The ﬂagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells

© 2002 Blackwell Science Ltd

The flagella of enteropathogenic Escherichia colimediate adherence to epithelial cells

bacteria and supports the existence of a molecularrelationship between the two existing type III secre-tion pathways of EPEC, the EPEC adherence factor(EAF) plasmid-encoded regulator, quorum sensingand epithelial cells.

Introduction

Several pathogenic bacteria, including diarrhoeagenicEscherichia coli strains have evolved sophisticated viru-lence-associated type III protein secretion systems thatare structurally and biochemically similar to the flagellartype III export apparatus involved in flagella assembly and motility (Menard et al., 1994; Wattiau et al., 1994;Jarvis et al., 1995; Rosqvist et al., 1995;Yahr et al., 1996;Roine et al., 1997; Hueck, 1998; Lory, 1998; Celli et al.,2000). These secretion machineries function as tran-slocons that deliver virulence factors across the bacterialcell envelope to their targeted sites in the host membranecell or into the cytosolic compartment, where they actdirectly or indirectly with components of the cellularcytoskeleton.

In the human small intestine, enteropathogenicEscherichia coli (EPEC), the most important bacterialcause of infant diarrhoeal disease in the developing world,causes characteristic attaching and effacing (AE) intesti-nal lesions (Staley et al., 1969; Moon et al., 1983; Nataroand Kaper, 1998). These lesions are manifested by criti-cal damage to the integrity of the enterocyte cytoskele-ton, which involves intimate attachment and activation of signal transduction pathways and rearrangements ofcytoskeletal proteins (reviewed in Frankel et al., 1998;Nataro and Kaper, 1998). The effector molecules respon-sible for these events are proteins encoded by chromo-somal genes that map to a pathogenicity island called thelocus of enterocyte effacement (LEE) (McDaniel et al.,1995). The LEE encodes an adhesin called intimin (Jerseet al., 1990), its translocated intimin receptor (Tir) (Kennyet al., 1997a) and components of the type III secretorycomplex, which is responsible for export of the secretedEsp proteins (EspA, EspB, EspD and EspF), and the LEE-encoded regulator (Ler), which controls the expression ofLEE genes (Elliott et al., 1998; Mellies et al., 1999). EspAis thought to form a hollow filamentous structure thatserves to mobilize virulence factors across the cell mem-brane (Frankel et al., 1998; Knutton et al., 1998; Sekiyaet al., 2001). In addition, EPEC strains contain a large

Molecular Microbiology (2002) 44(2), 361–379

Jorge A. Girón*1,2, Alfredo G. Torres2, Enrique Freer3

and James B. Kaper2

1Centro de Investigaciones en Ciencias Microbiológicas,Instituto de Ciencias, Benemérita UniversidadAutónoma de Puebla, México.2Department of Microbiology and Immunology, andCenter for Vaccine Development, University ofMaryland School of Medicine, Baltimore, MD 21201,USA.3Unidad de Microscopía Electrónica, Universidad deCosta Rica.

Summary

Enteropathogenic Escherichia coli (EPEC) utilizes atype III protein secretion system to target effectormolecules into the host cell leading to effacement of the intestinal mucosa. This secretion apparatusshares many structural features of the flagellar typeIII export system involved in flagella assembly andmotility. We report here that fliC insertional mutantsconstructed in two wild-type EPEC strains weremarkedly impaired in adherence and microcolony for-mation on cultured cells. An E. coli K-12 strain har-bouring the EPEC H6 fliC gene on a plasmid showeddiscrete adhering clusters on HeLa cells, albeit toless extent than the wild-type EPEC strain. Flagellapurified from EPEC bound to cultured epithelial cellsand antiflagella antibodies blocked adherence ofseveral EPEC serotypes. We determined that eukary-otic cells in culture stimulate expression of flagella by motile and non-motile EPEC. Isogenic strainsmutated in perA (a transcriptional activator), bfpA(a type IV pilin), luxS (a quorum-sensing autoinducergene) and in the type III secretion genes were reducedfor motility in Dulbecco’s modified Eagle medium(DMEM) motility agar and produced none or few flagella when associated with epithelial cells. Growthof these mutants in preconditioned tissue culturemedium restored motility and their ability to produceflagella, suggesting the influence of a signal providedby mammalian cells that triggers flagella production.This study shows for the first time that the flagella ofEPEC are directly involved in the adherence of these

Accepted 21 January, 2002. *For correspondence: [email protected]; Tel. (+1) 410 706 3004; Fax (+1) 410 706 6205.

92 kb plasmid that codes for a type IV bundle-formingpilus (BFP) (Girón et al., 1991) associated with bacterialclustering and formation of tight microcolonies on tissueculture cells and human intestinal cells, a phenotypereferred to as the localized adherence pattern (LA)(Cravioto et al., 1979; Scaletsky et al., 1984). This plasmidalso encodes Per (plasmid-encoded regulator), a tran-scriptional regulator that is required for optimal activationand function of LEE-encoded genes and BFP expression(Gómez and Kaper, 1995; Tobe et al., 1996). The Per reg-ulator is encoded by three open reading frames, perABC,and the predicted PerA protein shares homology withmembers of the AraC family of transcriptional activators.Quorum sensing has also been shown to influence LEE gene expression via Ler in a regulatory cascade(Sperandio et al., 1999).

Epidemiological studies of EPEC infection haverevealed that EPEC strains isolated throughout the worldbelong to a restricted number of O antigen serogroups,and notably to a limited number of flagellar (H) antigentypes (Nataro and Kaper, 1998). However, the role of flagella and motility in the pathogenic scheme of EPEChas been largely ignored. A growing number of studieshave incriminated flagella-mediated motility in virulence(for example, adherence, invasion and proinflammatoryresponse) in several Gram-negative pathogens such asSalmonella enterica serovars Typhimurium and Enteritidis(Allen-Vercoe and Woodward, 1999; Allen-Vercoe et al., 1999; Wyant et al., 1999; Dibb-Fuller et al., 1999;Robertson et al., 2000; Gewirtz et al., 2001a, b), E. colistrains pathogenic for chickens (La Ragione et al., 2000),Helicobacter pylori (Eaton et al., 1996), Proteus mirabilis(Mobley et al., 1996), Vibrio cholerae (Gardel andMekalanos, 1996; Postnova et al., 1996; Correa et al.,

2000), Clostridium difficile (Tasteyre et al. 2001) andYersinia enterocolitica (Young et al. 2001), as well as indevelopment of biofilm by Pseudomonas aeruginosa andVibrio cholerae (Pratt and Kolter, 1998; O’Toole andKolter, 1998; Watnick and Kolter, 1999). In this study, weinvestigated the involvement of flagella as an adhesin ofEPEC and the biological relevance of flagella expressionby bacteria adhering to cultured epithelial cells. Wedemonstrate here that the flagella produced by EPECcontribute to the adherence properties of the bacteria andthat a molecule secreted by eukaryotic cells induces theirexpression. Furthermore, data are provided that supportthe existence of a molecular relationship between flagel-lar and virulence-associated type III secretion systems,Per, and quorum sensing.

Results

Flagella mutants are defective in adherence

Several EPEC adhesins, including BFP, EspA-containingfibres and the intimin–Tir complex, are associated withadherence to mammalian cells. The role of flagella in thiscontext has not been fully explored, and therefore in thisstudy we investigated the contribution of flagella to theadhesive properties of the bacteria. To achieve this aim,we employed several approaches, including geneticmanipulations, biochemical and antigenic characteriza-tion of flagella and ultrastructural studies by high-resolution emission microscopy. Our working hypotheseswere that flagella are required for efficient bacterial adher-ence and that epithelial cells trigger expression of fla-gella in EPEC serotypes, including strains classified asnon-motile (H–).

Insertional mutations were introduced into the flagellin

© 2002 Blackwell Science Ltd, Molecular Microbiology, 44, 361–379

362 J. A. Girón et al.

Motility expression

Strain Mutation LB MEM PC-DMEM

E2348/69 w.t. + + +JPN15 Plasmid-cured + – +JPN15 (pMAR7) + + +OG127 perA + +/– +CVD206 eae + – –CVD452 escN + –/+ +UMD872 espA + –/+ +UMD864 espB + –/+ +UMD870 espD + –/+ +VS102 luxS + – +AC7 tir + +/– +AGT01 fliC – – –AGT02 fliC p(FliC) + + +AGT03 motB – – –E10 w.t. + + +AGT04(E10::fliC) fliC – – –

Except for E10 and its fliC mutant (AGT04), all other strains are derivates of E2348/69. AGT02is AGT01 complemented with pFliC (pBR322 harbouring EPEC H6 fliC); PC-DMEM, pre-conditioned DMEM; w.t., wild type. +, motile; +/–, moderately motile; –/+, weakly motile; –, non-motile.

Table 1. Bacterial strains and motility ofE2348/69, E10 and derived mutants.

Flagella-mediated adherence of EPEC 363

structural fliC gene of two wild-type EPEC strains to inves-tigate the role of flagella in adherence to epithelial cells inculture. The resulting fliC-minus strains AGT01 (derivedfrom E2348/69) and AGT04 (derived from E10) wereimpaired in motility in Luria–Bertani broth (LB) or in Dul-becco’s minimum essential medium (DMEM) motility agar(Fig. 1A), and flagella production (Fig. 1B) (Table 1). Ashypothesized, the flagella-less strains were less adherentand were not able to produce typical large microcolonies

on HeLa cells after 3 h of infection compared with the wild-type strain (Fig. 1F). Quantification of bacterial colony-forming units revealed 60% less adherence of fliC mutantAGT01 compared with wild-type E2348/69 (Fig. 1J)(P < 0.003). Complementation of the fliC gene in pBR322plasmid (pFliC) in AGT01 resulted in strain AGT02, whichshowed restoration of motility at 18 h of growth (Fig. 1A),flagella production (Fig. 1C) and adherence (Fig. 1G)although not at the wild-type levels (Fig. 1E and J).


Fig. 1. Phenotypes of wild-type E2348/69 and isogenic mutants.A. Motility of (1) E2348/69, (2) AGT01 (E2348/69fliC –), (3) AGT03 (E2348/69motB –) and (4) AGT02 (AGT01 complemented with pFliC)incubated for 18 h at 37°C. The motility was visualized as halos of radial diffusion of bacteria around the primary inoculum. AGT02 showedincreased motility after 18 h of incubation (not shown). Expression of flagella by the isogenic mutants was assessed by EM: (B) AGT01, (C)AGT02 and (D) AGT03. Photomicrographs of Giemsa-stained E2348/69 (E) and isogenic mutants (F) AGT01, (G) AGT02 and (H) AGT03adhering to HeLa cells. E2348/69 shows typical LA bacterial clusters on HeLa cells while adherence by AGT01 is remarkedly reduced.Adherence in AGT02 was partially complemented while AGT03 appeared to show numerous microcolonies.I. Immunoblot reacted with anti-H6 antibodies showing lack of expression of flagellin in isogenic mutant AGT01.J. Quantification of adherence. Although AGT01 is significantly impaired in adherence (P < 0.003), AGT02 has partially restored adherenceproperties (P < 0.16) and AGT03 showed increased adherence. K and L. Adherence phenotype of E. coli K-12 ORN172 transformed with H6 fliC gene on a plasmid after 6 h of incubation. Note the formationof discrete localized clusters reminiscent of the localized adherence pattern shown by EPEC.



Fig. 2. Synthesis and assembly of BFP andAE lesion by the fliC mutant.A. The strains indicated were grown in DMEMand reacted in immunoblots with anti-BFPserum. Except for the parent strain, no BfpAwas detected in the isogenic mutants orcomplemented fliC strain.B. Detection of BFP fibres upon adherence ofthe indicated strains to HeLa cells. fliC mutantand its complemented strain are able toproduce BFP at levels below the parentstrain. We hypothesize that BFP production inthese strains is stimulated by the presence ofepithelial cells.C. The fliC mutant is able to form pedestals(arrows) on epithelial cells after extendedperiods of incubation (>3 h).

Fig. 3. Binding of purified H6 flagella to HeLa cells in culture and inhibition of adherence. HeLa cells were incubated with purified flagella fromE2348/69 (A) or EHEC 86–24 (O157:H7) (B) for 3 h. After washing, the flagella bound to the cells were visualized by IF using type-specificanti-H6 and anti-H7 flagella antibodies. Fluorescent fragmented H6 flagella filaments (A) but not H7 flagella (B) are seen bound to the cellmonolayer, confirming the adhesive properties of H6 flagella. HeLa cells were stained with propidium iodine.C. Inhibition of adherence by anti-H6 antibodies at 1:10 (P < 0.01) and 1:100 (P < 0.03) dilutions and not by anti-H7 antibodies.

However, extended halos representing bacterial motilitywere seen beyond 24 h of incubation. Similarly, largerbacterial aggregates on HeLa cells were produced byAGT02 beyond 3 h of incubation (data not shown). Todetermine whether motility or flagellation was required forEPEC adhesion we constructed a mutation in motB

(a gene involved in rotation of the flagella) (MacNab,1996), resulting in strain AGT03. Although AGT03 still pro-duced flagella (Fig. 1D) it was unable to swim in motilityagar (Fig. 1A) but adhered to HeLa cells, forming clusterssimilar in size to those produced by the parent strain(Fig. 1H). Except for AGT01, all other strains synthesized


flagellin subunits as shown by immunoblotting (Fig. 1I). Toinvestigate further if flagella had adhesive properties, non-adherent E. coli K-12 strain ORN172, which has beendeleted of the fim type I pili genes, was transformed withthe H6 fliC gene from E2348/69 harboured on a plasmid.ORN172 carrying the fliC H6 gene was able to adhere toHeLa cells, forming discrete localized bacterial clusters ona few epithelial cells (Fig. 1K and L) only after 6 h of infec-tion. The level of adherence was, however, much lowerthan that produced by EPEC E2348/69 (Fig. 1E). Theseare compelling data that show that the flagella filamentsare required for efficient adherence of EPEC and micro-colony formation.

As the fliC mutant was unable to adhere efficiently, anobvious question to address was whether the productionof BFP was affected in this mutant. Interestingly, no BfpAsynthesis occurred in AGT01 and AGT02 upon growth in DMEM (a condition favourable for BFP synthesis) asdetected by immunoblotting of whole-cell extracts reactedwith anti-BFP antiserum (Fig. 2A). However, when AGT01and AGT02 were allowed to adhere to HeLa cells, BFPfibres were detected by immunofluorescence (IF),although not at wild-type levels (Fig. 2B). The bfpA mutantdid not show BfpA or BFP fibres in either condition(Fig. 2A and B). Thus, it is apparent that BFP expressionin the fliC mutant is enhanced by the presence of epithe-lial cells.

The ability of fliC mutants to recruit cellular actinbeneath adhering bacteria tested negative at 3 h of incu-bation in the fluorescent actin staining test (data notshown) probably because of the poor adherence shownin these strains. Nevertheless, some pedestal formation(a marker for AE lesion) was observed with scanning elec-tron microscopy when the bacteria were allowed to infectfor longer periods (6 h) (Fig. 2C). These results suggestthat fliC mutants are still able to cause AE lesions and that the presence of flagella and BFP favours adherenceand AE lesion formation. No apparent differences wereobserved in the profile of known secreted proteinsbetween wild-type and fliC mutants (data not shown). Arecent report describes the unfocused actin nucleation ina Tir–intimin and type III secretion system-independentmanner without pedestal formation (Hartland et al., 1999).

Flagella possess adhesive properties

On the basis of the experimental data obtained above, wesuspected that the flagella of EPEC per se could possessadhesive attributes. To address this issue, flagella (H6and H2) purified from EPEC strains and EHEC (H7) wereincubated with HeLa cells for 3 h and the bound flagelladetected by immunofluorescence (IF) assay using type-specific anti-H antibodies. The H6 (Fig. 3A) and H2 flagella (data not shown) but not the H7 flagella (Fig. 3B)

bound to this cell line, indicating that the flagella per seare potential adhesive structures. To test this interestinghypothesis further, we used antibodies against H6, H7and P. mirabilis flagella in experiments of inhibition ofadherence. An inhibition effect was noted using H6 anti-bodies (P < 0.01) but not with antibodies against H7(P < 0.19) (Fig. 3C) or P. mirabilis flagella (data notshown). A 65% and 60% reduction in adherence wasobtained with anti-H6 antibodies at 1:10 and 1:100 dilu-tions respectively (Fig. 3C). Interestingly, anti-H6 anti-bodies also inhibited adherence of non-H6 serotypes(data not shown).

Flagella are highly produced by adhering bacteria

Based on the data obtained above, it was tempting tospeculate that the expression of flagella by EPEC isinduced in vitro, and perhaps in vivo, in the presence ofmammalian cells in culture. Ultrastructural studies ofEPEC E2348/69 showed that this strain produces few flagella filaments when grown in LB medium (Fig. 4A).These observations have been reported before (Farriset al., 1998). Unlike BFP, which is favourably expressedafter growth in tissue culture media (e.g. DMEM) (Girónet al., 1991; Puente et al., 1996), E2348/69 produceseven fewer flagella filaments under these conditions(Fig. 4B). However, bacteria recovered from the super-natants of monolayers of HeLa cells infected for 3–5 h ofincubation produced numerous longer flagellar filaments(Fig. 4C). We examined by electron microscopy equalnumber (100) of bacteria grown under the different conditions employed. Only 10% and 5% of the E2348/69bacteria examined after growth in LB and DMEM, respectively, showed flagella, whereas most of the bacte-ria (~ 80%) grown in the presence of cultured epithelialcells, produced numerous flagella. These data suggestedthat the presence of eukaryotic cells enhanced expres-sion of flagella. However, variations in the degree of flagella produced in LB, DMEM and in the presence ofcultured cells between and within serotypes were noted(data not shown). The identity of the flagella producedwas confirmed by immunogold labelling using anti-H6antibodies (Fig. 4D). The synthesis of flagellin was mon-itored by immunoblotting to determine differential expres-sion of flagellin under the growth conditions describedabove. As expected, E2348/69 produced lower levels offlagellin in DMEM than in LB (Fig. 4E, lane 2). However,the bacteria recovered from the supernatants of infectedHeLa cells showed increased synthesis of flagellin(Fig. 4E, lane 3). For this comparison, the number of bac-teria in the different growth conditions was normalized to the same optical density. In contrast, a non-adherentE. coli K-12 HB101 produced low amounts of flagellinregardless of the presence or absence of eukaryotic cells


in culture (Fig. 4E), demonstrating that the induction of flagella by the presence of HeLa cells is not a generalattribute of all E. coli. The purified flagella were composedof flagellin monomers of 60 kDa (Fig. 4F), which weredemonstrated by amino-terminal amino acid sequencinganalysis to be flagellin subunits.

These intriguing results prompted us to study the pres-ence of flagella on bacteria adhering to tissue culturecells. We analysed a subset of well-characterized EPECstrains belonging to classical H serotypes (H2, H6, H34and H40) associated with diarrhoeal disease, includingseveral non-motile (H–) strains. Upon formation of thetypical LA bacterial clusters on HeLa or HEp-2 cells andusing anti-H6 antibodies and IF, we observed a strikingfluorescent pattern showing numerous long fluorescent filaments protruding from the adhering organisms, whichappeared to be distributed peritrichously (Fig. 5). Allmotile EPEC strains studied produced flagella to differentextents while adhering to tissue culture cells, as vis-ualized by IF (Fig. 5A–F). The fluorescent filamentsappeared to extend within and between bacterial micro-

colonies. An interesting phenomenon arises from theseobservations, which is the fact that different flagellaserotypes were detected with anti-H6 antibodies. The flagellins of enterobacteria share extended amino- andcarboxy-terminal homologies, with considerable diver-gence existing within the middle region of the proteins.The basis for H serotyping of E. coli strains relies, in fact,on the antigenic differences that exist among their fla-gellins (MacNab, 1996). However, flagella of different Htypes were identified using anti-H6 antibodies, suggest-ing the presence of a common native epitope(s) presentamong these flagellins, albeit reactivities were weakerwith antibodies against H2, H34 and H40 flagellins (datanot shown). The degree of flagella expression variedamong H types and strains (Fig. 5A–F), and while somestrains produced abundant wavy filaments others mani-fested few such structures. These observations suggestthat other functions besides motility are inherent to theseappendages. Among a limited number of H– strainstested, including B171 (O111:NM), a non-motile EPECstrain used in recent volunteer studies (Bieber et al.,



Fig. 4. Identification of flagella by electron microscopy. E2348/69 produces few flagella filaments when grown overnight in LB medium (A) and even fewer in DMEM (B), whereas numerous longer flagella are seen when the bacteria are recovered from the supernatants of HeLacells infected for 3 h (C). We examined by electron microscopy an equal number (100) of bacteria grown under the different conditionsemployed. Only 10% and 5% of the E2348/69 bacteria examined after growth in LB and DMEM, respectively, showed flagella, whereas mostof the bacteria (~80%) grown in the presence of cultured epithelial cells produced numerous flagella. Images are representatives of suchobservations.D. Immunogold labelling of E2348/69 flagella using anti-H6 serum.E. Synthesis of flagellin FliC by equal numbers of EPEC E2348/69 and E. coli K-12 HB101 bacteria grown overnight in LB (lane 1) andDMEM (lane 2), and in the presence of HeLa cells for 3 h (lane 3), as determined by immunoblotting.F. SDS-PAGE and Coomasie blue staining of the 60 kDa flagellin obtained from purified H6 flagella used in our studies. Position of massstandards is indicated on the left.Scale bars: A, 0.7 mm; B, 0.53 mm; C, 0.5 mm; and D, 0.5 mm.


1998), some produced from one to several flagella per one to five fields examined when adhering to HeLacells (Fig. 5G–I). The fact that EPEC strains such asE2348/69 produce abundant flagella when adhering tocultured cells, and that non-motile strains were also ableto produce flagella in the course of infection, strongly suggests that flagella expression in EPEC is triggered by cell contact or by an external signal of eukaryoticorigin. IF assays of EHEC (O157:H7) and ETEC (O8:H9)

bacteria adhering to HeLa cells showed no detection offlagella when employing specific H7 or H9 flagella anti-bodies (Fig. 5J and K), suggesting that enhanced expres-sion of flagella during infection is not a generalizedphenomenon among pathogenic E. coli. Likewise, IF ofEPEC E2348/69 adhering to cultured cells employingantibodies raised against flagella from enteric pathogensS. typhi, S. flexneri, S. sonnei, P. mirabilis and EHECO157:H7 showed no fluorescent filaments, demonstrating


Fig. 5. Detection of flagella produced by bacteria adhering to HeLa cells.A and B. E28 (O86:H34).C and D. E2348/69 (O127:H6).E. E10 (O119:H6).F. E18 (O128:H2).G. E7 (O127:H40).H. B171 (O111:NM).I. E26 (O55:H–).J. EHEC EDL933.K. ETEC E9034A.L. E2348/69.Note the abundant flagella filaments produced by EPEC strains that extend within and between the microcolonies (A–G). B and D are phase-contrast images of A and C showing typical LA bacterial clusters on HeLa cells. Non-motile EPEC strains (H and I) are able to produce only afew flagella upon adherence. It is apparent that EHEC EDL933 (J) and ETEC E9034A (K) do not produce flagella when associated with HeLacells. No reactivity is seen between E2348/69 flagella and anti-P. mirabilis flagella antibodies (L). After 3 h infection of HeLa cells, themonolayers were fixed with 2% formalin and reacted with antibodies against H6 (A–I), H7 (J), H9 (K) and P. mirabilis flagella (L) andsecondary FITC-labelled antibodies. Cellular and bacterial DNA are stained with propidium iodine (red) (in G, H, I and L) or with Hoechst stain(blue) (in K).

the specificity of the IF reaction (Fig. 5L and data notshown).

The striking fluorescent flagella profile differs from thepreviously described fluorescent patterns observed whenantibodies directed against the BFP (Fig. 6A and B) (Girónet al., 1991; Knutton et al., 1999; Tobe and Sasakawa,2001) or EspA-containing filaments (Knutton et al., 1998)are used in similar assays. In addition, we demonstratedby confocal microscopy that the BFP and flagella aresimultaneously produced during infection of HeLa cells(Fig. 6C–E). Based on the fluorescent pattern, the BFPfilaments appeared to closely tether bacteria within themicrocolony (Fig. 6A–C), whereas the typical wavy fla-gella produced appeared to extend outward within andbetween LA bacterial clusters (Figs 5 and 6D and E).

Demonstration of flagella on adhering bacteria

To further confirm the presence of these appendages onthe adhering bacteria, we performed scanning electronmicroscopy (SEM) of infected HeLa cells. Figure 7 depictswavy, thick (approximately 40 nm wide) intertwisting fla-gella-like filaments protruding from the bacteria and whichcan extend several microns away. These morphologicalfeatures are very suggestive of flagella filaments becausethey are thicker than pili (3–7 nm wide) and, unlike BFP, do

not typically associate into rope-like structures. In particu-lar, strains E10 and E28 (Fig. 7C, E and F) formed fila-ments that associated into a spider web-like meshworkthat covered the microcolony. These observations corre-late with the extensive fluorescent flagella seen in Fig. 5.From these descriptive micrographs we are tempted tohypothesize that these structures are assisting in micro-colony formation and possibly mediating direct interactionwith epithelial cells (Fig. 7E and F). We showed above byIF that BFP structures are also present within the bacter-ial microcolonies. Observation of the flagella structures byhigh-resolution emission SEM revealed wavy filamentswith beaded extrusions (Fig. 8A and B). EPEC inducesproliferation of microvilli-like processes (MLP) upon adher-ence to tissue culture cells, causing remodelling of theeukaryotic cell surface (Phillips et al., 2000). In order toconfirm that the filaments were bacterial in nature and notMLP produced by host cells, we performed the IF adher-ence assay using HeLa cells that were prefixed withmethanol. The use of prefixed cells dramatically reducedthe size and numbers of adherent microcolonies com-pared with non-fixed cells, although the bacteria were stillable to produce, albeit fewer, flagella under these condi-tions (Fig. 8C and D). This suggests that adherence andflagella expression are favourably manifested when live



Fig. 6. Coexpression of BFP and flagella by adhering bacteria. E2348/69 adhering to HeLa cells produces BFP filaments that are tightlyconfined to the bacterial cluster (A, B and C). Double staining with mouse monoclonal anti-BFP and rabbit polyclonal anti-H6 antibodiesdemonstrated that EPEC co-produces BFP (red) (C and E) and flagella (green) (D and E). The flagella appeared to extend outwards from the microcolony, tethering bacteria within and outside the bacterial clusters. The merged image of C and D is shown in E.



Fig. 7. Scanning electron microscopy (SEM) of EPEC-infected HeLa cells. E2348/69 (A), B171 (B), E10 (C), E7 (D) and E28 (E and F)adhering to HeLa cells showing flagella-like structures (arrows) protruding from the adhering bacteria. It is apparent that flagella serve asbridges between bacteria, and in some areas it appears that flagella are inserting into the cell membrane, serving as anchoring devices.Arrowheads point at microvilli-like processes. Scale bars: A, 1.1 mm; B, 1.0 mm; C, 1.2 mm; D, 2.0 mm; E, 1.5 mm; and F, 1.5 mm.

eukaryotic cells are employed, and that the fluorescent filaments detected by anti-H6 antibodies are of bacterialorigin. The identity of the wavy structures observed wasconfirmed to be flagella by immunogold labelling and SEM

using anti-H6 antibodies and anti-rabbit IgG conjugated to30 nm gold particles (Fig. 8E and F). In all, these resultsconclusively demonstrate that these appendages are flagella.

Influence of EAF plasmid and type III secretion genes inflagella expression and motility

The role of the EPEC adherence factor (EAF) plasmid inadherence of EPEC has been well established (Nataroand Kaper, 1998). In an effort to investigate the influenceof the EAF plasmid in the motility of EPEC and its corre-lation with adherence, we employed wild-type E2348/69,JPN15 (EAF plasmid-cured), JPN15(pMAR7) [JPN15

complemented with the EAF plasmid] and OG127(E2348/69 perA–). No apparent difference in motility wasobserved with any of the strains at 30°C (data not shown)or 37°C in LB motility agar (Fig. 9A). However, when themotility assay was performed in DMEM motility agar,E2349/69 and JPN15(pMAR7) strains, but not JPN15, diffused efficiently through the agar (Fig. 9A and Table 1).The inability of JPN15 to swim in DMEM correlated withits inability to efficiently produce flagella upon growth in



Fig. 8. High-resolution field emission SEM of EPEC adhering to HeLa cells. High magnification (>100 000¥) of the flagella-like appendagesproduced by E28 (A and B) reveals beaded (nodule-like) extrusions. Long wavy filaments resembling typical flagella (arrows) are seentethering bacteria and short filaments, probably representing EspA-containing organelles (Knutton et al., 1998), are also seen protruding fromthe bacterial cell surface. The two structures appeared to be of equal width but differed in length. SEM (C) and IF (D) of EPEC adhering toprefixed HeLa cells after 3 h of infection confirmed that the filaments are bacterial in nature. The flagellum is indicated by an arrow in C andas fluorescent filaments in D. Cellular nuclei were stained with propidium iodine in D.E and F. Immunogold labelling of flagella with anti-H6 antibodies and visualization by SEM demonstrated the aggregation of 30 nm goldparticles (arrows) to the 50-nm-wide wavy filaments. Scale bars: A, 0.18 mm; B, 0.25 mm; C, 1.5 mm; E and F, 1mm.


DMEM as determined by negative staining and electronmicroscopy (data not shown), and by immunoblottingusing whole-cell extracts and anti-H6 antibodies (Fig. 9D).Figure 9 depicts the synthesis of flagellin by E2348/69and derivative mutants and, as expression of flagellin isweaker in DMEM than in LB, for the purpose of compar-ison the blot was overexposed with the appropriate sub-strate (see Experimental procedures). In fact, JPN15produced barely detectable amounts of the 60 kDa fla-gellin compared with the parent strain. The perA mutantwas moderately motile in DMEM (Table 1), which corre-lated with its ability to still produce flagellin (Fig. 9D). We

had shown above that the fliC-minus strain was deficientin BFP production; thus, we became interested in deter-mining whether a bfpA-minus strain would be altered in synthesis of flagella and motility. Likewise, the bfpA-minusstrain was deficient in flagellin synthesis (Fig. 9D) andmotility (Table 1). These results expand our knowledge ofthe phenotypes controlled by the EAF plasmid genes andimplicate a modulatory relationship between perA, bfpAand flagella expression and motility.

Recently, the participation of the flagellar sigma factorFliA in regulation of expression of genes associated with the type III translocon of Salmonella was reported


Fig. 9. Motility of wild-type E2348/69 and isogenic mutants. Glass vials containing LB or DMEM medium supplemented with 0.3% agar wereinoculated with a needle with (A) E2348/69, JPN15 (plasmid-cured) and JPN15(pMAR7) or (B) isogenic mutants escN, espA, espB and espD.Motility was read after incubation for 16–18 h at 37°C.C. DMEM alone or PC-DMEM (preconditioned DMEM) containing 0.3% agar was inoculated with JPN15 strain and incubated for 16–18 h at37°C. PC-DMEM restored the ability of JPN15 to swim.D. Expression of flagellin by wild-type E2348/69 (w.t.) and isogenic mutants grown in DMEM. As E2348/69 expresses little flagellin aftergrowth in DMEM, this blot was overexposed for the purpose of comparison with the isogenic mutants. Note the lack or reduced synthesis offlagellin in all the mutants, except for the perA mutant. Mass standards are indicated with arrows.

(Eichelberg and Galán, 2000). Thus, we were then inter-ested in determining whether eae and the type III secre-tion genes encoded in the LEE region played any role in flagella production and motility. E2348/69 isogenicmutants defective in intimin and Tir production, in functionof the type III translocon (escN), and in synthesis of EspA, EspB and EspD were examined for the phenotypesunder investigation. All of these mutants were non-motileor weakly motile in DMEM motility agar (Table 1 andFig. 9B), suggesting a relationship between flagellation,motility and the type III secretion pathway. Except for the espD mutant, which was able to synthesize somedetectable flagellin and to display flagella in contact with cultured cells (Fig. 10), the eae, espA, espB andescN mutants were impaired in their ability to optimally

synthesize flagellin when grown in DMEM (Fig. 9D) andto produce abundant flagella when associated with HeLa cells (Fig. 10). Occasionally, one or two flagella fil-aments were observed in microcolonies formed by thesemutants. However, the tir mutant produced poor amountsof flagellin when tested in immunoblots (Fig. 9D) but wasable to produce flagella to levels similar to that of theparent strain when examined by microscopy (Fig. 10). Itis apparent from the adherence phenotypes shown inFig. 10 that all of the mutants tested are still able to inter-act with epithelial cells and to form microcolonies, anevent that was observed upon extended infection periods.Quorum sensing has been shown to influence the expres-sion of EPEC and enterohaemorrhagic E. coli LEE genesvia Ler in a regulatory cascade (Sperandio et al., 1999).



Fig. 10. Detection of flagella produced byisogenic mutants upon adherence to HeLacells. After infection with the indicatedmutants, the flagella were detected byimmunofluorescence as described in the text.Individual green bacteria adhered individuallyor formed microcolonies of different sizes. Incontrast to the perA, espD and tir mutants,which displayed several flagella (whitearrows) per field examined, the remainingmutants occasionally displayed one or twoflagella per field.


In agreement with previous observations (Sperandioet al., 2001), we noted here that a luxS mutant (VS102)was unable to swim and to produce flagella, even whenassociated in microcolonies with HeLa cells (Fig. 10 andTable 1).

The expression of EPEC flagella is triggered by asecreted eukaryotic molecule

To investigate the possible role of a molecule of eukary-otic origin involved in triggering flagella production andmotility, we prepared DMEM that was previously incu-bated for 24–48 h with monolayers of HeLa cells in theabsence of fetal bovine serum or antibiotics. This pre-conditioned medium was used in motility tests and todetect the synthesis of flagellin or production of flagellafilaments in the isogenic mutants. Except for the eaemutant, which was repeatedly negative for motility, thispreconditioned medium restored the ability of the remain-ing strains to swim (Table 1 and Fig. 9C) and to synthe-size and assemble flagella (data not shown). Thus, thebiosynthesis of flagella may be activated also by a per-independent mechanism. Further experiments demon-strated that the molecule present in the preconditionedmedium was heat stable and that high-pressure liquidchromatography fractions below 1 kDa still retained theability to induce flagellation and motility in non-motileEPEC mutants. While the nature of this molecule is underinvestigation, the present data strongly suggest that asoluble factor of eukaryotic origin was present in the preconditioned medium and that this factor bypassed regulation by PerA-activating genetic elements involvedin flagella production or regulation.

Discussion

Although the flagellar H antigen is one of the surface andepidemiological markers that identifies EPEC as a diar-rhoeagenic class of E. coli, in addition to expression ofBFP and possession of the LEE, neither previous norcurrent models of EPEC pathogenesis have implicatedflagella in adherence (Nataro and Kaper, 1998; Frankelet al., 1998). Here, we show for the first time that the flagella of EPEC are important adhesive structures highlyinduced upon interaction with epithelial cells and mostlikely by a secreted signalling molecule of eukaryoticorigin. EPEC H6 and H2, but not EHEC H7, purified flagella were demonstrated to directly mediate bacterialattachment to epithelial cells. Several different EPECserotypes were shown by IF to express flagella to different degrees when adhering to cultured cells. Theseresults were obtained using specific antisera againstEPEC flagella but not with antibodies against flagella ofother enteric pathogens. It is relevant to note that some

EPEC strains serologically classified as H– or non-motilewere also able to produce flagella, albeit less than motilestrains, when adhering to culture cells.

It is well documented that most EPEC plasmid- andchromosome-encoded virulence factors are environmen-tally and growth-phase regulated (Kenny et al., 1997b;Puente et al., 1996; Knutton et al., 1997). For example,intimin and EspA filaments are down-regulated as bacte-ria enter stationary phase, once the AE lesions haveformed (Puente et al., 1996; Rosenshine et al., 1996;Kenny et al., 1997; Knutton et al., 1997; 1998). Our datasuggest that flagella biosynthesis is not turned off evenwhen the bacteria enter stationary phase, and this patternindicates a constant stimulation of flagella expression. Weshowed that the production of flagella was reduced whenmethanol-fixed HeLa cells were employed compared with live cells. The possibility that a eukaryotic solubleproduct(s) signals the expression of flagella in EPEC wasthus addressed. We prepared DMEM preconditioned bygrowth of HeLa cells and showed that it activated motilityin E2348/69 isogenic strains unable to swim in DMEMmotility agar. Although the biochemical nature of thissoluble activator of flagella expression and motility is currently under investigation, it is tempting to speculatethat a molecule that triggers expression of flagella andpossibly other virulence genes, with a chemical structuresimilar to that secreted by HeLa cells, might be presentin the intestinal tract. In support of this speculation, thereare other data that suggest that the in vivo environmentinduces motility in S. enterica var. Gallinarum and Pullorum serovars, which are traditionally recognized asnon-motile (Chaubal and Holt, 1999). The effect of pH,temperature and surface contact on the elaboration of flagella by Salmonella serotype Enteritidis has also beenreported (Walker et al., 1999). Furthermore, the presenceof abundant flagella on adhering bacteria suggests thatother properties besides motility, such as adherence andpossibly translocation of proteins with potential virulenceattributes, could be inherent to these appendages.

It is obvious that most bacterial pathogens must over-come the viscous mucus that naturally bathes epithelialsurfaces in order to successfully reach their target niches.Thus, it is reasonable to propose that flagella expressionand motility properties of EPEC are of critical importanceto translocate bacteria across the intestinal mucus,thereby facilitating interaction with target sites on thesurface of enterocytes. Several studies have addressedthe role of flagella-mediated motility in virulence (adher-ence, colonization and invasion) and development ofbiofilm by several Gram-negative bacteria (Eaton et al.,1996; Mobley et al., 1996; Gardel and Mekalanos, 1996;Postnova et al., 1996; Booshardt et al., 1997; Pratt et al.,1998; Allen-Vercoe et al., 1999; Watnick and Kolter, 1999;Wyant et al., 1999; Correa et al., 2000; La Ragione et al.,


2000; Young et al., 2000; Gewirtz et al., 2001a). Weattempted to study the role of flagella in adherence ofEPEC by several approaches. We demonstrated that H6flagella purified from EPEC but not EHEC H7 flagella bindto HeLa cells. The data provided here do not explain themechanism of binding of flagella but it is possible that theH6 flagellin possesses unique binding domains presentalong the non-conserved region of the protein that isexposed on the flagella filament. It is possible that the heterogeneity at the amino acid sequence level of the flagellin protein accounts for the adhesive properties (for example with active sites of adherence) of EPEC flagella. Introduction of specific targeted mutations carriedin the flagellin protein, specifically the non-conservedregion, will be needed to test this interesting hypothesis.The flagellins of enterobacteria share extended sequencehomology in the amino- and carboxy-termini althoughconsiderable divergence exist within the middle region ofthe proteins (MacNab, 1996). The antigenic differencesresulting from divergent flagellin sequences (Reid et al.,1999) form the basis for H serotyping of E. coli. Interest-ingly however, the flagella of different H types producedby adhering EPEC strains were identified using anti-H6antibodies suggesting the presence of a common nativeepitope(s) among antigenically heterologous flagellins.Furthermore, the LA phenotype exhibited by differentEPEC serotypes was inhibited by anti-H6 antibodies suggesting similar functions among EPEC flagella. Thisinhibition effect could be attributed presumably to stearic hindrance impeding flagella binding sites to recognize target sites on the host cell. This hypothesis isbased on the assumption that flagella per se possessgenuine adherence moieties. Recent reports suggest thatflagellin of S. Typhimurium is translocated through theepithelial cell to basolateral membrane inducing a pro-inflammatory response (Gewirtz et al., 2001a, b). Thus,another possible explanation is that flagella induce hostcells responses that then mediate adherence. These are interesting issues that will be addressed in futureinvestigations.

Structural analysis by SEM of EPEC of differentserotypes adhering to HeLa cells revealed the presenceof structures resembling flagella that appeared to mediatedirect binding of the bacteria to the host cell thereby contributing to the formation of three-dimensional microcolonies. These structures are morphologically andantigenically different than the BFP structures that havebeen shown to promote aggregation of bacteria duringinitial stages of infection and dispersal of bacteria afterextended periods of incubation (Bieber et al., 1998;Knutton et al., 1999). We showed here that both flagellaand BFP are produced simultaneously during infection.The short EspA filaments produced during the early stageof AE lesion formation (Knutton et al., 1998) are also

structurally quite distinct from the >20 mm-long flagella filaments induced upon cell contact. The EspA filamentsare proposed to mobilize effector molecules into the hostcell thus providing an essential step in the molecular relationship between the bacterium and the host cell(Knutton et al., 1998; Frankel et al., 1998). The extendedsimilarities between flagellar and type III secretionsystems suggest common functional features that haveevolved for bacterial adaptation, survival and virulence(Hueck, 1998; Lory, 1998; Galán and Collmer, 1999).Recently, the translocation of virulence-associated non-flagellar proteins by the type III flagellar export machineryof Y. enterocolitica was reported (Young et al., 1999).Although no differences in the number of known secretedproteins were observed between wild-type EPEC and itsisogenic fliC mutant, it is tempting to speculate that eitherEsps or other yet unidentified virulence-associated pro-teins produced only upon contact with epithelial cells invitro or in vivo may be exported through the flagellar typeIII secretion pathway or alternatively through the EspAsecretory channel. Alternatively, it is possible that flagella-mediated attachment (specially during early stages ofinfection) is also required for efficient intimate attachmentand delivery and function of effector molecules throughthe type III secretion system.

An important role of flagella in adherence was furtherdemonstrated when two EPEC fliC mutants unable to syn-thesize flagella were impaired in their ability to adhere andform LA at 3 h of infection. Prolonged incubation of flagella-minus bacteria with HeLa cells for 6 h showedindividual adherent bacteria and small clusters with noapparent flagella filaments protruding from them. A motBmutant was still able to produce flagella and to adhere tocultured cells after 3 h of infection. From this observation,it appears that motility is not essential for adherencealthough there is a need to clearly delineate betweenadherence and chemotaxis. Future studies will be aimedat discriminating between these possibilities and toexpand our knowledge on the role of motB. Both mutantswere able however, to recruit cellular actin and to formpedestals but only after 6 h of infection. When theE2348/69 fliC mutant was tested for BfpA and BFPexpression, we found that this strain was deficient in syn-thesis of BfpA after growth in D-MEM, a condition that pro-motes BFP expression. Nevertheless, once the fliC wasallowed to interact with cultured cells for over 3 h of infec-tion, detectable amounts of BFP fibres were noted. Like-wise, the bfpA mutant was shown to be deficient in motilityand in flagellin synthesis after growth in D-MEM or in thepresence of cultured cells. We have shown here that boththe flagella and BFP are co-produced when the bacteriaare adhering and forming microcolonies on cultured cells.These results expand our knowledge of the phenotypescontrolled by the EAF plasmid genes and highlight a




modulatory relationship between perA, bfpA, and flagellaexpression and motility.

Our data strongly indicate that flagella are adhesivestructures directly involved in LA formation, promoting short and long-range physical bridges between bac-teria, and that their expression contributes to AE lesion formation.

The synthesis, assembly and function of the flagellarsystem of E. coli and S. enterica serovar Typhimurium isunder the control of a flagellar regulon that comprisesmore than 50 genes that are divided among at least 17operons (Liu and Matsumara, 1994; MacNab, 1996;Chilcott and Hughes, 2000). In EPEC, several virulence-associated genes appear to be required for adequate syn-thesis and function of flagella. We have demonstrated thatmutations in genes located in the EAF plasmid (e.g. perAand bfpA), in the chromosomal LEE region (escN, eae,tir, espA, esp and espD) and in the gene encoding thequorum-sensing autoinducer synthetase (luxS) substan-tially affect the expression of flagella and motility in EPECwhen grown in DMEM but not in LB. In agreement withthis observation, a relationship between genes involvedin flagella biosynthesis, motility and quorum sensing wasrecently noted in enterohaemorrhagic E. coli (Sperandioet al., 2001). When these mutants were assayed for flagella expression during the course of infection of HeLacells, only the espD and tir mutants appeared to producemore flagella than the remaining mutants, but less thanthe parent strain. The present data cannot explain whyonly these two mutants were not fully affected in flagellaproduction when interacting with mammalian cells, but itis certainly an interesting issue, and one which we areinvestigating further. Except for the eae mutant, all of themutants studied were restored for motility in the presenceof preconditioned medium, supporting our hypothesis thata signal of eukaryotic origin triggers flagella expression inEPEC. Efforts to induce motility in the eae mutant in thepresence of the preconditioned medium were unsuc-cessful, and this result is indicative of complicated feed-back regulatory mechanisms. This observation raises thepossibility that the eae gene or its product (intimin) mayplay some yet undefined role in flagella function and motil-ity. In this regard, recent work on virulent E. coli strainsbelonging to serogroup O26 show a strong correlationbetween the presence of the eae gene and the posses-sion of the H11 flagellar antigen (Zhang et al., 2000). Themotility results obtained with the LEE and EAF plasmidmutants support the hypothesis that a eukaryotic signalinduces flagella expression and adherence in EPEC. Inall, these data suggest the existence of a molecular rela-tionship between epithelial cells, the flagellar regulon andEPEC virulence genes, and strengthen the notion of theevolutionary relationship between flagellar and proteinsecretion type III pathways (Hueck, 1998; Lory, 1998;

Galán and Collmer, 1999; Chilcott et al., 2000; MacNab,2000).

Why distinct flagella were not identified in previousEPEC–host cell interaction studies is an obvious questionthat arises from this paper. For many years, researchersbelieved EPEC strains did not elaborate fimbriae (Scotland et al., 1983) until the BFP was discovered instrain B171 (Girón et al., 1991; Giron et al., 1993). Thisfinding was possible as a result of modifications in the invitro growth conditions. For almost half a century now, wehave known that the flagellar H antigen of EPEC is a cri-terion for defining epidemic strains, but researchers wereapparently focusing on the flagella not as an appendageinvolved in adherence, but as a serotyping antigen.Intimin, the BFP, the EspA filaments, the flagella and pos-sibly other unknown factors have always been there forus to discover when the bacteria interact with their hostcells. Knowing how to discriminate among these struc-tures and how their functions are orchestrated is certainlya challenge for EPEC researchers. Based on the presentresults and the most recent published data, it is reason-able to propose that the interaction of EPEC with hostcells entails the participation of several adhesins: intimin,type IV bundle-forming pili, an EspA-containing organelleassociated with the type III secretion system and flagella.Directly or indirectly, all of these bacterial componentsappear to be regulated by Per and are apparently stimu-lated by an eukaryotic factor(s) released into the super-natant. How these elements are synchronized duringinfection of the gut mucosa remains an interesting andimportant issue to address.

Experimental procedures

Bacterial strains

EPEC strains and isogenic derivatives employed are listed inTable 1. Wild-type EPEC strains employed were E2348/69(O127:H6), a prototypic strain whose virulence has beendemonstrated in volunteers studies; JPN15, a derivative ofE2348/69 cured of the EAF plamid; JPN15 (pMAR7), JPN15complemented with a transposon-marked EAF plasmidpMAR7; E10 (O119:H6), which was obtained from our bacterial collection; B171 (O111:NM) 19; E3 (O114:H2), E18 (O128:H2), E24 (O142:H34), E28 (O86:H34), E7(O127:H40), E23 (O55:H–) and E26 (O55:H–), which werekindly donated by Luiz R. Trabulsi (Instituto Butanta, Brazil);and enterohaemorrhagic E. coli (EHEC) EDL933 (O157:H7),enterotoxigenic E. coli (ETEC) E9034A (O8:H9) and E. coliK-12 HB101 and ORN172 (Dfim), which were obtained fromour collection.

Construction of isogenic fliC and motB mutants

Mutants defective in flagella production and motility were con-structed in the chromosome of E2348/69 and E10 strains by


marker exchange as follows. The 5¢ and 3¢ regions of the fliCflagellin gene of E2348/69 were amplified by polymerasechain reaction (PCR) with two set of primers: 5HFLIC (5¢-CCAAGCTTATGCAGTCTGCGCTGTCGA-3¢) and 3RvFLIC(5¢-GCGCTGGAGATATCATCAGAA-3¢); and 3BFLIC (5¢-CCGGATCCTCATACCTGGTTGGCTTTTGC-3¢) and5RvFLIC (5¢-TTCTGATGATATCTCCAGCGC-3¢). The ampli-cons obtained were ligated together with the chloramphenicol(cat) cassette, which was used to interrupt the flagellinsequence. The fliC::cat gene was cloned into suicide vectorpCVD442 and marker exchange was performed as previouslydescribed (Sperandio et al., 2001). Similarly, a mutation in the flagella motor rotation system (motB) was introduced inE2348/69 using the cat gene. motB was amplified by PCRusing primer pairs – 5HMOTB (5¢-ACCTTCGAAATCGAAGCTTTGA-3¢) and 3RvMOTB (5¢-GCGATATCCTTTCTCACCGC-3¢); and 3BMOTB (5¢-AGCGGATCCGCCCCTTTCA-3¢)and 5RvMOTB (5¢-GCGGTGAGAAAGGATATCGC-3¢) – and cloned into pCVD442 to perform the allelic exchange(Sperandio et al., 2001). AGT03 (E2348/69motB –) and AGT01 (fliC –) were confirmed by PCR to contain an insertionmutation. To confirm the phenotypes of these mutants, motility assays were performed in glass vials or Petri dishesutilizing LB broth, DMEM or preconditioned DMEM supple-mented with 0.3% agar. Motility was typically read after 16–18h of incubation at 37°C.

Expression of H6 in E. coli K12

The H6 fliC gene was amplified from E2348/69 using primersderived from the reported fliC sequence (Reid et al., 1999),cloned into pBR322 yielding pFliC and transformed in E. coliK-12 ORN172 lacking the type I pili fim genes. The adher-ence phenotype of this transformant was tested as describedbelow.

Ultrastructural studies

For transmission electron microscopy, bacteria were nega-tively stained with 1% phosphotungstic acid (pH 7.4) oncarbon–Formvar copper grids (Girón et al., 1991). Forimmunogold labelling of flagella, bacteria were reacted withanti-H6 antiserum and 10 nm gold-labelled anti-rabbit IgGand negatively stained as before. HeLa cell monolayersseeded on glass coverslips were infected with EPEC, fixed in 2% formaldehyde, and processed for scanning electron microscopy (SEM) (Knutton et al., 1998) or immuno-gold labelling using anti-rabbit IgG conjugated to 30 nmgold particles. Specimens were examined in a JEOL 1200 EX scanning microscope at 25 kV. For observation of speci-mens at high magnification (>100 000¥) a Leo 1500 high-resolution field emission scanning electron microscope was used.

Isolation of flagella filaments and secreted proteins

Flagella were mechanically sheared from E10 (O119:H6) andE18 (O128:H2) bacteria grown in Dulbecco’s minimal essen-tial medium (DMEM) and separated by differential centrifu-

gation and a caesium chloride gradient to obtain relativelypure flagella filaments (Tacket et al., 1987). In addition, flagella from EHEC EDL933 (O157:H7) were purified in asimilar way. The flagella preparations were adjusted to sameprotein concentration and then subjected to sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE)(Laemmli, 1970) and electroblotting to polyvinylidene difluo-ride (PVDF) membranes (pore size 0.45 mm, Millipore). Thepresence of flagella was confirmed by electron microscopyas described above. The protein band of interest was excisedand subjected to N-terminal sequence analysis (Protein andNucleic Acid Facility at Stanford University). To analyse theprofiles of proteins secreted by wild-type E2348/69 and fliCmutants, supernatants of bacteria grown in DMEM wereobtained and concentrated by ultrafiltration (Jarvis et al.,1995). In all the experiments, equal amounts of proteins wereloaded on the SDS-PAGE gels.

Western blotting and antisera

To determine production of flagellin, the bacteria were grownovernight in LB and DMEM, and those obtained from super-natants of infected cells for 3 h were adjusted to anabsorbance of 0.7 at OD600. Thus, equal numbers of bacte-ria were used to prepare whole-cell extracts by denaturationin SDS-PAGE sample buffer and separated in 14% SDS-PAGE gels. Proteins were transferred onto PVDF mem-branes and reacted with antiflagella antibodies and second-ary anti-rabbit IgG conjugated to horseradish peroxidase(Sigma). As expression of flagellin is weaker in DMEM thanin LB, for the purpose of comparing the synthesis of flagellinby E2348/69 and derivative mutants the blots were overex-posed with the appropriate substrate (see Fig. 9). Antiseraagainst EPEC H2, H6, H34 and H40, EHEC H7, and ETECH9 flagella were raised by immunization of rabbits with puri-fied flagella. Antibodies against S. typhi, S. flexneri, and S.sonnei flagella, and monoclonal antibodies against BFP wereavailable from previous studies (Girón 1995; Girón et al.,1995). Anti-P. mirabilis flagella serum was kindly donated byHarry Mobley (University of Maryland). Other anti-H6 andanti-H2 antibodies were a kind gift of Carlos Eslava (UNAM,México).

Interaction with eukaryotic cells

The adherence and immunofluorescence (IF) assays wereperformed using HeLa and HEp-2 cells, utilizing DMEM con-taining 1% D-mannose, with or without fetal bovine serum as described previously (Cravioto et al., 1979; Girón et al.,1991). After 3 h infection and thorough washing with phos-phate buffer, the cells were fixed with 2% formalin. The spec-imens were stained with Giemsa or prepared for IF asfollows. Primary rabbit antibodies against flagella or BFPwere added for 1 h at the appropriate dilutions in 10% normalhorse serum. After washing, the cells were incubated for 1hwith secondary anti-rabbit IgG FITC-conjugated antibodiesdiluted 1:3000. The cells were washed extensively andmounted in glycerol–PBS and visualized under a UV light and phase contrast using a Zeiss Axiolab microscope. Whenneeded, HeLa cells were prefixed with methanol for




20 min before the IF assay described above. HeLa cell mono-layers or cell suspensions were also incubated with or withoutpurified H6 or H7 flagella (adjusted to the same protein concentration) for 3 h at 37°C to test the binding propertiesof flagella. Bound flagella were detected by IF using similartitres of antiflagella antibodies. Polymerized actin wasassayed by the fluorescent actin staining (FAS) test as previously described (Knutton et al., 1989). For adherenceinhibition experiments, 107 CFU of an overnight bacterialculture grown in Luria–Bertani (LB) broth were incubated with1:10 and 1:100 dilutions of anti-H6 and anti-H7 antibodies inDMEM at room temperature for 30 min and then added to theHeLa cell monolayers. These antisera contained similar titresof anti-H antibodies as determined by immunoblotting. Afterincubation for 3 h, the cells were lysed with PBS–0.5% TritonX-100, diluted 10-fold, and plated onto MacConkey agar toestimate the number of bacteria adhering to the cell mono-layers. Replica samples were stained with Giemsa andexamined by light microscopy. Antibodies against P. mirabilisflagella were included as a negative control.

Preconditioned medium

Monolayers of HeLa cells that had been extensively washedwith PBS were incubated with DMEM without antibiotics or fetal bovine serum for 24–48 h. The supernatant referredas to ‘preconditioned medium’ was collected and the pHadjusted to 7.4, filtered through a 0.2 mm membrane, andsupplemented with 0.3% agar for motility tests or used togrow EPEC mutants and to detect the synthesis of flagellinor production of flagella filaments in these mutants.

Except for the cloning experiments, all the above experi-ments were repeated at least five times to confirm the resultsobtained. Unless otherwise indicated, the results shown hereare representative of a particular assay or observation underthe microscope.

Acknowledgements

We thank Jennifer Abbott, Vanessa Sperandio and SooanShin for critical discussions, and Jane Michalski for helpfulassistance. We thank Harry Mobley, Luiz Trabulsi and CarlosEslava for kindly providing antisera and strains; Adam Crawford for the tir-minus strain; Erasmo Negrete, MónicaRosales, and Renato Cappello for their valuable technicalassistance. This work was supported by NIH grant no.AI21657. J. A. Girón thanks Lilia Cedillo (BUAP), Conacyt(Grant 32777-M) and the Japan International CooperationAgency for support. A. G. Torres was supported by a researchsupplement for underrepresented minorities from the NIAID,NIH.

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The ﬂagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells

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