Integration host factor (IHF) mediates repression of flagella in enteropathogenic and enterohaemorrhagic Escherichia coli

Chen Yona-Nadler1, Tatiania Umanski1, Shin-Ichi Aizawa2, Devorah Friedberg1 and Ilan Rosenshine1

1 Department of Molecular Genetics and Biotechnology, The Hebrew University, Faculty of Medicine, POB 12272, Jerusalem 91120, Israel
2 Department of Biosciences, Teikyo University, 1-1 Toyosatodai, Utsunomiya 320-8551, Japan

Correspondence
Ilan Rosenshine
ilanro{at}cc.huji.ac.il


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The flagellar apparatus consists of components that function as a type III secretion system (TTSS). Enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC, respectively) produce an additional TTSS, which is involved in virulence via the translocation of effector proteins into infected host cells. This system is encoded by the locus of enterocyte effacement (LEE). The authors observed that EPEC and EHEC grown in Dulbecco's modified Eagle's medium to the mid- and late-exponential growth phase at 37 °C are non-motile. At the same time these conditions trigger the expression of the LEE-encoded TTSS. Furthermore, it was found that EPEC with an inactivated ihfA, which encodes the IHF{alpha} subunit of the integration host factor (IHF), becomes hyperflagellated and motile. Similar hypermotility was seen upon inactivation of the ihfA of EHEC strains. IHF-mediated repression of the EPEC flagella involves down-regulation of flhDC, which encodes a positive regulator of the flagellar regulon. IHF indirectly mediates flhDC repression, via a putative EPEC-unique regulator which is not encoded by LEE.


Abbreviations: DMEM, Dulbecco's modified Eagle's medium; EHEC, enterohaemorrhagic E. coli; EPEC, enteropathogenic E. coli; IHF, integration host factor; LEE, locus of enterocyte effacement; TTSS, type III secretion system


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Flagella play a role in bacterial adaptation to environmental conditions and have often been associated with the virulence of various pathogens (Ottemann & Miller, 1997). The flagellar system of Escherichia coli and Salmonella typhimurium is encoded by over 40 genes (Aldridge & Hughes, 2002). These genes are organized into several co-regulated operons. The flhDC operon encodes FlhD and FlhC, which act as positive regulators of the flagellar regulon. The pattern of flhDC expression varies in different bacterial species and strains and is modulated in response to environmental and physiological signals, according to the adaptability characteristics of a given strain. flhDC expression is influenced by signals such as variations in temperature and osmolarity (Li et al., 1993; Shi et al., 1993; Shin & Park, 1995), by regulators such as cAMP-catabolite activator protein (CAP), H-NS, HU, DnaK, DnaJ, GrpE, Fis and Lrp, and by a quorum-sensing factor (Silverman & Simon, 1974; Soutorina et al., 1999; Nishida et al., 1997; Shi et al., 1992; Osuna et al., 1995; Hay et al., 1997; Sperandio et al., 2002a, b).

The flagellum consists of components that function as a type III secretion system (TTSS), facilitating the export of flagellar proteins and flagellar assembly (Aldridge & Hughes, 2002). Enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC) contain an additional TTSS. This system is encoded by several operons located in the locus of enterocyte effacement (LEE) (McDaniel & Kaper, 1997). The LEE-encoded TTSS mediates the injection of virulence factors into infected mammalian cells. Most of the LEE operons are positively regulated by the Ler regulator, which is encoded by the LEE1 operon (Friedberg et al., 1999; Mellies et al., 1999; Sperandio et al., 2000; Sanchez-SanMartin et al., 2001). Integration host factor (IHF) directly activates the expression of Ler (Friedberg et al., 1999). Additional factors, including Fis, Per and a quorum sensing regulator, are also involved in the modulation of LEE1 expression (Goldberg et al., 2001; Kanamaru et al., 2000; Mellies et al., 1999; Sperandio et al., 1999, 2002a, b).

The optimal condition for expression of LEE genes by EPEC is growth of the cells to mid-exponential phase in Dulbecco's modified Eagle's medium (DMEM; Sigma) at 37 °C (Rosenshine et al. 1996). We show here that under these conditions EPEC cells are non-motile and do not express flagella. We found that repression of flagellar expression in EPEC involves silencing of flhDC by IHF. We further demonstrate that IHF mediates flhDC repression indirectly.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, culture conditions and oligonucleotide primers.
The bacterial strains were grown overnight as standing cultures in LB at 37 °C, diluted 1 : 50 in LB or buffered DMEM, as indicated, and grown to a density of OD600 0·3–0·35. When necessary, IPTG (1 mM) was added 1 h before harvesting the cultures. Antibiotics were added at the following concentrations: ampicillin (Amp), 100 µg ml-1; kanamycin (Kan), 40 µg ml-1; chloramphenicol (Cm), 25 µg ml-1; and streptomycin (Str) 100 µg ml-1. The bacterial strains and plasmids used in this study are listed in Table 1; the oligonucleotides are listed in Table 2.


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Table 1. Bacterial strains and plasmids

 

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Table 2. Oligonucleotides

 
Construction of plasmids.
Plasmid pDF12, containing the transcriptional fusion of flhDCEPEC with gfp-mut3 (Cormack et al., 1996), was constructed as follows. A DNA fragment starting 415 bp upstream from the putative flhDCEPEC translational start point, and 220 bp upstream from a putative transcriptional start point (Soutourina et al., 1999) including the flhDCEPEC coding region, was amplified using primers flh-F5 and flh-R5 with EPEC DNA serving as a template. The amplified DNA fragment was digested with BamHI and XbaI and cloned into the corresponding sites in pIR1 (Friedberg et al., 1999).

Plasmid pDF13, expressing flhDCEPEC from the T5-lac promoter, was constructed as follows. A fragment including flhDCEPEC was amplified using primers flh-F31 and flh-R1 and cloned into a pACYC184 derivative containing lacIq and the T5-lac promoter/operator element, ribosome-binding site and cloning site derived from the expression vector pQE31 (Qiagen). The fragment was cloned into the BamHI and HindIII sites of the vector, in-frame with the hexahistidine (6xhis) tag.

To construct plasmid pDF14, expressing ihfAB from the lac promoter, an AatII–PstI DNA fragment carrying the ihfAB genes expressed from the lac promoter was isolated from plasmid pDRC171 and ligated into the corresponding sites in pACYC177.

Construction of EPEC lacking the IHF-binding site upstream of LEE1.
A 632 bp DNA fragment was amplified using primers 18F1 and 17R, digested with BamHI and XbaI, and ligated into pBS to form pBS-fragment 1. A 656 bp DNA fragment was amplified by PCR using primers 16F and 19R, digested with HindIII and EcoRI, and ligated into pBS-fragment 1 to form pDF9. The insert in pDF9 consists of an EPEC DNA fragment extending from -724 to +540 bp relative to the LEE1 transcription start point, but including a deletion of 20 nt extending from -66 to -87 which is part of an IHF-binding site (Friedberg et al., 1999). The insert was recovered by digestion with XbaI and Eco0109I; the fragment ends were filled in using the Klenow fragment and ligated into the SmaI site of pCVD442 to generate pDF10. We used pDF10 to construct an EPEC strain (designated DF4), with a deleted IHF-binding site from -66 to -87 bp upstream to the LEE1 transcription start point. The allelic exchange was carried out as described by Donnenberg & Kaper (1991).

Measurement of gene expression by flow cytometry.
Gene expression of bacterial strains containing gfp fusions was monitored by flow cytometry as previously described (Friedberg et al., 1999).

Protein extraction and immunoblot analysis.
Overnight EPEC cultures were diluted 1 : 50 in DMEM and grown at 37 °C to a density of OD600 0·35–0·4. When necessary, IPTG (1 mM) was added immediately upon inoculation to induce ihfA, or 2·5 h after DMEM inoculation to induce ler expression. The cultures were centrifuged and the bacteria were lysed by boiling in SDS loading buffer, as previously described (Rosenshine et al., 1996). The protein concentration in the samples was adjusted, and the samples were subjected to SDS-PAGE and transferred to nitrocellulose membranes (AB-S 83; Schleicher & Schuell). Blots were incubated with polyclonal antibodies in TBS (150 mM NaCl, 20 mM Tris/HCl, pH 7·5) containing 1 % BSA. Binding of secondary anti-rabbit IgG antibody conjugated to alkaline phosphatase (Sigma) was detected with BCIP/NBT (Promega).

Motility tests.
Motility was screened by microscopy and on swarm plates containing a bottom layer of LB-1·5 % agar and a top layer of LB-0·15 % agar. Colonies grown overnight on LB agar plates were inoculated onto swarm plates and incubated for 5–7 h at 37 °C.

Invasion assay.
Invasion of HeLa cells was monitored by the gentamicin protection assay as previously described (Rosenshine et al., 1996). When necessary, IPTG (1 mM) was added.

Fluorescence microscopy.
HeLa cells were seeded and grown overnight on glass coverslips in 24-well plates containing 1 ml DMEM per well. The cells were then infected with 5 µl of an EPEC culture grown overnight at 37 °C in standing cultures of LB. Infection was terminated after 3·5 h and 6 h by fixation of the cells for 30 min in PBS containing 4 % paraformaldehyde. The fixed cells were washed with PBS, permeabilized for 5 min with 0·1 % Triton X-100 in PBS and washed as before. The actin filaments were stained by overlaying the coverslips with 20 µl (1 : 100 in TBS) phalloidin-rhodamine (Sigma). Flagellin was identified using anti-H6(FliC) antiserum (Israeli Ministry of Health; IMH), and secondary anti-rabbit-Alexa-488 conjugated antibody (1 : 100, Sigma).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
IHF represses flagellar expression in EPEC
EPEC can be ‘pre-activated’ to express the LEE-encoded TTSS by growth in DMEM to mid-exponential phase, at 37 °C. Microscopy showed that the activated EPEC cells were typically non-motile. We also found that the EPEC ihfA : : kan mutant was highly motile. The motility of the mutant appeared to be constitutive, independent of growth phase or medium. Repression of motility was restored upon transforming the EPEC ihfA : : kan with a plasmid encoding the wild-type ihfA allele.

To further validate these observations we grew EPEC strains in DMEM to mid-exponential phase, extracted the bacterial proteins and subjected them to Western blotting, using antibodies raised against flagellar components FliC, FliG and FliA and against the TTSS component EspA. We compared the expression of these proteins in three EPEC strains: wild-type, ihfA : : kan, and ihfA : : kan containing a plasmid expressing ihfA from the lac promoter (Fig. 1A). The results confirmed previous observations that IHF is required for expression of the TTSS components (Friedberg et al., 1999). In contrast, IHF suppressed production of the flagellar proteins. When we compared the motility of the above three EPEC strains on a swarm plate (Fig. 1B), the results were in agreement with the Western blot analysis. The EPEC ihfA : : kan mutant was highly motile and the motility of the strains expressing IHF, either genomic or in trans from a plasmid, was attenuated (Fig. 1B). Electron micrographs of the wild-type cells and the ihfA : : kan mutant grown in DMEM showed that the EPEC wild-type cells either do not possess flagella or rarely (about 5 % of the bacteria) show a single flagellum. In contrast, the ihfA : : kan mutant produced numerous peritrichous flagella (Fig. 1C).



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Fig. 1. Inactivation of IHF in EPEC induces flagellar expression and motility. (A) Immunoblot analysis. Proteins were extracted from wild-type EPEC, the EPEC ihfA : : kan mutant and the EPEC ihfA : : kan mutant complemented with pDF14 (EPEC ihfA : : kan/ihfA). Proteins were analysed by immunoblotting, using antibodies raised against flagellar components (FliC, FliG and FliA) and the LEE TTSS component EspA. (B) Swarm plates were used to test the motility of wild-type EPEC, the EPEC ihfA : : kan mutant and the genetically complemented mutant EPEC ihfA : : kan/ihfA. (C) Electron micrographs, negatively stained, of wild-type EPEC and the EPEC ihfA : : kan mutant. Bars, 1 µm.

 
Next, we tested whether similar IHF-mediated repression of the flagella operates in EHEC strains. To this end, we inactivated ihfA in two EHEC O157 isolates: EDL933 and 85-170. Using microscopy of cultures grown in DMEM, the wild-type strains were compared with the mutated strains and with the mutated strains complemented by a plasmid expressing ihfA. The results showed that, like EPEC, EHEC ihfA : : kan become hypermotile, whereas the wild-type strain and the complemented mutants were typically non-motile (data not shown).

IHF does not repress flagellar expression in E. coli K-12 and N99 strains
We examined whether the role of IHF as a suppressor of flagellar synthesis is unique to EPEC and EHEC or is a general characteristic of E. coli strains. We compared E. coli K-12 W3110 and E. coli N99 with their isogenic ihfA mutants. In contrast to EPEC, the ihfA mutation of W3110 did not result in increased expression of fliC (Fig. 2A) or in increased motility, as seen under the microscope (data not shown) and confirmed by swarm plates (Fig. 2B). In the N99 strain, the ihfA mutation resulted in the opposite effect: i.e. the ihfA mutant exhibited reduced flagellin synthesis (Fig. 2A) and showed reduced motility as compared with wild-type N99, as revealed by microscopy (data not shown) and by swarm plates (Fig. 2B). These findings indicate that IHF does not mediate flagellar repression in E. coli strains W3110 or N99.



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Fig. 2. IHF does not mediate the repression of motility and flagellin production in E. coli strains W3110 and N99. (A) Immunoblot analysis. Proteins were extracted from wild-type and ihfA : : kan mutants of E. coli strains W3110 and N99 grown in DMEM to mid-exponential phase. The extracted proteins were subjected to SDS-PAGE followed by immunoblot analysis, using anti-flagellin antibodies. (B) Swarm plates were used to test the motility of W3110 and N99 wild-type and isogenic ihfA : : kan mutants.

 
IHF indirectly mediates repression of flhDCEPEC
Based on the results shown in Fig. 1(A), we hypothesized that in EPEC, IHF represses the expression of the flhDC operon, which encodes the flagellar master regulator. To test this prediction, we constructed a plasmid carrying a transcriptional fusion (designated pDF12 or pflhDCEPEC-gfp) between the gfp reporter gene and an EPEC DNA fragment containing the complete flhDC coding region and its upstream region. Introduction of the plasmid into the EPEC wild-type resulted in marked activation of motility, as revealed by microscopy and swarm plates (data not shown). The multiple copy-number of flhDCEPEC-gfp probably overcame the repression mediated by the chromosomal IHF. To test the effect of IHF on the plasmid-expressed flhDCEPEC-gfp, we constructed a compatible plasmid based on pACYC177 that encodes the ihfAB genes expressed from the lac promoter (designated pDF14 or pihfAB). Introduction of pihfAB into EPEC/pflhDCEPEC-gfp resulted in strong inhibition of EPEC motility, observed by microscopy (data not shown) and by swarm plates (Fig. 3A). To analyse the role of IHF on flhDCEPEC expression, we compared gfp expression in EPEC/pflhDCEPEC-gfp containing or not containing pihfAB (Fig. 3B). The results clearly indicate that in EPEC, supplying IHF in trans strongly repressed expression of flhDCEPEC-gfp.



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Fig. 3. IHF represses the EPEC flhDC operon in EPEC but not in E. coli W3110. (A) Motility test. EPEC containing plasmid pflhDCEPEC-gfp expressing the flhDCEPEC-gfp fusion, in the absence or presence of plasmid pihfAB expressing IHF{alpha} and IHF{beta}. (B) Flow cytometry. EPEC/pflhDCEPEC-gfp and W3110/pflhDCEPEC-gfp containing or lacking pihfAB were grown in DMEM. Fluorescence intensity, which reflects the levels of flhDC expression, was measured by flow cytometry. The values are presented as means of three experiments, with standard error bars.

 
It is expected that if IHF represses flagellar expression by direct interaction with the regulatory region of flhDCEPEC, the inhibition should also take place in W3110. To test this possibility, we transformed E. coli W3110 with pflhDCEPEC-gfp or with both pflhDCEPEC-gfp and pihfAB and compared gfp expression in these strains. In contrast to EPEC, supplying IHF in trans to W3110 did not affect flhDCEPEC-gfp expression (Fig. 3B). These results suggest that: (1) IHF mediates flhDC expression in EPEC indirectly by affecting the expression of a putative regulator and (2) this putative regulator does not function or is absent from W3110 and may be unique to EPEC.

LEE genes are not involved in IHF-mediated flagellar repression
The LEE genes are unique to EPEC, EHEC and closely related strains, and their expression is positively regulated by IHF. This raises the possibility that the putative flhDC repressor of EPEC is encoded by the LEE. To test this hypothesis, we supplied ler in trans to the EPEC ihfA : : kan mutant, using a plasmid expressing ler from the ptac promoter. Expression of the recombinant Ler activated the expression of most of the LEE genes including LEE2, LEE3, LEE4, LEE5 and the EspG transcriptional units. However, EPEC ihfA : : kan exhibited a high motility and efficient flagellin production even upon expression of the recombinant Ler (Figs 4C and 4A). This indicates that the Ler-regulated LEE genes do not encode the putative flhDC regulator.



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Fig. 4. Expression of TTSS LEE genes does not affect flagellin expression and motility of EPEC strains. (A) Immunoblot analysis of proteins extracted from the EPEC strains grown in DMEM to mid-exponential phase: wild-type (lane 1), EPEC/pflhDC (lane 2), ihfA : : kan (lane 3), ihfA : : kan/pler (lane 4) and a mutant with a deletion in the IHF binding site – {Delta}IHF-BS (lane 5). The blot was developed with anti-flagellin antibodies. MW, size markers (kDa). (B–D) Relative motility of EPEC strains (strain numbers, indicated in parentheses, correspond to the lane numbers of the EPEC strains used for immunoblot analysis in (A). (E) The {Delta}IHF-BS mutant is deficient in Ler expression. The mutant and wild-type EPEC, as positive control, were grown to a density of OD600 0·35–0·4 in DMEM and the extracted proteins were analysed by Western blotting with anti-Ler antibody.

 
To further exclude the involvement of LEE genes in flhDC regulation, we constructed a strain (DF4) including a deletion of 20 nt, consisting of the IHF-binding site upstream from the LEE1 promoter. We confirmed by Western blot analysis, using anti-Ler antibodies, that the mutant does not produce Ler (Fig. 4E). This mutant did not express any of the tested LEE operons, including LEE1, LEE2, LEE3, LEE4, LEE5 and espG (data not shown). However, motility was repressed and there was a low level of flagellin production, similar to that of wild-type EPEC (Figs 4A and 4D). The results obtained from the swarm plates were confirmed by microscopy. Cumulatively, these results indicate that the LEE genes are not involved in the repression of flagellar expression.

Flagella and the LEE TTSS are both functional upon co-expression
The biological significance of IHF-mediated flagellar repression in EPEC is not clear. One possibility is that the flagellar system is repressed because it interferes with the function of the LEE-encoded TTSS. To test this hypothesis, a plasmid expressing flhDCEPEC from the ptac promoter (pDF13 or ptac-flhDC) was constructed and transformed into the EPEC wild-type and into EPEC bfpA : : TnphoA strain 36-6-1(1). The latter mutant strain does not express the bundle-forming pili mediating bacterial aggregation (Donnenberg et al., 1992). The two isogenic pairs of strains were used to infect HeLa cells and tested for several functions. These included motility, BFP-mediated aggregation, expression and secretion of EspA, invasion, and formation of actin pedestals. Microscopy indicated that the strains containing ptac-flhDC were highly motile, whereas the corresponding wild-type and bfpA : : TnphoA mutant, lacking ptac-flhDC, were non-motile. In contrast, expression of recombinant flhDC had little effect on all the other tested functions (Fig. 5A, B, and data not shown).



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Fig. 5. Co-expression of the LEE TTSS and the flagellar TTSS. (A) Expression of EspA by wild-type EPEC and EPEC/ptac-flhDC was compared by immunoblot analysis using anti-EspA antibody. (B) Comparison of the invasiveness of wild-type EPEC, EPEC/ptac-flhDC, EPEC bfpA : : TnphoA, bfpA : : TnphoA/ptac-flhDC using the gentamicin protection assay. The invasion assays were repeated twice with similar results and the results of one of these experiments are shown. The assays were carried out in quadruplicate. Standard deviation values are shown. (C) HeLa cells were infected with EPEC bfpA : : TnphoA/ptac-flhDC, fixed, stained with phalloidin-rhodamine (red) and anti-H6 antibody (green), and analysed by fluorescence microscopy. The fluorescent image (right panels) and the corresponding phase-contrast image (left panels) are shown. White arrowheads point to flagellated bacteria associated with actin pedestals; white arrows (middle panels) point to non-flagellated bacteria associated with actin pedestals; pink arrows point to flagellated bacteria associated with the HeLa cell surface but not with actin pedestals. Bar (below figure), 1 µm.

 
Using fluorescence microscopy, we tested whether a single bacterium can simultaneously express flagella and a functional TTSS. Formation of flagellar filaments was used as an indicator of the activity of the flagellar TTSS, and generation of actin pedestals was used as an indicator of functional LEE TTSS. The EPEC bfpA : : TnphoA mutant was used to prevent microcolony formation, which complicates the analysis of a single bacterium. We infected HeLa cells with a bfpA : : TnphoA strain containing pflhDC to drive flagellar expression. Infection was carried out for 3·5 and 6 h and infected cells were fixed and double stained with anti-H6-flagellin antibodies and phalloidin-rhodamine, which stains the actin pedestals. At 3·5 h post-infection most of the bacterial cells attached to the actin pedestals were flagellated (Fig. 5C). This indicates that upon co-expression both the flagellar TTSS and the LEE TTSS are functional.

Interestingly, at 6 h post-infection many of the pedestal-associated bacteria appeared to be non-flagellated, whereas the bacteria associated with HeLa cells, but not with actin pedestals, were mostly flagellated (Fig. 5C). The unattached bacteria were motile and flagellated as well, implying a homogeneous bacterial population. The differential flagellar expression is probably not due to differential flhDC expression, as in these experiments flhDC was expressed from the ptac promoter. The significance of the disappearance of the flagella from EPEC bacteria associated with actin pedestals during late stages of HeLa cell-infection is not clear.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study we report that expression of flagella in EPEC is repressed by IHF. We further show that IHF represses the flhDC operon, which encodes the master positive regulator of the flagellar regulon. In addition, we demonstrate that IHF-mediated repression of flhDC is unique to EPEC and EHEC and is not found in all E. coli strains. Moreover, the EPEC flhDC operon is repressed in EPEC but not in other E. coli strains. Cumulatively our results indicate that in EPEC, IHF mediates repression of flhDC transcription indirectly via a putative regulator. This putative regulator appears to be unique to EPEC and not present in E. coli K-12. Alternatively, it may be encoded but not expressed in E. coli K-12.

The difference in motility between the non-motile wild-type EPEC and the highly motile EPEC ihfA mutant was particularly enhanced in EPEC grown in DMEM at 37 °C to mid-exponential phase. Interestingly, these conditions are optimal for expression of the LEE1 operon, including ler, the positive regulator of the LEE region. These observations, as well as the positive effect of IHF on ler expression, raised the possibility that the putative unique EPEC factor that represses the flagella is encoded by LEE. We examined this possibility using several approaches, but could not find any evidence in favour of this hypothesis. Therefore, we are now using genomic screening to identify the putative EPEC factor that represses flhDC.

The EPEC ihfA mutant fails to induce actin rearrangement upon interaction with epithelial cells, and becomes highly motile. This phenotype is similar to that of the EPEC bipA mutant previously reported by Farris et al. (1998). Therefore, it is conceivable that, like IHF, BipA is also required for expression of the LEE genes and repression of flhDC. It remains to be seen whether IHF and BipA interact in some way to bring about such regulation. A recent report by Girón et al. (2002) suggests that a factor released by the epithelial cells induces flagellar expression in infecting EPEC cells and that the flagella mediate EPEC adherence to epithelial cells. The identity of this putative host factor and its mechanism of function remain obscure. According to our analyses, wild-type EPEC is non-flagellated and non-motile in the absence as well as in the presence of host cells. Perhaps the cell line that we are using does not produce the putative host factor which induces expression of flagella. It is not evident whether IHF, BipA and the putative host factor utilize the same regulatory cascade to regulate flhDC expression.

What is the biological rationale of flagellar repression in EPEC? One possibility is that the flagella interfere with the function of the LEE-encoded TTSS. We tested this concept by constructing an EPEC strain co-expressing the flagella and the LEE-encoded TTSS. Our results indicate that they coexist within the same EPEC cell. Another possibility is that flagellar repression in EPEC acts to lower the level of flagellin, which is a TLR5 ligand (Hayashi et al., 2001), thereby reducing the host's inflammatory response.


   ACKNOWLEDGEMENTS
 
We thank N. Takahashi for her helpful assistance, and A. Oppenheim for the plasmids. The research was supported by grants from the Israel Ministry of Science, the Israel Ministry of Health, the Israel–United States Binational Foundation and the European Union Fifth Framework Quality of Life Program (grant QLK2-2000-00600), T. Umanski is supported by a grant from the Bohringer Foundation.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 29 August 2002; revised 6 January 2003; accepted 10 January 2003.