SN63 both had an insertion in micC encoding a small RNA that negatively regulated the adjacent ompC porin (5) (Table 4). In SN64, the mini-Tn10kan was localized between two open reading frames, yehI and yehL, in a truncated gene, similar to the E. coli K-12 yehK gene. This is a variable region in different E. coli isolates, and the role of the genes flanking it has not been determined. The insertion site in SN116 was mapped between ydeP and its promoter. Among the non-LEE mutants only SN116 was completely deficient in inhibition of host cell attachment and in EspB secretion. In further studies, therefore, we focused on this mutant.
Overexpression of either evgA, ydeP, or ydeO negatively regulates the production of TTSS and BFP. The mini-Tn10kan insertion in SN116 introduced a constitutive promoter (the kan promoter) that can direct expression of three downstream genes, ydeP, b1500, and ydeO (Table 4). In E. coli K-12 the ydeP gene and the ydeO promoter are activated upon EvgA overexpression (29, 36). Therefore, we tested the hypothesis that the insertion in SN116 mimics activation of the evgAS cascade to downregulate LEE gene expression. To this end, we cloned evgA, ydeP, b1500, or ydeO under control of the IPTG-inducible promoter in pSA10. The wild-type EPEC was transformed with the different plasmids, and the production of different LEE proteins was tested in each strain (Fig. 3A). Overexpression of evgA, ydeP, or ydeO caused decreased production of EscJ, EspB, Tir, intimin, and Ler. Overexpression of ydeP also repressed BfpA production. In contrast, overexpression of b1500 affected neither LEE nor BFP protein production (Fig. 3A).
EvgA-mediated repression of LEE genes is not Ler dependent. Ler is a positive regulator of most of the LEE operons (19, 38). Thus, EvgA-mediated ler repression (Fig. 3A) might be necessary and sufficient for EvgA-mediated repression of the other LEE genes. The other possibility is that EvgA mediates the repression of LEE promoters, regardless of the presence of Ler. To distinguish between these two options, the wild-type EPEC was transformed with two compatible plasmids, pCNY2441 and pTU14, allowing simultaneous evgA and ler expression from a tac promoter. Expression of LEE proteins was compared in wild-type EPEC, in a strain overexpressing evgA, and in a strain overexpressing both evgA and ler. We found that overexpression of the recombinant Ler did not rescue the evgA-mediated repression of Tir and EspB production (Fig. 3B). These results suggest that although EvgA represses ler expression, this repression is not required for the EvgA-mediated downregulation of Tir and EspB production.
Search for EvgAS-activating conditions. We hypothesized that under EvgA-activating conditions an EPEC evgA mutant should overexpress the TTSS genes. In spite of extensive efforts, we could not define EvgA-activating conditions in EPEC or a TTSS-related phenotype in evgA, ydeP, ydeO, or ydeP-ydeO mutants (data not shown). Similarly, neither the environmental conditions that activate EvgA nor a phenotype for the evgAS mutants was found in E. coli K-12 (42, 64). The GadXW regulatory cascade was reported to overlap with the regulatory cascade of EvgAS (34) and to indirectly repress the LEE genes (48). Therefore, double mutants, including evgA/gadX, evgA/gadXW, ydeO/gadX, and ydeO/gadXW mutants, were constructed and tested for differences in expression of TTSS genes under various conditions. However, all of these mutants exhibited a phenotype similar to that of the wild-type strain (data not shown). In conclusion, the environmental conditions that stimulate EvgA activation remain elusive.
EvgA-mediated downregulation of the LEE genes is dependent on the ydeP-ydeO region. We next tested whether EvgA-mediated repression of LEE genes is dependent on ydeP or ydeO. The evgA-expressing vector pCNY2441 was introduced into wild-type EPEC and into mutants CNY2092, CNY2077, and CNY2075 (ydeP::kan, ydeO::kan, and ydePO::kan, respectively). According to an immunoblot analysis, evgA expression resulted in repression of EscJ, EspB, Tir, intimin, Ler, and BfpA production in the wild-type strain but not in the ydePO::kan mutant. Expression of evgA in the ydeP::kan mutant or in the ydeO::kan mutant caused only partial repression of the production of LEE proteins (Fig. 4A). As a negative control, we examined the production of single-strand-binding protein (SSB), a housekeeping protein. SSB expression was not altered in the various strains, indicating that there was a specific effect. These results suggest that EvgA activates the expression of both ydeP and ydeO, which are required for full efficiency of LEE repression.
To substantiate these results, we tested the abilities of the different strains to induce the formation of actin pedestals in infected HeLa cells (Fig. 4B to K). In these experiments, we used wild-type EPEC and a ler mutant as positive and negative controls, respectively (Fig. 4B, I, and K). Overexpression of recombinant evgA, ydeP, or ydeO caused a reduction in the efficiency of actin pedestal formation (Fig. 4C to E and K). However, actin pedestal formation was evident when evgA was expressed in the ydeP::kan and ydeO::kan mutants, and deletion of the entire ydeP-ydeO region (in the ydePO::kan mutant) completely suppressed the ability of EvgA to repress the formation of actin pedestals (Fig. 4F to H and K).
DISCUSSION
EPEC caused host cell detachment in a TTSS-dependent manner (47). Mutants with mutations in the TTSS effector genes (tir, map, espH, and espF) still induce host cell detachment (47; unpublished results). Using a different approach, we found here that although wild-type EPEC allows initial attachment of suspended fibroblasts, EPEC ultimately interferes with host cell spreading and attachment in a TTSS-dependent manner. Presumably, EPEC injects a putative effector(s), which may disrupt the focal adhesion, blocking cell spreading (47). A screening method to identify mutants with insertions in the gene(s) coding for the putative effector(s) was designed and used. We expected that inactivation of the putative effector gene would cause a reduction in the inhibition of host cell spreading and attachment. However, such mutants were not identified upon screening. The mutagenesis method that we used resulted in some "hot spots," where several insertions were found in the same gene. Future analyses may combine the use of several transposons, which would result in more randomized mutagenesis. It is also possible that we could not identify the putative effector because EPEC encodes more than one effector capable of mediating inhibition of host cell attachment.
Nevertheless, mutants with insertions in other genes were recovered, and most of the genes were localized in the LEE. These insertions fully or partially inactivated the TTSS. The mutations of the few non-LEE mutants were localized to genes that included micC and were upstream of bfpP, yehL, and ydeP. Among these mutants, only the mutant with an insertion upstream of ydeP was completely deficient in inhibition of host cell attachment and was therefore analyzed further. The insertion upstream of bfpP probably interfered with biogenesis of the BFP, which is required for efficient host cell infection (4, 13). Furthermore, the BFP mediates bacterium-bacterium interactions (12, 24), which may be necessary for inducing host cell detachment. The insertion upstream of ydeP was associated with reduced inhibition of cell attachment. The ydeP promoter is positively regulated by EvgAS (36), which is highly homologous to BvgAS (57), which is known to regulate Bordetella pertussis virulence. Recently, evgS and ydeP mutants of EHEC serotype O26:H- were found to be attenuated in colonization of calf intestines (58). We thus focused on the role of the EvgAS cascade in regulating production of the LEE and BFP proteins.
Overexpression of the unphosphorylated response regulator mimics two-component system activation, resulting in altered expression of the target genes in the absence of the environmental signals responsible for their phosphorylation (41). This strategy, together with microarray analysis of E. coli K-12, was used to identify five operons that include 15 genes that are directly activated by EvgA. Among these genes is ydeO, which encodes a member of the AraC/XylS family of transcriptional regulators (20, 36). YdeO further activates, directly or indirectly, expression of an additional 18 genes (36), including gadE (yhiE), encoding a LuxR family transcriptional activator (33), and yhiF, encoding a DNA-binding response regulator homolog, which negatively regulates several LEE operons in EHEC O157 (52). Our findings show that evgA overexpression in EPEC represses both the TTSS genes and the BFP genes. The evgA-mediated TTSS and BFP repression is fully suppressed in a strain with a deletion in the ydePO region. In addition, overexpression of ydeO and ydeP is sufficient to cause repression. These results indicate that evgA-mediated repression is dependent on YdeO and YdeP. These proteins might act indirectly, perhaps by inducing production of YhiF and/or YhiE (52). Interestingly, we found that EvgA-mediated repression of ler is not required for repression of the other LEE genes.
A spontaneous, constitutive, active evgS mutant (29) phosphorylates and activates EvgA (14). In addition, a highly specific interaction has been found between the histidine-containing phosphotransfer module of EvgS and EvgA, and their intermolecular phosphorelay has been found to be necessary for EvgA activation (44). Assuming that EvgA is activated upon EvgS stimulation, we predicted that under EvgS-stimulating conditions, the TTSS genes should be repressed in a wild-type EPEC but not in an evgA mutant. However, our comparative study of the EPEC evgA mutant and the wild-type strains under various conditions, including bvgAS-repressing conditions, did not reveal any phenotype. Therefore, the physiological conditions necessary for activation of the EvgS kinase in EPEC remain unknown. Similarly, the nature of the EvgAS activation conditions in E. coli K-12 also remains obscure (42, 64).
An alternative explanation for the lack of a phenotype for the evgA mutant is that the physiological function of the EvgAS system is redundant with respect to other regulatory cascades. Indeed, previous reports showed that there is a partial overlap between the EvgAS and GadXW cascades. This overlap includes activation of YhiE (GadE) and YhiF (36, 55). Moreover, GadX overexpression in EPEC represses the expression of TTSS and BFP genes via expression of the perABC operon (48). The reports mentioned above and the lack of a phenotype in the EPEC evgA mutant led us to construct double mutants with mutations in evgA or ydeO and in gadX or the gadX-gadW region. However, the four double mutants examined did not differ from the wild type.
In conclusion, many LEE regulators, most of which function at the transcriptional level, have been discovered. Here we report that the EvgAS two-component system, apparently activated by an unknown external signal, represses TTSS expression. Identification of the nature of the elusive EvgS activating signal should elucidate the physiological role of EvgAS-mediated repression of the TTSS genes in EPEC.
ACKNOWLEDGMENTS
We thank O. Amster-Choder for providing the anti-SSB antibody and G. Frankel for providing the anti-EspB, -Tir, -Int, -EscJ, and -BfpA antibodies.
This work was supported by grants from The Israel Science Foundation founded by The Israel Academy of Science and Humanities and from the United States-Israel Binational Science Foundation. I.R. is the Etta Rosensohn Professor of Bacteriology.
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ABSTRACT
Upon infection of host cells, enteropathogenic Escherichia coli (EPEC) delivers a set of effector proteins into the host cell cytoplasm via the type III secretion system (TTSS). The effectors subvert various host cell functions. We found that EPEC interferes with the spreading and ultimately with the attachment of suspended fibroblasts or epithelial cells, and we isolated mini-Tn10kan insertion mutants that failed to similarly affect host cells. In most mutants, the insertion sites were mapped to genes encoding TTSS components, including cesD, escC, escJ, escV, espD, sepL, espB, and escF. Other mutants contained insertions in micC or upstream of bfpP, yehL, or ydeP. The insertion upstream of ydeP was associated with a reduction in TTSS protein production and was studied further. To determine whether the apparent repression was due to constitutive expression of the downstream encoded genes, ydeP and ydeO expression vectors were constructed. Expression of recombinant YdeP, YdeO, or EvgA, a positive regulator of both ydeP and ydeO, repressed TTSS protein production. Our results suggest that upon activation of the EvgAS two-component system, EvgA (the response regulator) activates both ydeP and ydeO expression and that YdeP and YdeO act conjointly, directly or indirectly repressing expression of the TTSS genes.
INTRODUCTION
Enteropathogenic Escherichia coli (EPEC) causes infantile diarrhea, which results in the death of several hundred thousand children each year in developing countries (10). EPEC infection is characterized by intimate binding to intestinal enterocytes, localized effacement of absorptive microvilli, transient filopodium formation, and accumulation of polymerized actin beneath the bacteria (26, 46, 54). This histopathology is termed the attaching and effacing lesion (18, 28). The genes responsible for attaching and effacing lesion formation are clustered in the locus of enterocyte effacement (LEE) (37). The LEE consists of 41 genes encoding a type III secretion system (TTSS), as well as several effector proteins. The LEE genes are organized in five major operons, LEE1 to LEE5 (16, 17). Operons LEE2 to LEE5 are positively regulated by Ler, which is encoded by the LEE1 operon (19, 38). Regulation of the LEE1 operon is complex and involves many factors, including H-NS (3, 56), integration host factor (19), Fis (21), PerC (3, 38), BipA (23), GadX (48), GrlA, and GrlR (9), as well as quorum sensing (27, 49). In the closely related organism enterohemorrhagic E. coli (EHEC), the LEE operons are regulated by EtrA, EivF, RpoS, and ClpX (25, 63). For efficient attachment, EPEC uses the plasmid-encoded bundle-forming pilus (BFP) (18, 46). The BFP is encoded by two operons, perABC (bfpTVW) and bfpA-L, which contains 13 genes encoding the pilus structural genes (22, 53).
E. coli K-12 has 32 two-component systems (39), one of which is the EvgAS system, whose physiological role is obscure. EvgS is the sensor histidine kinase, and EvgA is the corresponding response regulator (40, 43, 44). The transcriptome of an E. coli K-12 evgAS mutant has been compared with that of the wild type, using DNA array analysis (42). In addition, the attempts to identify the evgAS mutant phenotype have included a high-throughput phenotype microarray analysis (64), in which nearly 2,000 different phenotypes were examined. However, neither study showed that there was a significant difference between the evgAS mutant and the wild-type strain. Additionally, an in vitro examination revealed low levels of EvgS autophosphorylation and EvgA transphosphorylation (59). The results of these studies may imply that the EvgAS two-component system does not function in E. coli or that the input signal for evgAS activation has not been found.
Here we show that EPEC prevents spreading and ultimately interferes with the attachment of suspended fibroblasts, or epithelial cells, to the substratum. Furthermore, we isolated mini-Tn10kan insertion mutants deficient in inhibition of host cell attachment. These mutants contain insertions in LEE genes, including cesD, escC, escJ, escV, espD, sepL, espB, and escF, in non-LEE genes, including micC, and in the regions upstream of bfpP, yehL, and ydeP. Further characterization of the insertion upstream of ydeP indicated that it is associated with a reduction in TTSS protein production, probably as a result of constitutive expression of ydeP and ydeO. Our results suggest that upon EvgA activation, ydeP and ydeO are expressed and possibly act jointly, directly or indirectly, to repress the expression of the TTSS genes.
MATERIALS AND METHODS
Bacterial strains, plasmids, and oligonucleotides. The strains and plasmids used in this study are listed in Table 1, and the oligonucleotides used are listed in Table 2. Bacteria were grown in either LB broth, M9 minimal medium (35), or Dulbecco's modified Eagle's medium (DMEM) (Sigma). When indicated below, the medium was supplemented with either streptomycin (100 μg/ml), tetracycline (10 μg/ml), ampicillin (50 μg/ml), kanamycin (50 μg/ml), or chloramphenicol (25 μg/ml).
Analysis of inhibition of host cell spreading and attachment. Bacterial cultures that were grown overnight at 37°C in LB broth without shaking were diluted 1:50 in DMEM and grown without shaking under conditions known to stimulate TTSS expression for 3.5 h in the presence of 5% CO2 at 37°C to the mid-logarithmic growth phase (optical density at 600 nm, 0.3), which created preactivated cultures (19). Prior to infection, DU17 fibroblasts (47) were trypsinized, washed with phosphate-buffered saline (PBS), and resuspended in DMEM supplemented with 10% fetal calf serum. A 500-μl suspension of DU17 cells (105 fibroblasts) was infected with 500 μl of a culture of preactivated bacteria (5 x 107 bacteria) at a multiplicity of infection (MOI) of 1:500. The infected cells were seeded onto coverslips in wells of 24-well plates. After 15, 60, or 180 min of infection, cells were fixed for 10 min using 3.7% paraformaldehyde in PBS, washed, permeabilized for 2 min with 0.1% Triton X-100, rewashed, and actin stained using phalloidin-rhodamine (Sigma). Slides were prepared, and micrographs were obtained by fluorescence microscopy.
Mutagenesis and isolation of mutants deficient in blocking host cell attachment. EPEC strain E2348/69 was mutated using a mini-Tn10kan transposon that was delivered into EPEC strain E2348/69 (Str) by mating with E. coli SM10pir (Kanr) containing pLOF/Km (Ampr Kanr), as previously described (8). Transconjugate EPEC colonies were selected on LB medium plates supplemented with kanamycin and streptomycin and tested for ampicillin sensitivity to confirm the lack of plasmid integration into the EPEC chromosome. Each mutated colony was inoculated into LB broth in 96-well tissue plates (master plates) and grown overnight at 37°C. The cultures were then preactivated, maintaining the 96-well format. Concurrently, DU17 fibroblast cultures were trypsinized, washed, and resuspended in DMEM supplemented with 10% fetal calf serum. Using a 96-well plate, 50 μl of suspended DU17 cells (3.4 x 104 fibroblasts) was inoculated with 50-μl portions of different mutants (5 x 106 bacteria) (MOI, 1:66). DU17 was infected with each mutant in duplicate. Infection with wild-type EPEC and infection with the escN mutant served as positive and negative controls, respectively. After 1 h of infection at 37°C, the wells were washed three times with PBS, stained with 100 μl Giemsa stain (Sigma) for 10 min, and then washed with PBS to remove the excess Giemsa stain. The wells were then examined. An inability to block cell attachment resulted in blue staining of a well. Mutants that were unable to block host cell attachment were recovered from the master plates.
Quantitative attachment assay. Using 24-well tissue culture plates, 500 μl of suspended DU17 cells (2.5 x 105 fibroblasts) was infected with 500 μl of preactivated cells of different mutants (5 x 107 bacteria) (MOI, 1:50). Specific wells in each plate in which the cells were infected with wild-type EPEC and with the escN mutant served as positive and negative controls, respectively. After 1 h of infection at 37°C, the wells were washed three times with DMEM and incubated for 20 min with 200 μl lysis buffer (0.1% Triton X-100, 20 mM MgCl2), which specifically lysed the DU17 cells. Next, 180 μl of the lysate was cleared by 5 min of centrifugation (174 x g). The protein concentration of the cleared lysate was directly correlated with the number of attached DU17 cells (data not shown). The protein concentration was measured using a BCA protein assay kit (Sigma). Assays were carried out in triplicate, and standard errors were calculated. The effect of each mutant was compared with the effects of the controls; the effect of wild-type EPEC was defined as 0%, and the effect of the escN mutant was defined as 100%.
Determination of the mini-Tn10kan insertion sites. All mutants were analyzed by PCR using primers for various LEE genes (6) and for the mini-Tn10kan cassette (C1_Tn10_out_R and C4_Tn10_out_F), followed by sequencing (Table 2). DNA fragments containing insertions in non-LEE mutants were cloned. To this end, the total DNA of each mutant was digested with either EcoRI, PstI, MfeI, or SalI and subjected to Southern analysis. As a probe, we used a digoxigenin-labeled kanamycin cassette generated by PCR, using primers C2_Tn10_in_F and C3_Tn10_in_R (Table 2) and a DIG kit (Roche). Digested fragments smaller than 5 kbp were purified from the agarose gel and ligated into pUC18, using either EcoRI, PstI, MfeI, or SalI depending on the enzyme used for genomic DNA fragmentation. Clones containing mini-Tn10kan were selected using LB agar plates supplemented with kanamycin. Sequencing using primers M13_F and M13_R revealed the exact location of the mini-Tn10kan insertion.
Construction of mutants and plasmids. Specific genes were deleted with the aid of the red system, using the DY378 strain or the pKD46 plasmid, as previously described (7, 62). We used pKD3, pKD4 (7), and the chromosomal DNA of Salmonella containing Tn10d-Tet (15) as chloramphenicol, kanamycin, and tetracycline DNA template-resistant cassettes, respectively. The primers used for mutant analysis are shown in Table 2. In some cases, mutations were first generated in DY378 and then transferred into wild-type EPEC harboring pKD46. To generate the expression plasmids, genes were amplified with specific primers (Table 2) and cloned into the pSA10 cloning vector (45) as previously described (2). The plasmid sequence was verified by sequencing, using analysis primers pSA10_F and pSA10_R (Table 2).
Analysis of protein production and secretion under conditions stimulating TTSS and BFP production. One milliliter of preactivated bacteria was centrifuged, and supernatants containing the secreted proteins, as well as pellets containing total proteins, were recovered. Where indicated below, isopropyl--D-thiogalactopyranoside (IPTG) was added 1 h after dilution in DMEM. The protein levels in the different samples were adjusted according to the cell density, and the samples were used for immunoblot analysis. Membranes were incubated with different primary polyclonal rabbit antibodies (anti-EspB, -Tir, -intimin, -Ler, -EscJ, -BfpA, or -SSB) diluted in Tris-buffered saline (150 mM NaCl, 20 mM Tris-HCl; pH 7.5) containing 1% bovine serum albumin. Binding of secondary anti-rabbit immunoglobulin G conjugated to alkaline phosphatase (Sigma) was detected using BCIP (5-bromo-4-chloro-3-indolylphosphate)-nitroblue tetrazolium (Promega).
Conditions used for comparison of the evgA mutant with wild-type EPEC. To create TTSS-repressing conditions, we compared EspB production in overnight cultures grown without shaking in LB broth, either at 37°C or at 27°C, or in M9 minimal medium. To create bvgAS-repressing conditions (31), 5 mM nicotinic acid or 20 mM MgSO4 was added to the LB broth.
Qualitative and quantitative analysis of actin pedestal formation in infected HeLa cells. HeLa cells were infected, fixed, stained with phalloidin-rhodamine, and analyzed by fluorescence microscopy as previously described (61). To quantify pedestal formation efficacy, the number of HeLa cells with more than four actin pedestals in several randomly chosen fields was divided by the total number of HeLa cells, which yielded the percentage of HeLa cells with actin pedestals. About 100 cells were examined for each strain.
RESULTS
EPEC inhibits cell spreading and ultimately interferes with cell attachment by a TTSS-dependent mechanism. EPEC induces infected host cells to detach from the substratum, and the detachment is particularly enhanced in DU17 fibroblasts (1, 47). We tested whether EPEC also prevents the attachment of suspended host cells to the substratum. Adherent DU17 fibroblasts were trypsinized and infected with activated wild-type EPEC or the EPEC escN mutant or not infected. Immediately upon infection, infected cells were seeded, and at different times the unattached cells were washed off. The remaining cells were fixed, actin stained, and examined microscopically (Fig. 1). At 15 min after seeding, infected and uninfected cells were found to be similarly attached. At 60 min after seeding, uninfected cells and cells infected with the escN mutant began to spread. Cells infected with wild-type EPEC failed to spread and later (180 min) detached and were washed away (Fig. 1). In contrast, uninfected cells and cells infected with the escN mutant remained attached and spread. These findings indicate that wild-type EPEC allows initial host cell attachment to the substratum. However, EPEC ultimately interferes with host cell attachment and spreading in a TTSS-dependent manner. We called this sequence of events "attachment inhibition."
Isolation of mutants deficient in inhibiting cell attachment. Using Giemsa staining in a 96-well format, cell attachment was visible to the naked eye as bluish turbidity at the bottom of a well, whereas a lack of attachment resulted in clear wells (Fig. 2B). We used this assay to screen 4,000 mini-Tn10kan mutants of EPEC for a deficiency in preventing fibroblast attachment (Fig. 2). Mutants that repeatedly failed to prevent host cell attachment were analyzed further by (i) mapping the insertion site, (ii) quantifying the inhibition of cell attachment, (iii) testing EspB secretion, and (iv) microscopically observing the infected HeLa cells. The latter procedure was used to analyze the formation of actin pedestals in infected cells and to determine the pattern of bacterial attachment. We identified 26 different mutants; 21 of these mutants contained insertions located within the LEE, and 5 mutants contained insertions located outside the LEE. The 21 LEE mutants had insertions in cesD, escC, escJ, escV, espD, sepL, espB, and escF (Table 3).
In most cases, insertion within the LEE genes resulted in mutants deficient in EspB secretion and in inducing actin pedestal formation. These characteristics were also observed for five of the escV::mini-Tn10kan mutants but not for SN59, which secreted EspB and only partially inhibited cell attachment (26%) (Table 3). The insertion in SN59 was mapped 9 bp upstream of that of SN50, flanking the sequence CGTTATGCG (the boldface C and G are mini-Tn10kan insertion sites in SN59 and SN50, respectively, and the underlined methionine codon represents M79). We speculate that in SN59, the mini-Tn10kan sequence incidentally forms a ribosomal binding site, which allows translation initiation from M79. This may lead to synthesis of truncated EscV that is sufficient for assembly of a partially active TTSS, which may explain the differences in the escV mutant phenotypes.
Another interesting observation was an observation made with the SN67 mutant, which despite its active TTSS, suggested by its EspB secretion and its actin pedestal induction, allowed only partial cell attachment (50%) (Table 3). This may suggest a role for a possible effector; however, the SN67 insertion has been localized to escD, which is known to code for a structural protein that together with EscJ forms a ring-like structure in the TTSS inner membrane (30, 50, 51). The insertion in SN67 is located 12 bp upstream of the M14 codon, which may allow translation initiation from M14, as described for SN59. The putative truncated EscD may support the formation of a partially active TTSS. This suggests that high translocation efficiency may be required to block host cell attachment.
Similar phenotypes were observed in SN6 and SN51 mutants, both of which contained insertions in cesD encoding a TTSS chaperone for EspD and EspB. These mutants exhibited attenuated TTSS activity and allowed cell attachment, like an escN mutant, again suggesting that high translocation efficiency is required to block host cell attachment. In conclusion, aside from mutants with insertions in cesD and escD all LEE mutants exhibited similar phenotypes, which supports the notion that ultimately EPEC inhibition of host cell spreading and attachment is TTSS dependent. In addition, the results validate our screening methodology.
Five non-LEE mutants were isolated, SN61, SN63, SN64, SN66, and SN116 (Table 4). Strain SN66, in which the insertion was in the bfpF-P intergenic region, failed to form microcolonies on infected HeLa cells compared to the wild type (data not shown). Mutants SN61 and SN63 both had an insertion in micC encoding a small RNA that negatively regulated the adjacent ompC porin (5) (Table 4). In SN64, the mini-Tn10kan was localized between two open reading frames, yehI and yehL, in a truncated gene, similar to the E. coli K-12 yehK gene. This is a variable region in different E. coli isolates, and the role of the genes flanking it has not been determined. The insertion site in SN116 was mapped between ydeP and its promoter. Among the non-LEE mutants only SN116 was completely deficient in inhibition of host cell attachment and in EspB secretion. In further studies, therefore, we focused on this mutant.
Overexpression of either evgA, ydeP, or ydeO negatively regulates the production of TTSS and BFP. The mini-Tn10kan insertion in SN116 introduced a constitutive promoter (the kan promoter) that can direct expression of three downstream genes, ydeP, b1500, and ydeO (Table 4). In E. coli K-12 the ydeP gene and the ydeO promoter are activated upon EvgA overexpression (29, 36). Therefore, we tested the hypothesis that the insertion in SN116 mimics activation of the evgAS cascade to downregulate LEE gene expression. To this end, we cloned evgA, ydeP, b1500, or ydeO under control of the IPTG-inducible promoter in pSA10. The wild-type EPEC was transformed with the different plasmids, and the production of different LEE proteins was tested in each strain (Fig. 3A). Overexpression of evgA, ydeP, or ydeO caused decreased production of EscJ, EspB, Tir, intimin, and Ler. Overexpression of ydeP also repressed BfpA production. In contrast, overexpression of b1500 affected neither LEE nor BFP protein production (Fig. 3A).
EvgA-mediated repression of LEE genes is not Ler dependent. Ler is a positive regulator of most of the LEE operons (19, 38). Thus, EvgA-mediated ler repression (Fig. 3A) might be necessary and sufficient for EvgA-mediated repression of the other LEE genes. The other possibility is that EvgA mediates the repression of LEE promoters, regardless of the presence of Ler. To distinguish between these two options, the wild-type EPEC was transformed with two compatible plasmids, pCNY2441 and pTU14, allowing simultaneous evgA and ler expression from a tac promoter. Expression of LEE proteins was compared in wild-type EPEC, in a strain overexpressing evgA, and in a strain overexpressing both evgA and ler. We found that overexpression of the recombinant Ler did not rescue the evgA-mediated repression of Tir and EspB production (Fig. 3B). These results suggest that although EvgA represses ler expression, this repression is not required for the EvgA-mediated downregulation of Tir and EspB production.
Search for EvgAS-activating conditions. We hypothesized that under EvgA-activating conditions an EPEC evgA mutant should overexpress the TTSS genes. In spite of extensive efforts, we could not define EvgA-activating conditions in EPEC or a TTSS-related phenotype in evgA, ydeP, ydeO, or ydeP-ydeO mutants (data not shown). Similarly, neither the environmental conditions that activate EvgA nor a phenotype for the evgAS mutants was found in E. coli K-12 (42, 64). The GadXW regulatory cascade was reported to overlap with the regulatory cascade of EvgAS (34) and to indirectly repress the LEE genes (48). Therefore, double mutants, including evgA/gadX, evgA/gadXW, ydeO/gadX, and ydeO/gadXW mutants, were constructed and tested for differences in expression of TTSS genes under various conditions. However, all of these mutants exhibited a phenotype similar to that of the wild-type strain (data not shown). In conclusion, the environmental conditions that stimulate EvgA activation remain elusive.
EvgA-mediated downregulation of the LEE genes is dependent on the ydeP-ydeO region. We next tested whether EvgA-mediated repression of LEE genes is dependent on ydeP or ydeO. The evgA-expressing vector pCNY2441 was introduced into wild-type EPEC and into mutants CNY2092, CNY2077, and CNY2075 (ydeP::kan, ydeO::kan, and ydePO::kan, respectively). According to an immunoblot analysis, evgA expression resulted in repression of EscJ, EspB, Tir, intimin, Ler, and BfpA production in the wild-type strain but not in the ydePO::kan mutant. Expression of evgA in the ydeP::kan mutant or in the ydeO::kan mutant caused only partial repression of the production of LEE proteins (Fig. 4A). As a negative control, we examined the production of single-strand-binding protein (SSB), a housekeeping protein. SSB expression was not altered in the various strains, indicating that there was a specific effect. These results suggest that EvgA activates the expression of both ydeP and ydeO, which are required for full efficiency of LEE repression.
To substantiate these results, we tested the abilities of the different strains to induce the formation of actin pedestals in infected HeLa cells (Fig. 4B to K). In these experiments, we used wild-type EPEC and a ler mutant as positive and negative controls, respectively (Fig. 4B, I, and K). Overexpression of recombinant evgA, ydeP, or ydeO caused a reduction in the efficiency of actin pedestal formation (Fig. 4C to E and K). However, actin pedestal formation was evident when evgA was expressed in the ydeP::kan and ydeO::kan mutants, and deletion of the entire ydeP-ydeO region (in the ydePO::kan mutant) completely suppressed the ability of EvgA to repress the formation of actin pedestals (Fig. 4F to H and K).
DISCUSSION
EPEC caused host cell detachment in a TTSS-dependent manner (47). Mutants with mutations in the TTSS effector genes (tir, map, espH, and espF) still induce host cell detachment (47; unpublished results). Using a different approach, we found here that although wild-type EPEC allows initial attachment of suspended fibroblasts, EPEC ultimately interferes with host cell spreading and attachment in a TTSS-dependent manner. Presumably, EPEC injects a putative effector(s), which may disrupt the focal adhesion, blocking cell spreading (47). A screening method to identify mutants with insertions in the gene(s) coding for the putative effector(s) was designed and used. We expected that inactivation of the putative effector gene would cause a reduction in the inhibition of host cell spreading and attachment. However, such mutants were not identified upon screening. The mutagenesis method that we used resulted in some "hot spots," where several insertions were found in the same gene. Future analyses may combine the use of several transposons, which would result in more randomized mutagenesis. It is also possible that we could not identify the putative effector because EPEC encodes more than one effector capable of mediating inhibition of host cell attachment.
Nevertheless, mutants with insertions in other genes were recovered, and most of the genes were localized in the LEE. These insertions fully or partially inactivated the TTSS. The mutations of the few non-LEE mutants were localized to genes that included micC and were upstream of bfpP, yehL, and ydeP. Among these mutants, only the mutant with an insertion upstream of ydeP was completely deficient in inhibition of host cell attachment and was therefore analyzed further. The insertion upstream of bfpP probably interfered with biogenesis of the BFP, which is required for efficient host cell infection (4, 13). Furthermore, the BFP mediates bacterium-bacterium interactions (12, 24), which may be necessary for inducing host cell detachment. The insertion upstream of ydeP was associated with reduced inhibition of cell attachment. The ydeP promoter is positively regulated by EvgAS (36), which is highly homologous to BvgAS (57), which is known to regulate Bordetella pertussis virulence. Recently, evgS and ydeP mutants of EHEC serotype O26:H- were found to be attenuated in colonization of calf intestines (58). We thus focused on the role of the EvgAS cascade in regulating production of the LEE and BFP proteins.
Overexpression of the unphosphorylated response regulator mimics two-component system activation, resulting in altered expression of the target genes in the absence of the environmental signals responsible for their phosphorylation (41). This strategy, together with microarray analysis of E. coli K-12, was used to identify five operons that include 15 genes that are directly activated by EvgA. Among these genes is ydeO, which encodes a member of the AraC/XylS family of transcriptional regulators (20, 36). YdeO further activates, directly or indirectly, expression of an additional 18 genes (36), including gadE (yhiE), encoding a LuxR family transcriptional activator (33), and yhiF, encoding a DNA-binding response regulator homolog, which negatively regulates several LEE operons in EHEC O157 (52). Our findings show that evgA overexpression in EPEC represses both the TTSS genes and the BFP genes. The evgA-mediated TTSS and BFP repression is fully suppressed in a strain with a deletion in the ydePO region. In addition, overexpression of ydeO and ydeP is sufficient to cause repression. These results indicate that evgA-mediated repression is dependent on YdeO and YdeP. These proteins might act indirectly, perhaps by inducing production of YhiF and/or YhiE (52). Interestingly, we found that EvgA-mediated repression of ler is not required for repression of the other LEE genes.
A spontaneous, constitutive, active evgS mutant (29) phosphorylates and activates EvgA (14). In addition, a highly specific interaction has been found between the histidine-containing phosphotransfer module of EvgS and EvgA, and their intermolecular phosphorelay has been found to be necessary for EvgA activation (44). Assuming that EvgA is activated upon EvgS stimulation, we predicted that under EvgS-stimulating conditions, the TTSS genes should be repressed in a wild-type EPEC but not in an evgA mutant. However, our comparative study of the EPEC evgA mutant and the wild-type strains under various conditions, including bvgAS-repressing conditions, did not reveal any phenotype. Therefore, the physiological conditions necessary for activation of the EvgS kinase in EPEC remain unknown. Similarly, the nature of the EvgAS activation conditions in E. coli K-12 also remains obscure (42, 64).
An alternative explanation for the lack of a phenotype for the evgA mutant is that the physiological function of the EvgAS system is redundant with respect to other regulatory cascades. Indeed, previous reports showed that there is a partial overlap between the EvgAS and GadXW cascades. This overlap includes activation of YhiE (GadE) and YhiF (36, 55). Moreover, GadX overexpression in EPEC represses the expression of TTSS and BFP genes via expression of the perABC operon (48). The reports mentioned above and the lack of a phenotype in the EPEC evgA mutant led us to construct double mutants with mutations in evgA or ydeO and in gadX or the gadX-gadW region. However, the four double mutants examined did not differ from the wild type.
In conclusion, many LEE regulators, most of which function at the transcriptional level, have been discovered. Here we report that the EvgAS two-component system, apparently activated by an unknown external signal, represses TTSS expression. Identification of the nature of the elusive EvgS activating signal should elucidate the physiological role of EvgAS-mediated repression of the TTSS genes in EPEC.
ACKNOWLEDGMENTS
We thank O. Amster-Choder for providing the anti-SSB antibody and G. Frankel for providing the anti-EspB, -Tir, -Int, -EscJ, and -BfpA antibodies.
This work was supported by grants from The Israel Science Foundation founded by The Israel Academy of Science and Humanities and from the United States-Israel Binational Science Foundation. I.R. is the Etta Rosensohn Professor of Bacteriology.
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