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
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ABSTRACT |
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INTRODUCTION |
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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.
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METHODS |
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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 AatIIPstI 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 0·350·4. 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 57 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).
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RESULTS |
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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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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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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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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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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.
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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Cormack, B. P., Valdivia, R. H. & Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 3338.[CrossRef][Medline]
Donnenberg, M. S. & Kaper, J. B. (1991). Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect Immun 59, 43104317.[Medline]
Donnenberg, M. S., Calderwood, S. B., Donohue-Rolfe, A., Keusch, G. T. & Kaper, J. B. (1990). Construction and analysis of TnphoA mutants of enteropathogenic Escherichia coli unable to invade HEp-2 cells. Infect Immun 58, 15651571.[Medline]
Donnenberg, M. S., Giron, J. A., Nataro, J. P. & Kaper, J. B. (1992). A plasmid-encoded type IV fimbrial gene of enteropathogenic Escherichia coli associated with localized adherence. Mol Microbiol 6, 34273437.[Medline]
Farris, M., Grant, A., Richardson, T. B. & O'Connor, C.D. (1998). BipA: a tyrosine-phosphorylated GTPase that mediates interactions between enteropathogenic Escherichia coli (EPEC) and epithelial cells. Mol Microbiol 28, 265279.[CrossRef][Medline]
Friedberg, D., Umanski, T., Fang, Y. & Rosenshine, I. (1999). Hierarchy in the expression of the locus of enterocyte effacement genes of enteropathogenic Escherichia coli. Mol Microbiol 34, 941952.[CrossRef][Medline]
Girón, J. A., Torres, A. G., Freer, E. & Kaper, J. B. (2002). The flagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells. Mol Microbiol 44, 361379.[CrossRef][Medline]
Goldberg, M. D., Johnson, M., Hinton, J. C. & Williams, P. H. (2001). Role of the nucleoid-associated protein Fis in the regulation of virulence properties of enteropathogenic Escherichia coli. Mol Microbiol 41, 549559.[CrossRef][Medline]
Hay, N. A., Tipper, D. J., Gygi, D. & Hughes, C. (1997). A nonswarming mutant of Proteus mirabilis lacks the Lrp global transcriptional regulator. J Bacteriol 179, 47414746.[Abstract]
Hayashi, F., Smith, K. D., Ozinsky, A. & 7 other authors (2001). The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410, 10991103.[CrossRef][Medline]
Kanamaru, K., Tatsuno, I., Tobe, T. & Sasakawa, C. (2000). SdiA, an Escherichia coli homologue of quorum-sensing regulators, controls the expression of virulence factors in enterohaemorrhagic Escherichia coli O157 : H7. Mol Microbiol 38, 805816.[CrossRef][Medline]
Li, C., Louise, C. J., Shi, W. & Adler, J. (1993). Adverse conditions which cause lack of flagella in Escherichia coli. J Bacteriol 175, 22292235.[Abstract]
McDaniel, T. K. & Kaper, J. B. (1997). A cloned pathogenicity island from enteropathogenic Escherichia coli confers the attaching and effacing phenotype on E. coli K-12. Mol Microbiol 23, 399407.[Medline]
Mellies, J. L., Elliott, S. J., Sperandio, V., Donnenberg, M. S. & Kaper, J. B. (1999). The Per regulon of enteropathogenic Escherichia coli: identification of a regulatory cascade and a novel transcriptional activator, the locus of enterocyte effacement (LEE)-encoded regulator (Ler). Mol Microbiol 33, 296306.[CrossRef][Medline]
Nishida, S., Mizushima, T., Miki, T. & Sekimizu, K. (1997). Immotile phenotype of an Escherichia coli mutant lacking the histone-like protein HU. FEMS Microbiol Lett 150, 297301.[CrossRef][Medline]
Osuna, R., Lienau, D., Hughes, K. T. & Johnson, R. C. (1995). Sequence, regulation, and functions of fis in Salmonella typhimurium. J Bacteriol 177, 20212032.[Abstract]
Ottemann, K. M. & Miller, J. F. (1997). Roles for motility in bacterialhost interactions. Mol Microbiol 24, 11091117.[Medline]
Rosenshine, I., Ruschkowski, S. & Finlay, B. B. (1996). Expression of attaching/effacing activity by enteropathogenic Escherichia coli depends on growth phase, temperature, and protein synthesis upon contact with epithelial cells. Infect Immun 64, 966973.[Abstract]
Sanchez-SanMartin, C., Bustamante, V. H., Calva, E. & Puente, J. L. (2001). Transcriptional regulation of the orf19 gene and the tir-cesT-eae operon of enteropathogenic Escherichia coli. J Bacteriol 183, 28232833.
Shi, W., Zhou, Y., Wild, J., Adler, J. & Gross, C. A. (1992). DnaK, DnaJ, and GrpE are required for flagellum synthesis in Escherichia coli. J Bacteriol 174, 62566263.[Abstract]
Shi, W., Louise, C. J. & Adler, J. (1993). Mechanism of adverse conditions causing lack of flagella in Escherichia coli. J Bacteriol 175, 22362240.[Abstract]
Shin, S. & Park, C. (1995). Modulation of flagellar expression in Escherichia coli by acetyl phosphate and the osmoregulator OmpR. J Bacteriol 177, 46964702.[Abstract]
Silverman, M. & Simon, M. (1974). Characterization of Escherichia coli flagellar mutants that are insensitive to catabolite repression. J Bacteriol 120, 11961203.[Medline]
Soutourina, O., Kolb, A., Krin, E., Laurent-Winter, C., Rimsky, S., Danchin, A. & Bertin, P. (1999). Multiple control of flagellum biosynthesis in Escherichia coli: role of H-NS protein and the cyclic AMP-catabolite activator protein complex in transcription of the flhDC master operon. J Bacteriol 181, 75007508.
Sperandio, V., Mellies, J. L., Nguyen, W., Shin, S. & Kaper, J. B. (1999). Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic Escherichia coli. Proc Natl Acad Sci U S A 96, 1519615201.
Sperandio, V., Mellies, J. L., Delahay, R. M., Frankel, G., Crawford, J. A., Nguyen, W. & Kaper, J. B. (2000). Activation of enteropathogenic Escherichia coli (EPEC) LEE2 and LEE3 operons by Ler. Mol Microbiol 38, 781793.[CrossRef][Medline]
Sperandio, V., Torres, A. G. & Kaper, J. B. (2002a). Quorum sensing Escherichia coli regulators B and C (QseBC): a novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in E. coli. Mol Microbiol 43, 809821.[CrossRef][Medline]
Sperandio, V., Li, C. C. & Kaper, J. B. (2002b). Quorum-sensing Escherichia coli regulator A: a regulator of the LysR family involved in the regulation of the locus of enterocyte effacement pathogenicity island in enterohemorrhagic E. coli. Infect Immun 70, 30853093.
Tzipori, S., Karch, H., Wachsmuth, K. I., Robins-Browne, R. M., O'Brien, A. D., Lior, H., Cohen, M. L., Smithers, J. & Levine, M. M. (1987). Role of a 60-megadalton plasmid and Shiga-like toxins in the pathogenesis of infection caused by enterohemorrhagic Escherichia coli O157 : H7 in gnotobiotic piglets. Infect Immun 55, 31173125.[Medline]
Received 29 August 2002;
revised 6 January 2003;
accepted 10 January 2003.