Binding of intimin from enteropathogenic Escherichia coli to lymphocytes and its functional consequences

Intimin-conjugated fluorescent beads bind to spleen CD4 T cells and Peyer's patch, mesenteric lymph node, and cecal follicle lymphocytes, with less binding to lamina propria T cells and intraepithelial lymphocytes. Intimin costimulates proliferation of spleen CD4 T cells and cells from organized lymphoid tissues but does not costimulate cells from the lamina propria of normal or inflamed colon.     

Role of tir and intimin in the virulence of rabbit enteropathogenic Escherichia coli serotype O103:H2

Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.     

Polymorphisms within EspA filaments of enteropathogenic and enterohemorrhagic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) possess a filamentous type III secretion system (TTSS) employed to deliver effector proteins into host cells. EspA is a type III secreted protein which forms the filamentous extension to the TTSS and which interacts with host cells during early stages of attaching and effacing (A/E) lesion formation. By immunofluorescence, a polyclonal antibody previously raised to EspA from EPEC strain E2348/69 (O127:H6) stained approximately 12-nm-diameter EspA filaments produced by this strain but did not stain similar filaments produced by EHEC serotype O157:H7. Similarly, an antibody that we subsequently raised to EHEC strain 85-170 (O157:H7) EspA stained approximately 12-nm-diameter EspA filaments produced by strain 85-170 but did not stain E2348/69 EspA filaments. Given such heterogeneity between EPEC and EHEC EspA filaments, we examined polymorphisms of functional EspA filaments among different EPEC and EHEC serotypes. With use of the EPEC EspA antiserum, EspA filaments were observed only with EPEC serotypes O127:H6 and O55:H6, serotypes which encode an identical EspA protein. When stained with the EHEC EspA antiserum, EspA filaments were detected only on EHEC strains belonging to serotype O157:H7; the EHEC antiserum did, however, stain EspA filaments produced by the closely related EPEC serotype O55:H7 but not filaments of any other EPEC serotype tested. Such polymorphisms among functional EspA filaments of EPEC and EHEC would be expected to have important implications for the development of broad-range EspA-based vaccines.     

Biotypes of enteropathogenic Escherichia coli strains from rabbits

The Isolator 1.5 Microbial tube (E. I. du Pont de Nemours & Co., Inc., Wilmington, Del.) is a commercially available blood culture system for use in pediatrics. The methodology is based on blood lysis followed by direct plating of the sample on culture media to detect bacteria and fungi. Comparative recovery rates of pathogens from blood collected in this and a conventional broth system were similar. The Isolator detected 104 of 120 clinically significant isolates, whereas 106 of 120 isolates were detected by the broth system. The major advantage of the Isolator methodology was early detection of septicemia. Initial detection of gram-negative bacteria occurred an average of 14.2 h earlier by the Isolator system than by the conventional broth method. The Isolator also permitted quantitation of bacteremia and fungemia. Probable contaminants were recovered from 10.0% of the cultures processed by the Isolator, but steps which could be taken to minimize this problem were identified. The Isolator is a useful method for pediatric blood cultures.     

Characterization of two virulence proteins secreted by rabbit enteropathogenic Escherichia coli, EspA and EspB, whose maximal expression is sensitive to host body temperature.

Enteropathogenic Escherichia coli (EPEC) and rabbit EPEC (RDEC-1) cause unique histopathological features on intestinal mucosa, including attaching/effacing (A/E) lesions. Due to the human specificity of EPEC, RDEC-1 has been used as an animal model to study EPEC pathogenesis. At least two of the previously identified EPEC-secreted proteins, EspA and EspB, are required for triggering host epithelial signal transduction pathways, intimate adherence, and A/E lesions. However, the functions of these secreted proteins and their roles in pathogenesis have not been characterized. To investigate the function of EspA and EspB in RDEC-1, the espA and espB genes were cloned and their sequences were compared to that of EPEC O127. The EspA proteins showed high similarity (88.5% identity), while EspB was heterogeneous in internal regions (69.8% identity). However, RDEC-1 EspB was identical to that of enterohemorrhagic E. coli serotype O26. Mutations in RDEC-1 espA and espB revealed that the corresponding RDEC-1 gene products are essential for triggering of host signal transduction pathways and invasion into HeLa cells. Complementation with plasmids containing EPEC espA or/and espB genes into RDEC-1 mutant strains demonstrated that they were functionally interchangeable, although the EPEC proteins mediated higher levels of invasion. Furthermore, maximal expression of RDEC-1 and EPEC-secreted proteins occurred at their respective host body temperatures, which may contribute to the lack of EPEC infectivity in rabbits.     

Protection against hemorrhagic colitis in an animal model by oral immunization with isogeneic rabbit enteropathogenic Escherichia coli attenuated by truncating intimin

Strains of Shiga toxin (Stx)-producing Escherichia coli, also called enterohemorrhagic E. coli (EHEC), are important food-borne pathogens for humans. Most EHEC strains intimately adhere to the intestinal mucosa in a characteristic attaching and effacing (A/E) pattern, which is mediated by the bacterial adhesin intimin. Subsequent release of Stx1 and/or Stx2 leads to the frequent development of hemorrhagic colitis and, less commonly, to hemolytic-uremic syndrome. The aim of the present study was to develop an attenuated A/E E. coli strain for use as a vaccine against EHEC infection encoding a truncated intimin lacking adhesive capacity, but which would still express somatic antigens, other products of the locus of enterocyte effacement pathogenicity island, and an immunogenic remnant of the intimin molecule. A single-nucleotide deletion was generated in the eae gene in the prototype rabbit A/E E. coli strain RDEC-1 (O15:H-), which resulted in truncation of intimin by 81 C-terminal residues (860 to 939 amino acids) containing a disulfide loop. Inoculation of rabbits with large doses of the truncated intimin mutant (RDEC-1Deltaeae(860-939)) was well tolerated, as observed by the absence of clinical signs of disease or evidence of intestinal A/E lesions. The efficacy of RDEC-1Deltaeae(860-939) as a vaccine was evaluated by orogastric inoculation of rabbits with RDEC-1Deltaeae(860-939) followed by challenge with the virulent strain RDEC-H19A, an Stx1-producing derivative of wild-type RDEC-1 capable of inducing hemorrhagic colitis in rabbits. Following RDEC-H19A challenge, nonimmunized control rabbits exhibited characteristic weight loss with watery to bloody diarrhea and demonstrated intimate bacterial attachment, effacement of microvilli, submucosal edema, mucosal heterophile infiltrates, and Shiga toxin-induced vascular lesions. In contrast, the RDEC-1Deltaeae(860-939)-immunized rabbits showed no clinical signs of disease, maintained normal weight gain, had reduced fecal shedding of challenge organisms, and showed an absence of gross or microscopic lesions in the intestinal mucosa. Serum antibodies specific to intimin were detected among rabbits immunized with RDEC-1Deltaeae(860-939), indicating that truncation of the intimin functional domain not only attenuated bacterial virulence, but also retained at least some of the immunogenicity of native intimin. Although it is not possible to gauge the exact contribution of residual intimin immunity to protection, this attenuation strategy for A/E E. coli strains shows promise for the development of effective vaccines to prevent EHEC infection in humans and animals.     

Molecular characterization of a carboxy-terminal eukaryotic-cell-binding domain of intimin from enteropathogenic Escherichia coli.

A eukaryotic cell-binding domain from the intimin (Int) polypeptide of enteropathogenic Escherichia coli O127 (EPEC) was investigated. Derivatives of the carboxy-terminal 280-amino-acid domains of Int (Int-EPEC280) and the Int homolog invasin (Inv) from Yersinia pseudotuberculosis (InvYP280) were fused to the E. coli maltose-binding protein (MBP), expressed, and purified. The smallest MBP-IntEPEC fusion protein that efficiently mediated binding to HEp-2 cells, monitored by using purified fusion proteins in fluorescence activated cell sorter analysis or by using fluorescent Covaspheres coated with purified fusions, contained the carboxy-terminal 150 amino acids of Int. Replacement of Cys-937 with Ser (IntEPEC280CS) destroyed the cell-binding activity of IntEPEC280. Covaspheres coated with MBP-IntEPEC280 were associated with HEp-2 cell microvilli but failed to induce actin accumulation underneath bound particles or cell spreading on coated plastic surfaces. MBP-IntEPEC280, but not MBP, MBP-IntEPEC280CS, or MBP-InvYP280, inhibited EPEC entry into HEp-2 cells.     

Isolation of recombinant antibodies against EspA and intimin of Escherichia coli O157:H7

Intimin, Tir, and EspA proteins are expressed by attaching-effacing Escherichia coli, which include enteropathogenic and enterohemorrhagic E. coli pathotypes. EspA proteins are part of the type three secretion system needle complex that delivers Tir to the host epithelial cell, while surface arrayed intimin docks the bacterium to the translocated Tir. This intimate attachment leads to attaching and effacing lesions. Recombinant forms of these effector proteins from enterohemorrhagic E. coli O157:H7 were produced by using E. coli expression vectors. Binding of intimin and Tir fragments in enzyme-linked immunosorbent assay (ELISAs) demonstrated the interaction of intimin fragments containing the C-terminal 282 or 188 amino acids to a Tir fragment containing amino acid residues 258 to 361. Recombinant intimin and EspA proteins were used to elicit immune responses in rabbits and immune phage-display antibody libraries were produced. Screening of these immune libraries by conventional phage-antibody panning and colony filter screening produced a panel of antibodies with specificity for EspA or intimin. Antibodies recognizing different C-terminal epitopes on intimin bound specifically to the gamma intimin of O157:H7 and not to other classes of intimin. Antibodies recognizing EspA from E. coli O157 also recognized the protein from the eae-deficient O157 mutant DM3 and from E. coli O111. Anti-intimin antibodies were also produced as fusion proteins coupled to the reporter molecule alkaline phosphatase, allowing the one-step detection of gamma intimin. The isolated recombinant monoclonal antibodies were functional in a range of assay formats, including ELISA, Western blotting, and dot blots, thus demonstrating their diagnostic potential.     

Contribution of plasmid-encoded fimbriae and intimin to capacity of rabbit-specific enteropathogenic Escherichia coli to attach to and colonize rabbit intestine

Attachment to the intestinal mucosa is an essential step in the pathogenesis of diarrhea caused by enteropathogenic Escherichia coli (EPEC). Fimbriae and intimin, the outer membrane protein product of the chromosomal eae gene, contribute to this process, but their relative roles and the nature of their interaction are not known. The aim of this study was to determine the relative contribution of plasmid-encoded fimbriae, termed Ral, and intimin to the capacity of rabbit-specific EPEC (REPEC) to attach to the intestinal mucosa of rabbits. To achieve this, we constructed a series of mutants in REPEC strain 83/39 (O15:H-), in which the ralE and eae genes were insertionally inactivated. These strains were then inoculated into ligated loops of rabbit ileum, which were resected 18 h later and examined by light and electron microscopy. The results showed that intimin, but not Ral, is essential for the elicitation of attaching-effacing lesions by REPEC. Nevertheless, a delta eae Ral-bearing mutant adhered to the intestinal epithelium to the same extent as its eae-positive parent and far more extensively than an eae(+) delta ral strain. To examine the contribution of Ral and intimin to colonization of rabbit intestine, we fed these strains to weanling rabbits, which were killed 4 days later, so that the number of bacteria in various regions of the intestine could be determined. The results indicated that strain 83/39 requires both Ral and intimin to colonize the intestine successfully and that a delta eae delta ralE double mutant was incapable of colonizing the intestine. Taken together, these findings indicate that Ral and intimin act independently as adhesion factors of REPEC strain 83/39 and that this strain carries no other significant colonization factor. When both Ral and intimin are present, they appear to act cooperatively, with Ral-mediated adhesion preceding that mediated by intimin.     

Typing of intimin genes in human and animal enterohemorrhagic and enteropathogenic Escherichia coli: characterization of a new intimin variant

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) produce the characteristic "attaching and effacing" (A/E) lesion of the brush border. Intimin, an outer membrane protein encoded by eae, is responsible for the tight association of both pathogens with the host cell. Several eae have been cloned from different EPEC and EHEC strains isolated from humans and animals. These sequences are conserved in the N-terminal region but highly variable in the last C-terminal 280 amino acids (aa), where the cell binding activity is localized. Based on these considerations, we developed a panel of specific primers to investigate the eae heterogeneity of the variable 3' region by using PCR amplification. We then investigated the distribution of the known intimin types in a large collection of EPEC and EHEC strains isolated from humans and different animal species. The existence of a yet-unknown family of intimin was suspected because several EHEC strains, isolated from human and cattle, did not react with any of the specific primer pairs, although these strains were eae positive when primers amplifying the conserved 5' end were used. We then cloned and sequenced the eae present in one of these strains (EHEC of serotype O103:H2) and subsequently designed a PCR primer that recognizes in a specific manner the variable 3' region of this new intimin type. This intimin, referred to as "epsilon," was present in human and bovine EHEC strains of serogroups O8, O11, O45, O103, O121, and O165. Intimin epsilon is the largest intimin cloned to date (948 aa) and shares the greatest overall sequence identity with intimin beta, although analysis of the last C-terminal 280 aa suggests a greater similarity with intimins alpha and gamma.     

Identification of eae sequences in enteropathogenic Escherichia coli strains from rabbits.

DNA sequences coding for attachment and for verotoxin production were investigated in a collection of enteropathogenic Escherichia coli strains from rabbits. All of the strains produced diarrhea after experimental infection, attached to the brush borders of the intestinal lining, and possessed homology to the eae probe, whereas strains isolated from healthy rabbits did not. Sequences homologous to the AF/R1 fimbriae of strain RDEC-1 were not found. One strain reacted with the probe for the Shiga-like toxin type I gene.     

Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa

The adhesion of classic enteropathogenic Escherichia coli (EPEC) strains of human origin to isolated human small intestinal enterocytes and cultured small intestinal mucosa was investigated. An adhesion assay with isolated human enterocytes prepared from duodenal biopsy samples was developed and tested with EPEC strains known to cause diarrhea in healthy adult volunteers. In the assay a mean of 53 and 55% of enterocytes had brush border-adherent E. coli E2348 (O127;H6) and E851 (O142:H6), respectively, whereas the value for a nonpathogenic control strain and a plasmid-cured derivative of strain E2348 was 0%. A collection of 17 EPEC strains was also tested for the ability to colonize cultured human duodenal mucosa. Extensive colonization occurred with 13 strains, including serogroups O55, O86, O111, O114, O119, O127, O128, and O142; and in each case electron microscopic examination of colonized mucosa revealed the characteristic histopathological lesion reported by others in natural and experimental EPEC infections. EPEC strains were seen to adhere intimately to the enterocyte surface, causing localized destruction of microvilli. The plasmid-cured derivative of strain E2348, which colonized cultured mucosa much less efficiently than the parent strain, nevertheless produced an identical lesion, indicating that plasmid-encoded factors are not essential for adhesion and the brush border-damaging property of EPEC.     

Pathogenicity of attaching effacing enteropathogenic Escherichia coli isolated from diarrheic suckling and weanling rabbits for newborn rabbits

The pathogenicity of six strains of Escherichia coli originating from different commercial rabbitries was tested in neonatal rabbits. Two strains isolated from healthy weaned rabbits (O7:H6 and O9:H?) did not induce any clinical sign or lesion. Two strains (O109:H2) isolated from diarrheic suckling rabbits caused yellow diarrhea 36 to 60 h after inoculation and high mortality between 60 and 72 h after infection. At 12 h after infection, light and electron microscopy showed attachment to epithelial cells and effacement of microvilli from proximal small intestine to colon. Bacteria were often present in the apical cytoplasm of epithelial cells. The two strains isolated from diarrheic weanling rabbits (O109:H2 and O15:H-) did not induce any clinical sign. Attachment to epithelial cells and effacement of microvilli was observed 48 h after inoculation in distal small intestine, cecum, and colon. These data are further evidence for the existence of two groups of attaching effacing enteropathogenic E. coli in rabbits, showing different preferences for age group and intestinal compartment.     

Serotypes, intimin subtypes, and antimicrobial resistance patterns of atypical enteropathogenic Escherichia coli isolated in England from 1993 to 1996

The aim of this study was to characterise the atypical enteropathogenic Escherichia coli (EPEC) strains isolated during a study of intestinal infectious disease in the UK by serotyping, intimin subtyping, and antimicrobial resistance typing. Serotypes, intimin subtypes, and resistance patterns of strains from cases were then compared with those from the control group. A wide range of serotypes, intimin subtypes, and antimicrobial resistance patterns was identified in isolates from both cases and controls, with O70:H11 and O111:H− being the most frequently detected serotypes. The most common intimin types were γ and γ₂. Thirty-six percent of the EPEC isolates were resistant to at least one antimicrobial agent. No significant differences in the characteristics of EPEC strains isolated from patients with symptoms of gastrointestinal disease versus those isolated from healthy controls were detected, although strains harbouring the β-intimin subtype were more commonly isolated from children under 5 years of age (p=0.002). The compilation of data on atypical EPEC strains presented here indicates the need for further study of their virulence and epidemiology in order to assess their significance as human pathogens.     

Lactobacillus casei DN-114 001 inhibits the increase in paracellular permeability of enteropathogenic Escherichia coli-infected T84 cells

Probiotics are living microorganisms which, when ingested in adequate amounts, exert health benefits toward the host. For instance, probiotics might act through reinforcement of the intestinal epithelial barrier function. The goal of the present study was to determine whether Lactobacillus casei DN-114 001 could abrogate the increase in paracellular permeability induced by enteropathogenic Escherichia coli. We used the human colon T84 cell line infected with a wild-type enteropathogenic E. coli (strain E2348/69). Paracellular permeability was followed by monitoring transepithelial electrical resistance variations and by observing zonula occludens-1 distribution. Two infection procedures were used: co-incubation (the pathogenic and probiotic strains were simultaneously incubated with T84 cells) and post-infection (the probiotic was added in the presence of pathogenic bacteria 3 h after the beginning of the infection). We also investigated the effect of L. casei on enteropathogenic E. coli adhesion. L. casei DN-114 001 inhibited, in a dose-dependent-manner, the decrease in enteropathogenic E. coli-induced transepithelial electrical resistance and zonula occludens-1 redistribution using two different infection procedures. However, L. casei did not inhibit pathogenic strain adhesion. L. casei DN-114 001 inhibited the increase in EPEC-induced paracellular permeability. This property could partially explain the previously observed health benefits of this probiotic for human natural defenses, such as those associated with prevention of diarrhea.     

Peyer's patch adherence of enteropathogenic Escherichia coli strains in rabbits.

RDEC-1 (serotype O15) is an attaching and effacing strain of rabbit enteropathogenic Escherichia coli (REPEC) that causes diarrhea in postweanling rabbits. It expresses AF/R1 pili that mediate Peyer's patch M-cell adherence. We investigated Peyer's patch adherence, the presence of virulence genes, ileal brush border aggregation, and pilus expression in 9 strains representing several serotypes of REPEC as well as in two commensal strains. Postweanling rabbits were inoculated with 10(6) organisms and sacrificed at 24 h, and tissues were prepared for examination by light microscopy. Strains B10 and RDEC-1 were also studied at 12 and 72 h postinoculation. All REPEC strains were eaeA positive, expressed pili, and adhered to ileal brush borders. Both commensal strains expressed pili, and one strain adhered to brush borders. All REPEC strains demonstrated some degree of Peyer's patch lymphoid follicle adherence, ranging from diffuse coverage to small patches covering two to three dome epithelial cells. Strains C102 and C110 had genes homologous with the structural subunit gene of the AF/R1 pilus (afrA) of RDEC-1, which correlated with greater degrees of lymphoid follicle adherence and lesser degrees of ileal villus adherence. The observation that all REPEC strains adhere to Peyer's patch epithelium suggests the possibility that human strains of enteropathogenic E. coli (EPEC) might do likewise. EPEC strains might thus serve as mucosal vaccine vectors in humans. Better understanding of the molecular mechanism of REPEC adherence should provide a model for the targeting of the Peyer's patch in humans.     

Saccharomyces boulardii preserves the barrier function and modulates the signal transduction pathway induced in enteropathogenic Escherichia coli-infected T84 cells

Use of the nonpathogenic yeast Saccharomyces boulardii in the treatment of infectious diarrhea has attracted growing interest. The present study designed to investigate the effect of this yeast on enteropathogenic Escherichia coli (EPEC)-associated disease demonstrates that S. boulardii abrogated the alterations induced by an EPEC strain on transepithelial resistance, [(3)H]inulin flux, and ZO-1 distribution in T84 cells. Moreover, EPEC-mediated apoptosis of epithelial cells was delayed in the presence of S. boulardii. The yeast did not modify the number of adherent bacteria but lowered by 50% the number of intracellular bacteria. Infection by EPEC induced tyrosine phosphorylation of several proteins in T84 cells, including p46 and p52 SHC isoforms, that was attenuated in the presence of S. boulardii. Similarly, EPEC-induced activation of the ERK1/2 mitogen-activated protein (MAP) kinase pathway was diminished in the presence of the yeast. Interestingly, inhibition of the ERK1/2 pathway with the specific inhibitor PD 98059 decreased EPEC internalization, suggesting that modulation of the ERK1/2 MAP pathway might account for the lowering of the number of intracellular bacteria observed in the presence of S. boulardii. Altogether, this study demonstrated that S. boulardii exerts a protective effect on epithelial cells after EPEC adhesion by modulating the signaling pathway induced by bacterial infection.     

Coiled-coil domain of enteropathogenic Escherichia coli type III secreted protein EspD is involved in EspA filament-mediated cell attachment and hemolysis

Many animal and plant pathogens use type III secretion systems to secrete key virulence factors, some directly into the host cell cytosol. However, the basis for such protein translocation has yet to be fully elucidated for any type III secretion system. We have previously shown that in enteropathogenic and enterohemorrhagic Escherichia coli the type III secreted protein EspA is assembled into a filamentous organelle that attaches the bacterium to the plasma membrane of the host cell. Formation of EspA filaments is dependent on expression of another type III secreted protein, EspD. The carboxy terminus of EspD, a protein involved in formation of the translocation pore in the host cell membrane, is predicted to adopt a coiled-coil conformation with 99% probability. Here, we demonstrate EspD-EspD protein interaction using the yeast two-hybrid system and column overlays. Nonconservative triple amino acid substitutions of specific EspD carboxy-terminal residues generated an enteropathogenic E. coli mutant that was attenuated in its ability to induce attaching and effacing lesions on HEp-2 cells. Although the mutation had no effect on EspA filament biosynthesis, it also resulted in reduced binding to and reduced hemolysis of red blood cells. These results segregate, for the first time, functional domains of EspD that control EspA filament length from EspD-mediated cell attachment and pore formation.     

Intimate adherence by enteropathogenic Escherichia coli modulates TLR5 localization and proinflammatory host response in intestinal epithelial cells

Enteropathogenic Escherichia coli (EPEC) causes diarrhoeal disease by altering enterocyte physiology and producing mucosal inflammation. Many details concerning the host response against EPEC remain unknown. We evaluated the role of EPEC virulence factors on the inflammatory response through an analysis of bacterial recognition, cell signalling, and cytokine production using an in vitro epithelial cell infection model. Interestingly, we found that EPEC infection recruits Toll-like receptor 5 (TLR5) to the cell surface. We confirmed that type 3 secretion system (T3SS) and flagellin (FliC) are necessary for efficient extracellular regulated kinases 1 and 2 (ERK1/2) activation and found that intimin could down-regulate this pathway. Besides flagellin, intimin was required to keep nuclear factor kappa B (NF-κB) activated during infection. EPEC infection activated tumour necrosis factor alpha (TNF-α) production and induced interleukin (IL)-1β and IL-8 release. Virulence factors such as intimin, T3SS, EspA and fliC were required for IL-1β secretion, whereas intimin and T3SS participated in IL-8 release. Flagellin was essential for late secretion of TNF-α and IL-8 and intimin stimulated cytokine secretion. Initial adherence limited TNF-α release, whereas late attachment sustained TNF-α secretion. We conclude that intimin modulates TLR5 activation and intimate adherence alters the proinflammatory response.