The ﬂagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells by Jose Carrasquero-Diaz

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

The flagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells Jorge A. Girón*1,2, Alfredo G. Torres2, Enrique Freer3 and James B. Kaper2 1 Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, México. 2 Department of Microbiology and Immunology, and Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD 21201, USA. 3 Unidad de Microscopía Electrónica, Universidad de Costa Rica. Summary Enteropathogenic Escherichia coli (EPEC) utilizes a type III protein secretion system to target effector molecules into the host cell leading to effacement of the intestinal mucosa. This secretion apparatus shares many structural features of the flagellar type III export system involved in flagella assembly and motility. We report here that fliC insertional mutants constructed in two wild-type EPEC strains were markedly impaired in adherence and microcolony formation on cultured cells. An E. coli K-12 strain harbouring the EPEC H6 fliC gene on a plasmid showed discrete adhering clusters on HeLa cells, albeit to less extent than the wild-type EPEC strain. Flagella purified from EPEC bound to cultured epithelial cells and antiflagella antibodies blocked adherence of several EPEC serotypes. We determined that eukaryotic cells in culture stimulate expression of flagella by motile and non-motile EPEC. Isogenic strains mutated in perA (a transcriptional activator), bfpA (a type IV pilin), luxS (a quorum-sensing autoinducer gene) and in the type III secretion genes were reduced for motility in Dulbecco’s modified Eagle medium (DMEM) motility agar and produced none or few flagella when associated with epithelial cells. Growth of these mutants in preconditioned tissue culture medium restored motility and their ability to produce flagella, suggesting the influence of a signal provided by mammalian cells that triggers flagella production. This study shows for the first time that the flagella of EPEC are directly involved in the adherence of these Accepted 21 January, 2002. *For correspondence: E-mail jagiron@yahoo.com; Tel. (+1) 410 706 3004; Fax (+1) 410 706 6205.

bacteria and supports the existence of a molecular relationship between the two existing type III secretion pathways of EPEC, the EPEC adherence factor (EAF) plasmid-encoded regulator, quorum sensing and epithelial cells. Introduction Several pathogenic bacteria, including diarrhoeagenic Escherichia coli strains have evolved sophisticated virulence-associated type III protein secretion systems that are structurally and biochemically similar to the flagellar type 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 translocons that deliver virulence factors across the bacterial cell envelope to their targeted sites in the host membrane cell or into the cytosolic compartment, where they act directly or indirectly with components of the cellular cytoskeleton. In the human small intestine, enteropathogenic Escherichia coli (EPEC), the most important bacterial cause of infant diarrhoeal disease in the developing world, causes characteristic attaching and effacing (AE) intestinal lesions (Staley et al., 1969; Moon et al., 1983; Nataro and Kaper, 1998). These lesions are manifested by critical damage to the integrity of the enterocyte cytoskeleton, which involves intimate attachment and activation of signal transduction pathways and rearrangements of cytoskeletal proteins (reviewed in Frankel et al., 1998; Nataro and Kaper, 1998). The effector molecules responsible for these events are proteins encoded by chromosomal genes that map to a pathogenicity island called the locus of enterocyte effacement (LEE) (McDaniel et al., 1995). The LEE encodes an adhesin called intimin (Jerse et al., 1990), its translocated intimin receptor (Tir) (Kenny et al., 1997a) and components of the type III secretory complex, which is responsible for export of the secreted Esp proteins (EspA, EspB, EspD and EspF), and the LEEencoded regulator (Ler), which controls the expression of LEE genes (Elliott et al., 1998; Mellies et al., 1999). EspA is thought to form a hollow filamentous structure that serves to mobilize virulence factors across the cell membrane (Frankel et al., 1998; Knutton et al., 1998; Sekiya et al., 2001). In addition, EPEC strains contain a large

362 J. A. Girón et al. 92 kb plasmid that codes for a type IV bundle-forming pilus (BFP) (Girón et al., 1991) associated with bacterial clustering and formation of tight microcolonies on tissue culture cells and human intestinal cells, a phenotype referred to as the localized adherence pattern (LA) (Cravioto et al., 1979; Scaletsky et al., 1984). This plasmid also encodes Per (plasmid-encoded regulator), a transcriptional regulator that is required for optimal activation and function of LEE-encoded genes and BFP expression (Gómez and Kaper, 1995; Tobe et al., 1996). The Per regulator is encoded by three open reading frames, perABC, and the predicted PerA protein shares homology with members 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 have revealed that EPEC strains isolated throughout the world belong to a restricted number of O antigen serogroups, and notably to a limited number of flagellar (H) antigen types (Nataro and Kaper, 1998). However, the role of flagella and motility in the pathogenic scheme of EPEC has been largely ignored. A growing number of studies have incriminated flagella-mediated motility in virulence (for example, adherence, invasion and proinflammatory response) in several Gram-negative pathogens such as Salmonella 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. coli strains pathogenic for chickens (La Ragione et al., 2000), Helicobacter pylori (Eaton et al., 1996), Proteus mirabilis (Mobley et al., 1996), Vibrio cholerae (Gardel and Mekalanos, 1996; Postnova et al., 1996; Correa et al.,

2000), Clostridium difficile (Tasteyre et al. 2001) and Yersinia enterocolitica (Young et al. 2001), as well as in development of biofilm by Pseudomonas aeruginosa and Vibrio cholerae (Pratt and Kolter, 1998; O’Toole and Kolter, 1998; Watnick and Kolter, 1999). In this study, we investigated the involvement of flagella as an adhesin of EPEC and the biological relevance of flagella expression by bacteria adhering to cultured epithelial cells. We demonstrate here that the flagella produced by EPEC contribute to the adherence properties of the bacteria and that a molecule secreted by eukaryotic cells induces their expression. Furthermore, data are provided that support the existence of a molecular relationship between flagellar and virulence-associated type III secretion systems, Per, and quorum sensing. Results Flagella mutants are defective in adherence Several EPEC adhesins, including BFP, EspA-containing fibres and the intimin–Tir complex, are associated with adherence to mammalian cells. The role of flagella in this context has not been fully explored, and therefore in this study we investigated the contribution of flagella to the adhesive properties of the bacteria. To achieve this aim, we employed several approaches, including genetic manipulations, biochemical and antigenic characterization of flagella and ultrastructural studies by highresolution emission microscopy. Our working hypotheses were that flagella are required for efficient bacterial adherence and that epithelial cells trigger expression of flagella in EPEC serotypes, including strains classified as non-motile (H–). Insertional mutations were introduced into the flagellin Table 1. Bacterial strains and motility of E2348/69, E10 and derived mutants.

Motility expression Strain E2348/69 w.t. JPN15 JPN15 (pMAR7) OG127 CVD206 CVD452 UMD872 UMD864 UMD870 VS102 AC7 AGT01 AGT02 AGT03 E10 w.t. AGT04(E10::fliC)

Mutation Plasmid-cured perA eae escN espA espB espD luxS tir fliC fliC p(FliC) motB fliC

MEM

PC-DMEM

+ + + + + + + + + + + – + – + –

+ – + +/– – –/+ –/+ –/+ –/+ – +/– – + – + –

+ + + + – + + + + + + – + – + –

Except for E10 and its fliC mutant (AGT04), all other strains are derivates of E2348/69. AGT02 is AGT01 complemented with pFliC (pBR322 harbouring EPEC H6 fliC); PC-DMEM, preconditioned DMEM; w.t., wild type. +, motile; +/–, moderately motile; –/+, weakly motile; –, non-motile. © 2002 Blackwell Science Ltd, Molecular Microbiology, 44, 361–379

Flagella-mediated adherence of EPEC 363 structural fliC gene of two wild-type EPEC strains to investigate the role of flagella in adherence to epithelial cells in culture. The resulting fliC-minus strains AGT01 (derived from E2348/69) and AGT04 (derived from E10) were impaired in motility in Luria–Bertani broth (LB) or in Dulbecco’s minimum essential medium (DMEM) motility agar (Fig. 1A), and flagella production (Fig. 1B) (Table 1). As hypothesized, the flagella-less strains were less adherent and were not able to produce typical large microcolonies

on HeLa cells after 3 h of infection compared with the wildtype strain (Fig. 1F). Quantification of bacterial colonyforming units revealed 60% less adherence of fliC mutant AGT01 compared with wild-type E2348/69 (Fig. 1J) (P < 0.003). Complementation of the fliC gene in pBR322 plasmid (pFliC) in AGT01 resulted in strain AGT02, which showed 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 showed increased 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) AGT03 adhering 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 adherence properties (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 formation of discrete localized clusters reminiscent of the localized adherence pattern shown by EPEC. © 2002 Blackwell Science Ltd, Molecular Microbiology, 44, 361–379

364 J. A. Girón et al. Fig. 2. Synthesis and assembly of BFP and AE lesion by the fliC mutant. A. The strains indicated were grown in DMEM and reacted in immunoblots with anti-BFP serum. Except for the parent strain, no BfpA was detected in the isogenic mutants or complemented fliC strain. B. Detection of BFP fibres upon adherence of the indicated strains to HeLa cells. fliC mutant and its complemented strain are able to produce BFP at levels below the parent strain. We hypothesize that BFP production in these strains is stimulated by the presence of epithelial cells. C. The fliC mutant is able to form pedestals (arrows) on epithelial cells after extended periods of incubation (>3 h).

However, extended halos representing bacterial motility were seen beyond 24 h of incubation. Similarly, larger bacterial aggregates on HeLa cells were produced by AGT02 beyond 3 h of incubation (data not shown). To determine whether motility or flagellation was required for EPEC adhesion we constructed a mutation in motB

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

Fig. 3. Binding of purified H6 flagella to HeLa cells in culture and inhibition of adherence. HeLa cells were incubated with purified flagella from E2348/69 (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-specific anti-H6 and anti-H7 flagella antibodies. Fluorescent fragmented H6 flagella filaments (A) but not H7 flagella (B) are seen bound to the cell monolayer, 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. © 2002 Blackwell Science Ltd, Molecular Microbiology, 44, 361–379

Flagella-mediated adherence of EPEC 365 flagellin subunits as shown by immunoblotting (Fig. 1I). To investigate further if flagella had adhesive properties, nonadherent E. coli K-12 strain ORN172, which has been deleted of the fim type I pili genes, was transformed with the H6 fliC gene from E2348/69 harboured on a plasmid. ORN172 carrying the fliC H6 gene was able to adhere to HeLa cells, forming discrete localized bacterial clusters on a few epithelial cells (Fig. 1K and L) only after 6 h of infection. The level of adherence was, however, much lower than that produced by EPEC E2348/69 (Fig. 1E). These are compelling data that show that the flagella filaments are required for efficient adherence of EPEC and microcolony formation. As the fliC mutant was unable to adhere efficiently, an obvious question to address was whether the production of BFP was affected in this mutant. Interestingly, no BfpA synthesis occurred in AGT01 and AGT02 upon growth in DMEM (a condition favourable for BFP synthesis) as detected by immunoblotting of whole-cell extracts reacted with anti-BFP antiserum (Fig. 2A). However, when AGT01 and AGT02 were allowed to adhere to HeLa cells, BFP fibres were detected by immunofluorescence (IF), although not at wild-type levels (Fig. 2B). The bfpA mutant did not show BfpA or BFP fibres in either condition (Fig. 2A and B). Thus, it is apparent that BFP expression in the fliC mutant is enhanced by the presence of epithelial cells. The ability of fliC mutants to recruit cellular actin beneath adhering bacteria tested negative at 3 h of incubation in the fluorescent actin staining test (data not shown) probably because of the poor adherence shown in these strains. Nevertheless, some pedestal formation (a marker for AE lesion) was observed with scanning electron microscopy when the bacteria were allowed to infect for longer periods (6 h) (Fig. 2C). These results suggest that fliC mutants are still able to cause AE lesions and that the presence of flagella and BFP favours adherence and AE lesion formation. No apparent differences were observed in the profile of known secreted proteins between wild-type and fliC mutants (data not shown). A recent report describes the unfocused actin nucleation in a Tir–intimin and type III secretion system-independent manner without pedestal formation (Hartland et al., 1999). Flagella possess adhesive properties On the basis of the experimental data obtained above, we suspected that the flagella of EPEC per se could possess adhesive attributes. To address this issue, flagella (H6 and H2) purified from EPEC strains and EHEC (H7) were incubated with HeLa cells for 3 h and the bound flagella detected by immunofluorescence (IF) assay using typespecific anti-H antibodies. The H6 (Fig. 3A) and H2 flagella (data not shown) but not the H7 flagella (Fig. 3B) © 2002 Blackwell Science Ltd, Molecular Microbiology, 44, 361–379

bound to this cell line, indicating that the flagella per se are potential adhesive structures. To test this interesting hypothesis further, we used antibodies against H6, H7 and P. mirabilis flagella in experiments of inhibition of adherence. An inhibition effect was noted using H6 antibodies (P < 0.01) but not with antibodies against H7 (P < 0.19) (Fig. 3C) or P. mirabilis flagella (data not shown). A 65% and 60% reduction in adherence was obtained with anti-H6 antibodies at 1:10 and 1:100 dilutions respectively (Fig. 3C). Interestingly, anti-H6 antibodies 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 to speculate that the expression of flagella by EPEC is induced in vitro, and perhaps in vivo, in the presence of mammalian cells in culture. Ultrastructural studies of EPEC E2348/69 showed that this strain produces few flagella filaments when grown in LB medium (Fig. 4A). These observations have been reported before (Farris et al., 1998). Unlike BFP, which is favourably expressed after growth in tissue culture media (e.g. DMEM) (Girón et al., 1991; Puente et al., 1996), E2348/69 produces even fewer flagella filaments under these conditions (Fig. 4B). However, bacteria recovered from the supernatants of monolayers of HeLa cells infected for 3–5 h of incubation produced numerous longer flagellar filaments (Fig. 4C). We examined by electron microscopy equal number (100) of bacteria grown under the different conditions employed. Only 10% and 5% of the E2348/69 bacteria examined after growth in LB and DMEM, respectively, showed flagella, whereas most of the bacteria (~ 80%) grown in the presence of cultured epithelial cells, produced numerous flagella. These data suggested that the presence of eukaryotic cells enhanced expression of flagella. However, variations in the degree of flagella produced in LB, DMEM and in the presence of cultured cells between and within serotypes were noted (data not shown). The identity of the flagella produced was confirmed by immunogold labelling using anti-H6 antibodies (Fig. 4D). The synthesis of flagellin was monitored by immunoblotting to determine differential expression of flagellin under the growth conditions described above. As expected, E2348/69 produced lower levels of flagellin in DMEM than in LB (Fig. 4E, lane 2). However, the bacteria recovered from the supernatants of infected HeLa cells showed increased synthesis of flagellin (Fig. 4E, lane 3). For this comparison, the number of bacteria in the different growth conditions was normalized to the same optical density. In contrast, a non-adherent E. coli K-12 HB101 produced low amounts of flagellin regardless of the presence or absence of eukaryotic cells

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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 HeLa cells infected for 3 h (C). We examined by electron microscopy an equal number (100) of bacteria grown under the different conditions employed. Only 10% and 5% of the E2348/69 bacteria examined after growth in LB and DMEM, respectively, showed flagella, whereas most of the bacteria (~80%) grown in the presence of cultured epithelial cells produced numerous flagella. Images are representatives of such observations. 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) and DMEM (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 mass standards is indicated on the left. Scale bars: A, 0.7 mm; B, 0.53 mm; C, 0.5 mm; and D, 0.5 mm.

in culture (Fig. 4E), demonstrating that the induction of flagella by the presence of HeLa cells is not a general attribute of all E. coli. The purified flagella were composed of flagellin monomers of 60 kDa (Fig. 4F), which were demonstrated by amino-terminal amino acid sequencing analysis to be flagellin subunits. These intriguing results prompted us to study the presence of flagella on bacteria adhering to tissue culture cells. We analysed a subset of well-characterized EPEC strains belonging to classical H serotypes (H2, H6, H34 and H40) associated with diarrhoeal disease, including several non-motile (H–) strains. Upon formation of the typical LA bacterial clusters on HeLa or HEp-2 cells and using anti-H6 antibodies and IF, we observed a striking fluorescent pattern showing numerous long fluorescent filaments protruding from the adhering organisms, which appeared to be distributed peritrichously (Fig. 5). All motile EPEC strains studied produced flagella to different extents while adhering to tissue culture cells, as visualized by IF (Fig. 5A–F). The fluorescent filaments appeared to extend within and between bacterial micro-

colonies. An interesting phenomenon arises from these observations, which is the fact that different flagella serotypes were detected with anti-H6 antibodies. The flagellins of enterobacteria share extended amino- and carboxy-terminal homologies, with considerable divergence 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 flagellins (MacNab, 1996). However, flagella of different H types were identified using anti-H6 antibodies, suggesting the presence of a common native epitope(s) present among these flagellins, albeit reactivities were weaker with antibodies against H2, H34 and H40 flagellins (data not shown). The degree of flagella expression varied among H types and strains (Fig. 5A–F), and while some strains produced abundant wavy filaments others manifested few such structures. These observations suggest that other functions besides motility are inherent to these appendages. Among a limited number of H– strains tested, including B171 (O111:NM), a non-motile EPEC strain used in recent volunteer studies (Bieber et al., © 2002 Blackwell Science Ltd, Molecular Microbiology, 44, 361–379

Flagella-mediated adherence of EPEC 367

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 phasecontrast 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 a few flagella upon adherence. It is apparent that EHEC EDL933 (J) and ETEC E9034A (K) do not produce flagella when associated with HeLa cells. No reactivity is seen between E2348/69 flagella and anti-P. mirabilis flagella antibodies (L). After 3 h infection of HeLa cells, the monolayers were fixed with 2% formalin and reacted with antibodies against H6 (A–I), H7 (J), H9 (K) and P. mirabilis flagella (L) and secondary 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).

1998), some produced from one to several flagella per one to five fields examined when adhering to HeLa cells (Fig. 5G–I). The fact that EPEC strains such as E2348/69 produce abundant flagella when adhering to cultured cells, and that non-motile strains were also able to 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 eukaryotic origin. IF assays of EHEC (O157:H7) and ETEC (O8:H9) © 2002 Blackwell Science Ltd, Molecular Microbiology, 44, 361–379

bacteria adhering to HeLa cells showed no detection of flagella when employing specific H7 or H9 flagella antibodies (Fig. 5J and K), suggesting that enhanced expression of flagella during infection is not a generalized phenomenon among pathogenic E. coli. Likewise, IF of EPEC E2348/69 adhering to cultured cells employing antibodies raised against flagella from enteric pathogens S. typhi, S. flexneri, S. sonnei, P. mirabilis and EHEC O157:H7 showed no fluorescent filaments, demonstrating

368 J. A. Girón et al. the specificity of the IF reaction (Fig. 5L and data not shown). The striking fluorescent flagella profile differs from the previously described fluorescent patterns observed when antibodies directed against the BFP (Fig. 6A and B) (Girón et 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 demonstrated by confocal microscopy that the BFP and flagella are simultaneously produced during infection of HeLa cells (Fig. 6C–E). Based on the fluorescent pattern, the BFP filaments appeared to closely tether bacteria within the microcolony (Fig. 6A–C), whereas the typical wavy flagella produced appeared to extend outward within and between LA bacterial clusters (Figs 5 and 6D and E). Demonstration of flagella on adhering bacteria To further confirm the presence of these appendages on the adhering bacteria, we performed scanning electron microscopy (SEM) of infected HeLa cells. Figure 7 depicts wavy, thick (approximately 40 nm wide) intertwisting flagella-like filaments protruding from the bacteria and which can extend several microns away. These morphological features are very suggestive of flagella filaments because they are thicker than pili (3–7 nm wide) and, unlike BFP, do

not typically associate into rope-like structures. In particular, strains E10 and E28 (Fig. 7C, E and F) formed filaments that associated into a spider web-like meshwork that covered the microcolony. These observations correlate with the extensive fluorescent flagella seen in Fig. 5. From these descriptive micrographs we are tempted to hypothesize that these structures are assisting in microcolony formation and possibly mediating direct interaction with epithelial cells (Fig. 7E and F). We showed above by IF that BFP structures are also present within the bacterial microcolonies. Observation of the flagella structures by high-resolution emission SEM revealed wavy filaments with beaded extrusions (Fig. 8A and B). EPEC induces proliferation of microvilli-like processes (MLP) upon adherence to tissue culture cells, causing remodelling of the eukaryotic cell surface (Phillips et al., 2000). In order to confirm that the filaments were bacterial in nature and not MLP produced by host cells, we performed the IF adherence assay using HeLa cells that were prefixed with methanol. The use of prefixed cells dramatically reduced the size and numbers of adherent microcolonies compared with non-fixed cells, although the bacteria were still able to produce, albeit fewer, flagella under these conditions (Fig. 8C and D). This suggests that adherence and flagella 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 tightly confined to the bacterial cluster (A, B and C). Double staining with mouse monoclonal anti-BFP and rabbit polyclonal anti-H6 antibodies demonstrated 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.

Flagella-mediated adherence of EPEC 369

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 as bridges 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 bacterial origin. The identity of the wavy structures observed was confirmed to be flagella by immunogold labelling and SEM ÂŠ 2002 Blackwell Science Ltd, Molecular Microbiology, 44, 361â&#x20AC;&#x201C;379

using anti-H6 antibodies and anti-rabbit IgG conjugated to 30 nm gold particles (Fig. 8E and F). In all, these results conclusively demonstrate that these appendages are flagella.

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Fig. 8. High-resolution field emission SEM of EPEC adhering to HeLa cells. High magnification (>100 000¥) of the flagella-like appendages produced by E28 (A and B) reveals beaded (nodule-like) extrusions. Long wavy filaments resembling typical flagella (arrows) are seen tethering bacteria and short filaments, probably representing EspA-containing organelles (Knutton et al., 1998), are also seen protruding from the bacterial cell surface. The two structures appeared to be of equal width but differed in length. SEM (C) and IF (D) of EPEC adhering to prefixed HeLa cells after 3 h of infection confirmed that the filaments are bacterial in nature. The flagellum is indicated by an arrow in C and as 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 gold particles (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.

Influence of EAF plasmid and type III secretion genes in flagella expression and motility The role of the EPEC adherence factor (EAF) plasmid in adherence of EPEC has been well established (Nataro and Kaper, 1998). In an effort to investigate the influence of the EAF plasmid in the motility of EPEC and its correlation 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 was observed with any of the strains at 30°C (data not shown) or 37°C in LB motility agar (Fig. 9A). However, when the motility 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 with its inability to efficiently produce flagella upon growth in © 2002 Blackwell Science Ltd, Molecular Microbiology, 44, 361–379

Flagella-mediated adherence of EPEC 371 DMEM as determined by negative staining and electron microscopy (data not shown), and by immunoblotting using whole-cell extracts and anti-H6 antibodies (Fig. 9D). Figure 9 depicts the synthesis of flagellin by E2348/69 and derivative mutants and, as expression of flagellin is weaker in DMEM than in LB, for the purpose of comparison the blot was overexposed with the appropriate substrate (see Experimental procedures). In fact, JPN15 produced barely detectable amounts of the 60 kDa flagellin compared with the parent strain. The perA mutant was moderately motile in DMEM (Table 1), which correlated with its ability to still produce flagellin (Fig. 9D). We

had shown above that the fliC-minus strain was deficient in BFP production; thus, we became interested in determining whether a bfpA-minus strain would be altered in synthesis of flagella and motility. Likewise, the bfpA-minus strain was deficient in flagellin synthesis (Fig. 9D) and motility (Table 1). These results expand our knowledge of the phenotypes controlled by the EAF plasmid genes and implicate a modulatory relationship between perA, bfpA and flagella expression and motility. Recently, the participation of the flagellar sigma factor FliA 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 were inoculated 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 at 37°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 after growth in DMEM, this blot was overexposed for the purpose of comparison with the isogenic mutants. Note the lack or reduced synthesis of flagellin in all the mutants, except for the perA mutant. Mass standards are indicated with arrows. © 2002 Blackwell Science Ltd, Molecular Microbiology, 44, 361–379

372 J. A. Girón et al. (Eichelberg and Galán, 2000). Thus, we were then interested in determining whether eae and the type III secretion genes encoded in the LEE region played any role in flagella production and motility. E2348/69 isogenic mutants defective in intimin and Tir production, in function of the type III translocon (escN), and in synthesis of EspA, EspB and EspD were examined for the phenotypes under investigation. All of these mutants were non-motile or weakly motile in DMEM motility agar (Table 1 and Fig. 9B), suggesting a relationship between flagellation, motility and the type III secretion pathway. Except for the espD mutant, which was able to synthesize some detectable flagellin and to display flagella in contact with cultured cells (Fig. 10), the eae, espA, espB and escN mutants were impaired in their ability to optimally

synthesize flagellin when grown in DMEM (Fig. 9D) and to produce abundant flagella when associated with HeLa cells (Fig. 10). Occasionally, one or two flagella filaments were observed in microcolonies formed by these mutants. However, the tir mutant produced poor amounts of flagellin when tested in immunoblots (Fig. 9D) but was able to produce flagella to levels similar to that of the parent strain when examined by microscopy (Fig. 10). It is apparent from the adherence phenotypes shown in Fig. 10 that all of the mutants tested are still able to interact with epithelial cells and to form microcolonies, an event that was observed upon extended infection periods. Quorum sensing has been shown to influence the expression of EPEC and enterohaemorrhagic E. coli LEE genes via Ler in a regulatory cascade (Sperandio et al., 1999). Fig. 10. Detection of flagella produced by isogenic mutants upon adherence to HeLa cells. After infection with the indicated mutants, the flagella were detected by immunofluorescence as described in the text. Individual green bacteria adhered individually or formed microcolonies of different sizes. In contrast to the perA, espD and tir mutants, which displayed several flagella (white arrows) per field examined, the remaining mutants occasionally displayed one or two flagella per field.

Flagella-mediated adherence of EPEC 373 In agreement with previous observations (Sperandio et al., 2001), we noted here that a luxS mutant (VS102) was unable to swim and to produce flagella, even when associated in microcolonies with HeLa cells (Fig. 10 and Table 1). The expression of EPEC flagella is triggered by a secreted eukaryotic molecule To investigate the possible role of a molecule of eukaryotic origin involved in triggering flagella production and motility, we prepared DMEM that was previously incubated for 24–48 h with monolayers of HeLa cells in the absence of fetal bovine serum or antibiotics. This preconditioned medium was used in motility tests and to detect the synthesis of flagellin or production of flagella filaments in the isogenic mutants. Except for the eae mutant, which was repeatedly negative for motility, this preconditioned medium restored the ability of the remaining strains to swim (Table 1 and Fig. 9C) and to synthesize and assemble flagella (data not shown). Thus, the biosynthesis of flagella may be activated also by a perindependent mechanism. Further experiments demonstrated that the molecule present in the preconditioned medium was heat stable and that high-pressure liquid chromatography fractions below 1 kDa still retained the ability to induce flagellation and motility in non-motile EPEC mutants. While the nature of this molecule is under investigation, the present data strongly suggest that a soluble factor of eukaryotic origin was present in the preconditioned medium and that this factor bypassed regulation by PerA-activating genetic elements involved in flagella production or regulation. Discussion Although the flagellar H antigen is one of the surface and epidemiological markers that identifies EPEC as a diarrhoeagenic class of E. coli, in addition to expression of BFP and possession of the LEE, neither previous nor current models of EPEC pathogenesis have implicated flagella in adherence (Nataro and Kaper, 1998; Frankel et al., 1998). Here, we show for the first time that the flagella of EPEC are important adhesive structures highly induced upon interaction with epithelial cells and most likely by a secreted signalling molecule of eukaryotic origin. EPEC H6 and H2, but not EHEC H7, purified flagella were demonstrated to directly mediate bacterial attachment to epithelial cells. Several different EPEC serotypes were shown by IF to express flagella to different degrees when adhering to cultured cells. These results were obtained using specific antisera against EPEC flagella but not with antibodies against flagella of other enteric pathogens. It is relevant to note that some © 2002 Blackwell Science Ltd, Molecular Microbiology, 44, 361–379

EPEC strains serologically classified as H– or non-motile were also able to produce flagella, albeit less than motile strains, when adhering to culture cells. It is well documented that most EPEC plasmid- and chromosome-encoded virulence factors are environmentally 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 bacteria enter stationary phase, once the AE lesions have formed (Puente et al., 1996; Rosenshine et al., 1996; Kenny et al., 1997; Knutton et al., 1997; 1998). Our data suggest that flagella biosynthesis is not turned off even when the bacteria enter stationary phase, and this pattern indicates a constant stimulation of flagella expression. We showed that the production of flagella was reduced when methanol-fixed HeLa cells were employed compared with live cells. The possibility that a eukaryotic soluble product(s) signals the expression of flagella in EPEC was thus addressed. We prepared DMEM preconditioned by growth of HeLa cells and showed that it activated motility in E2348/69 isogenic strains unable to swim in DMEM motility agar. Although the biochemical nature of this soluble activator of flagella expression and motility is currently under investigation, it is tempting to speculate that a molecule that triggers expression of flagella and possibly other virulence genes, with a chemical structure similar to that secreted by HeLa cells, might be present in the intestinal tract. In support of this speculation, there are other data that suggest that the in vivo environment induces motility in S. enterica var. Gallinarum and Pullorum serovars, which are traditionally recognized as non-motile (Chaubal and Holt, 1999). The effect of pH, temperature and surface contact on the elaboration of flagella by Salmonella serotype Enteritidis has also been reported (Walker et al., 1999). Furthermore, the presence of abundant flagella on adhering bacteria suggests that other properties besides motility, such as adherence and possibly translocation of proteins with potential virulence attributes, could be inherent to these appendages. It is obvious that most bacterial pathogens must overcome the viscous mucus that naturally bathes epithelial surfaces in order to successfully reach their target niches. Thus, it is reasonable to propose that flagella expression and motility properties of EPEC are of critical importance to translocate bacteria across the intestinal mucus, thereby facilitating interaction with target sites on the surface of enterocytes. Several studies have addressed the role of flagella-mediated motility in virulence (adherence, colonization and invasion) and development of biofilm 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.,

374 J. A. Girón et al. 2000; Young et al., 2000; Gewirtz et al., 2001a). We attempted to study the role of flagella in adherence of EPEC by several approaches. We demonstrated that H6 flagella purified from EPEC but not EHEC H7 flagella bind to HeLa cells. The data provided here do not explain the mechanism of binding of flagella but it is possible that the H6 flagellin possesses unique binding domains present along the non-conserved region of the protein that is exposed 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 carried in the flagellin protein, specifically the non-conserved region, will be needed to test this interesting hypothesis. The flagellins of enterobacteria share extended sequence homology in the amino- and carboxy-termini although considerable divergence exist within the middle region of the proteins (MacNab, 1996). The antigenic differences resulting from divergent flagellin sequences (Reid et al., 1999) form the basis for H serotyping of E. coli. Interestingly however, the flagella of different H types produced by adhering EPEC strains were identified using anti-H6 antibodies suggesting the presence of a common native epitope(s) among antigenically heterologous flagellins. Furthermore, the LA phenotype exhibited by different EPEC serotypes was inhibited by anti-H6 antibodies suggesting similar functions among EPEC flagella. This inhibition effect could be attributed presumably to stearic hindrance impeding flagella binding sites to recognize target sites on the host cell. This hypothesis is based on the assumption that flagella per se possess genuine adherence moieties. Recent reports suggest that flagellin of S. Typhimurium is translocated through the epithelial cell to basolateral membrane inducing a proinflammatory response (Gewirtz et al., 2001a, b). Thus, another possible explanation is that flagella induce host cells responses that then mediate adherence. These are interesting issues that will be addressed in future investigations. Structural analysis by SEM of EPEC of different serotypes adhering to HeLa cells revealed the presence of structures resembling flagella that appeared to mediate direct binding of the bacteria to the host cell thereby contributing to the formation of three-dimensional microcolonies. These structures are morphologically and antigenically different than the BFP structures that have been shown to promote aggregation of bacteria during initial stages of infection and dispersal of bacteria after extended periods of incubation (Bieber et al., 1998; Knutton et al., 1999). We showed here that both flagella and BFP are produced simultaneously during infection. The short EspA filaments produced during the early stage of 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 filaments are proposed to mobilize effector molecules into the host cell 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 extended similarities between flagellar and type III secretion systems suggest common functional features that have evolved for bacterial adaptation, survival and virulence (Hueck, 1998; Lory, 1998; Galán and Collmer, 1999). Recently, the translocation of virulence-associated nonflagellar proteins by the type III flagellar export machinery of Y. enterocolitica was reported (Young et al., 1999). Although no differences in the number of known secreted proteins were observed between wild-type EPEC and its isogenic fliC mutant, it is tempting to speculate that either Esps or other yet unidentified virulence-associated proteins produced only upon contact with epithelial cells in vitro or in vivo may be exported through the flagellar type III secretion pathway or alternatively through the EspA secretory channel. Alternatively, it is possible that flagellamediated attachment (specially during early stages of infection) is also required for efficient intimate attachment and delivery and function of effector molecules through the type III secretion system. An important role of flagella in adherence was further demonstrated when two EPEC fliC mutants unable to synthesize flagella were impaired in their ability to adhere and form LA at 3 h of infection. Prolonged incubation of flagella-minus bacteria with HeLa cells for 6 h showed individual adherent bacteria and small clusters with no apparent flagella filaments protruding from them. A motB mutant was still able to produce flagella and to adhere to cultured cells after 3 h of infection. From this observation, it appears that motility is not essential for adherence although there is a need to clearly delineate between adherence and chemotaxis. Future studies will be aimed at discriminating between these possibilities and to expand our knowledge on the role of motB. Both mutants were able however, to recruit cellular actin and to form pedestals but only after 6 h of infection. When the E2348/69 fliC mutant was tested for BfpA and BFP expression, we found that this strain was deficient in synthesis of BfpA after growth in D-MEM, a condition that promotes BFP expression. Nevertheless, once the fliC was allowed to interact with cultured cells for over 3 h of infection, detectable amounts of BFP fibres were noted. Likewise, the bfpA mutant was shown to be deficient in motility and in flagellin synthesis after growth in D-MEM or in the presence of cultured cells. We have shown here that both the flagella and BFP are co-produced when the bacteria are adhering and forming microcolonies on cultured cells. These results expand our knowledge of the phenotypes controlled by the EAF plasmid genes and highlight a © 2002 Blackwell Science Ltd, Molecular Microbiology, 44, 361–379

Flagella-mediated adherence of EPEC 375 modulatory relationship between perA, bfpA, and flagella expression and motility. Our data strongly indicate that flagella are adhesive structures directly involved in LA formation, promoting short and long-range physical bridges between bacteria, and that their expression contributes to AE lesion formation. The synthesis, assembly and function of the flagellar system of E. coli and S. enterica serovar Typhimurium is under the control of a flagellar regulon that comprises more than 50 genes that are divided among at least 17 operons (Liu and Matsumara, 1994; MacNab, 1996; Chilcott and Hughes, 2000). In EPEC, several virulenceassociated genes appear to be required for adequate synthesis and function of flagella. We have demonstrated that mutations in genes located in the EAF plasmid (e.g. perA and bfpA), in the chromosomal LEE region (escN, eae, tir, espA, esp and espD) and in the gene encoding the quorum-sensing autoinducer synthetase (luxS) substantially affect the expression of flagella and motility in EPEC when grown in DMEM but not in LB. In agreement with this observation, a relationship between genes involved in flagella biosynthesis, motility and quorum sensing was recently noted in enterohaemorrhagic E. coli (Sperandio et al., 2001). When these mutants were assayed for flagella expression during the course of infection of HeLa cells, only the espD and tir mutants appeared to produce more flagella than the remaining mutants, but less than the parent strain. The present data cannot explain why only these two mutants were not fully affected in flagella production when interacting with mammalian cells, but it is certainly an interesting issue, and one which we are investigating further. Except for the eae mutant, all of the mutants studied were restored for motility in the presence of preconditioned medium, supporting our hypothesis that a signal of eukaryotic origin triggers flagella expression in EPEC. Efforts to induce motility in the eae mutant in the presence of the preconditioned medium were unsuccessful, and this result is indicative of complicated feedback regulatory mechanisms. This observation raises the possibility that the eae gene or its product (intimin) may play some yet undefined role in flagella function and motility. In this regard, recent work on virulent E. coli strains belonging to serogroup O26 show a strong correlation between the presence of the eae gene and the possession of the H11 flagellar antigen (Zhang et al., 2000). The motility results obtained with the LEE and EAF plasmid mutants support the hypothesis that a eukaryotic signal induces flagella expression and adherence in EPEC. In all, these data suggest the existence of a molecular relationship between epithelial cells, the flagellar regulon and EPEC virulence genes, and strengthen the notion of the evolutionary relationship between flagellar and protein secretion type III pathways (Hueck, 1998; Lory, 1998; © 2002 Blackwell Science Ltd, Molecular Microbiology, 44, 361–379

Galán and Collmer, 1999; Chilcott et al., 2000; MacNab, 2000). Why distinct flagella were not identified in previous EPEC–host cell interaction studies is an obvious question that arises from this paper. For many years, researchers believed EPEC strains did not elaborate fimbriae (Scotland et al., 1983) until the BFP was discovered in strain B171 (Girón et al., 1991; Giron et al., 1993). This finding was possible as a result of modifications in the in vitro growth conditions. For almost half a century now, we have known that the flagellar H antigen of EPEC is a criterion for defining epidemic strains, but researchers were apparently focusing on the flagella not as an appendage involved in adherence, but as a serotyping antigen. Intimin, the BFP, the EspA filaments, the flagella and possibly other unknown factors have always been there for us to discover when the bacteria interact with their host cells. Knowing how to discriminate among these structures and how their functions are orchestrated is certainly a challenge for EPEC researchers. Based on the present results and the most recent published data, it is reasonable to propose that the interaction of EPEC with host cells entails the participation of several adhesins: intimin, type IV bundle-forming pili, an EspA-containing organelle associated with the type III secretion system and flagella. Directly or indirectly, all of these bacterial components appear to be regulated by Per and are apparently stimulated by an eukaryotic factor(s) released into the supernatant. How these elements are synchronized during infection of the gut mucosa remains an interesting and important issue to address.

Experimental procedures Bacterial strains EPEC strains and isogenic derivatives employed are listed in Table 1. Wild-type EPEC strains employed were E2348/69 (O127:H6), a prototypic strain whose virulence has been demonstrated in volunteers studies; JPN15, a derivative of E2348/69 cured of the EAF plamid; JPN15 (pMAR7), JPN15 complemented with a transposon-marked EAF plasmid pMAR7; 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 were kindly 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. coli K-12 HB101 and ORN172 (Dfim), which were obtained from our collection.

Construction of isogenic fliC and motB mutants Mutants defective in flagella production and motility were constructed in the chromosome of E2348/69 and E10 strains by

376 J. A. Girón et al. marker exchange as follows. The 5¢ and 3¢ regions of the fliC flagellin gene of E2348/69 were amplified by polymerase chain reaction (PCR) with two set of primers: 5HFLIC (5¢CCAAGCTTATGCAGTCTGCGCTGTCGA-3¢) and 3RvFLIC (5¢-GCGCTGGAGATATCATCAGAA-3¢); and 3BFLIC (5¢-CCGGATCCTCATACCTGGTTGGCTTTTGC-3¢) and 5RvFLIC (5¢-TTCTGATGATATCTCCAGCGC-3¢). The amplicons obtained were ligated together with the chloramphenicol (cat) cassette, which was used to interrupt the flagellin sequence. The fliC::cat gene was cloned into suicide vector pCVD442 and marker exchange was performed as previously described (Sperandio et al., 2001). Similarly, a mutation in the flagella motor rotation system (motB) was introduced in E2348/69 using the cat gene. motB was amplified by PCR using primer pairs – 5HMOTB (5¢-ACCTTCGAAATCGAA GCTTTGA-3¢) and 3RvMOTB (5¢-GCGATATCCTTTCTCAC CGC-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 A GT01 (fliC –) were confirmed by PCR to contain an insertion mutation. To confirm the phenotypes of these mutants, motility assays were performed in glass vials or Petri dishes utilizing LB broth, DMEM or preconditioned DMEM supplemented with 0.3% agar. Motility was typically read after 16–18 h of incubation at 37°C.

Expression of H6 in E. coli K12 The H6 fliC gene was amplified from E2348/69 using primers derived from the reported fliC sequence (Reid et al., 1999), cloned into pBR322 yielding pFliC and transformed in E. coli K-12 ORN172 lacking the type I pili fim genes. The adherence phenotype of this transformant was tested as described below.

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

Isolation of flagella filaments and secreted proteins Flagella were mechanically sheared from E10 (O119:H6) and E18 (O128:H2) bacteria grown in Dulbecco’s minimal essential medium (DMEM) and separated by differential centrifu-

gation and a caesium chloride gradient to obtain relatively pure flagella filaments (Tacket et al., 1987). In addition, flagella from EHEC EDL933 (O157:H7) were purified in a similar way. The flagella preparations were adjusted to same protein concentration and then subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970) and electroblotting to polyvinylidene difluoride (PVDF) membranes (pore size 0.45 mm, Millipore). The presence of flagella was confirmed by electron microscopy as described above. The protein band of interest was excised and subjected to N-terminal sequence analysis (Protein and Nucleic Acid Facility at Stanford University). To analyse the profiles of proteins secreted by wild-type E2348/69 and fliC mutants, supernatants of bacteria grown in DMEM were obtained and concentrated by ultrafiltration (Jarvis et al., 1995). In all the experiments, equal amounts of proteins were loaded on the SDS-PAGE gels.

Western blotting and antisera To determine production of flagellin, the bacteria were grown overnight in LB and DMEM, and those obtained from supernatants of infected cells for 3 h were adjusted to an absorbance of 0.7 at OD600. Thus, equal numbers of bacteria were used to prepare whole-cell extracts by denaturation in SDS-PAGE sample buffer and separated in 14% SDSPAGE gels. Proteins were transferred onto PVDF membranes and reacted with antiflagella antibodies and secondary anti-rabbit IgG conjugated to horseradish peroxidase (Sigma). As expression of flagellin is weaker in DMEM than in LB, for the purpose of comparing the synthesis of flagellin by E2348/69 and derivative mutants the blots were overexposed with the appropriate substrate (see Fig. 9). Antisera against EPEC H2, H6, H34 and H40, EHEC H7, and ETEC H9 flagella were raised by immunization of rabbits with purified flagella. Antibodies against S. typhi, S. flexneri, and S. sonnei flagella, and monoclonal antibodies against BFP were available from previous studies (Girón 1995; Girón et al., 1995). Anti-P. mirabilis flagella serum was kindly donated by Harry Mobley (University of Maryland). Other anti-H6 and anti-H2 antibodies were a kind gift of Carlos Eslava (UNAM, México).

Interaction with eukaryotic cells The adherence and immunofluorescence (IF) assays were performed using HeLa and HEp-2 cells, utilizing DMEM containing 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 phosphate buffer, the cells were fixed with 2% formalin. The specimens were stained with Giemsa or prepared for IF as follows. Primary rabbit antibodies against flagella or BFP were added for 1 h at the appropriate dilutions in 10% normal horse serum. After washing, the cells were incubated for 1h with secondary anti-rabbit IgG FITC-conjugated antibodies diluted 1:3000. The cells were washed extensively and mounted in glycerol–PBS and visualized under a UV light and phase contrast using a Zeiss Axiolab microscope. When needed, HeLa cells were prefixed with methanol for © 2002 Blackwell Science Ltd, Molecular Microbiology, 44, 361–379

Flagella-mediated adherence of EPEC 377 20 min before the IF assay described above. HeLa cell monolayers or cell suspensions were also incubated with or without purified H6 or H7 flagella (adjusted to the same protein concentration) for 3 h at 37°C to test the binding properties of flagella. Bound flagella were detected by IF using similar titres of antiflagella antibodies. Polymerized actin was assayed by the fluorescent actin staining (FAS) test as previously described (Knutton et al., 1989). For adherence inhibition experiments, 107 CFU of an overnight bacterial culture grown in Luria–Bertani (LB) broth were incubated with 1:10 and 1:100 dilutions of anti-H6 and anti-H7 antibodies in DMEM at room temperature for 30 min and then added to the HeLa cell monolayers. These antisera contained similar titres of anti-H antibodies as determined by immunoblotting. After incubation for 3 h, the cells were lysed with PBS–0.5% Triton X-100, diluted 10-fold, and plated onto MacConkey agar to estimate the number of bacteria adhering to the cell monolayers. Replica samples were stained with Giemsa and examined by light microscopy. Antibodies against P. mirabilis flagella were included as a negative control.

Preconditioned medium Monolayers of HeLa cells that had been extensively washed with PBS were incubated with DMEM without antibiotics or fetal bovine serum for 24–48 h. The supernatant referred as to ‘preconditioned medium’ was collected and the pH adjusted to 7.4, filtered through a 0.2 mm membrane, and supplemented with 0.3% agar for motility tests or used to grow EPEC mutants and to detect the synthesis of flagellin or production of flagella filaments in these mutants. Except for the cloning experiments, all the above experiments were repeated at least five times to confirm the results obtained. Unless otherwise indicated, the results shown here are representative of a particular assay or observation under the microscope.

Acknowledgements We thank Jennifer Abbott, Vanessa Sperandio and Sooan Shin for critical discussions, and Jane Michalski for helpful assistance. We thank Harry Mobley, Luiz Trabulsi and Carlos Eslava for kindly providing antisera and strains; Adam Crawford for the tir-minus strain; Erasmo Negrete, Mónica Rosales, and Renato Cappello for their valuable technical assistance. This work was supported by NIH grant no. AI21657. J. A. Girón thanks Lilia Cedillo (BUAP), Conacyt (Grant 32777-M) and the Japan International Cooperation Agency for support. A. G. Torres was supported by a research supplement for underrepresented minorities from the NIAID, NIH.

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