Enteropathogenic Escherichia Coli Infection Research Articles

PKA-mediated phosphorylation of EPEC-Tir at serine residues 434 and 463

Type-III or type-IV secretion systems of many Gram-negative bacterial pathogens inject effector proteins into host cells that modulate cellular functions in their favour. A preferred target of these effectors is the actin-cytoskeleton as shown by studies using the gastric pathogens Helicobacter pylori (H. pylori) and enteropathogenic Escherichia coli (EPEC). We recently developed a co-infection approach to study effector protein function and molecular mechanisms by which they highjack cellular signalling cascades. This is exemplified by our observation that EPEC profoundly blocks H. pylori-induced epithelial cell scattering and elongation, a disease-related event requiring the activity of small Rho GTPase Rac1. While this suppressive effect is dependent on the effector protein Tir and the outer-membrane protein Intimin, it unexpectedly revealed evidence for Tir-signalling independent of phosphorylation of Tir at tyrosine residues 454 and 474. Instead, our studies revealed a previously unidentified function for protein kinase A (PKA)-mediated phosphorylation of Tir at serine residues 434 and 463. We demonstrated that EPEC infection activates PKA for Tir phosphorylation. Activated PKA then phosphorylates Rac1 at its serine residue 71 associated with reduced GTP-load and inhibited cell elongation. Phosphorylation of Rho GTPases such as Rac1 might be an interesting novel strategy in microbial pathogenesis.

Associations between Mucosal Innate and Adaptive Immune Responses and Resolution of Diarrheal Pathogen Infections

The identification of immune response mechanisms that contribute to the control of diarrheal disease in developing countries remains an important priority. We addressed the role of fecal chemokines and cytokines in the resolution of diarrheal Escherichia coli and Giardia lamblia infections. Stools collected from 127 Mexican children 5 to 15 months of age enrolled in a randomized, double-blind, placebo-controlled, vitamin A supplementation trial were screened for enteropathogenic Escherichia coli (EPEC), enterotoxigenic E. coli (ETEC), and Giardia lamblia. Fecal concentrations of tumor necrosis factor alpha (TNF-alpha), monocyte chemoattractant protein-1 (MCP-1), interleukin-4 (IL-4), IL-5, IL-6, IL-8, IL-10, and interferon-gamma (IFN-gamma) were determined. Hazard models incorporating cytokine variables were fit to durations of asymptomatic and symptomatic pathogen infections, controlling for treatment group. Increased levels of TNF-alpha and IL-6 were associated with decreased durations of EPEC infection and increased ETEC durations. Increased IL-4 and IFN-gamma levels were associated with decreased and increased durations, respectively, of both EPEC and ETEC infections. Increased IL-10 levels were associated with increased and decreased durations of asymptomatic and symptomatic EPEC infections, respectively, and increased durations of both asymptomatic and symptomatic ETEC infections. Increased levels of MCP-1, IFN-gamma, IL-4, and IL-5 were associated with increased G. lamblia infection duration, while increased IL-8 levels were associated with decreased durations. Differences in proinflammatory and Treg cytokine levels are associated with differences in the resolution of inflammatory and noninflammatory pathogen infections.

The T3SS Effector EspT Defines a New Category of Invasive Enteropathogenic E. coli (EPEC) Which Form Intracellular Actin Pedestals

Enteropathogenic Escherichia coli (EPEC) strains are defined as extracellular pathogens which nucleate actin rich pedestal-like membrane extensions on intestinal enterocytes to which they intimately adhere. EPEC infection is mediated by type III secretion system effectors, which modulate host cell signaling. Recently we have shown that the WxxxE effector EspT activates Rac1 and Cdc42 leading to formation of membrane ruffles and lamellipodia. Here we report that EspT-induced membrane ruffles facilitate EPEC invasion into non-phagocytic cells in a process involving Rac1 and Wave2. Internalized EPEC resides within a vacuole and Tir is localized to the vacuolar membrane, resulting in actin polymerization and formation of intracellular pedestals. To the best of our knowledge this is the first time a pathogen has been shown to induce formation of actin comets across a vacuole membrane. Moreover, our data breaks the dogma of EPEC as an extracellular pathogen and defines a new category of invasive EPEC.

Feedback effects of host-derived adenosine on enteropathogenicEscherichia coli

Enteropathogenic E. coli (EPEC) is a common cause of diarrhea in children in developing countries. After adhering to intestinal cells, EPEC secretes effector proteins into host cells, causing cell damage and eventually death. We previously showed that EPEC infection triggers the release of ATP from host cells and that ATP is broken down to ADP, AMP, and adenosine. Adenosine produced from the breakdown of extracellular ATP triggers fluid secretion in intestinal monolayers and may be an important mediator of EPEC-induced diarrhea. Here we examined whether adenosine has any effects on EPEC bacteria. Adenosine stimulated EPEC growth in several types of media in vitro. Adenosine also altered the pattern of EPEC adherence to cultured cells from a localized adherence pattern to a more diffuse pattern. Adenosine changed the expression of virulence factors in EPEC, inhibiting the expression of the bundle-forming pilus (BFP) and enhancing expression of the EPEC secreted proteins (Esps). In vivo, experimental manipulations of adenosine levels had strong effects on the outcome of EPEC infection in rabbit intestinal loops. In addition to its previously reported effects on host tissues, adenosine has strong effects on EPEC bacteria, stimulating EPEC growth, altering its adherence pattern, and changing the expression of several important virulence genes. Adenosine, like noradrenaline, is a small, host-derived molecule that is utilized as a signal by EPEC.

Enteropathogenic Escherichia coli changes distribution of occludin and ZO-1 in tight junction membrane microdomains in vivo

Diarrhea is a disease caused by enteropathogenic Escherichia coli (EPEC) infection, which caused the deaths of several hundred thousand children each year. However, the molecular mechanisms underlying EPEC infection in vivo are not fully understood. In the present study, we used the C57BL/6J mouse as an in vivo model of EPEC infection and investigated the effect of EPEC on tight junction (TJ) structure and barrier function. TJ ultrastructure was studied by transmission electron microscopy and a small molecule tracer biotin was used to examine the paracellular permeability of the colon. The distribution of TJ proteins occludin and ZO-1 in the epithelium was investigated by immunofluorescence microscopy. Our results demonstrated that TJ structure was disrupted following EPEC infection. And the morphological changes of TJ were accompanied by increased paracellular permeability which led to impairment of TJ barrier function. Immunofluorescency analysis revealed that occludin and ZO-1 were translocated from villous membrane to the cytoplasm in intestinal epithelial cells during EPEC invasion. Moreover, wild-type EPEC and the mutant EPEC strain, Δ espF, had similar effects on barrier function and TJ protein localization at 5 days postinfection. Our findings demonstrate that EPEC infection in vivo led to disruption of tight junction barrier function. These results may provide insights into the molecular mechanism of the pathogenesis of EPEC infection.

Enteropathogenic Escherichia coli Infection Inhibits Intestinal Serotonin Transporter Function and Expression

Serotonin transporter (SERT) plays a critical role in regulating serotonin (5-hydroxytryptamine [5-HT]) availability in the gut. Elevated 5-HT levels are associated with diarrheal conditions such as irritable bowel syndrome and enteric infections. Whether alteration in SERT activity contributes to the pathophysiology of diarrhea induced by the food-borne pathogen enteropathogenic Escherichia coli (EPEC) is not known. The present studies examined the effects of EPEC infection on SERT activity and expression in intestinal epithelial cells and elucidated the underlying mechanisms. Caco-2 cells as a model of human intestinal epithelia and EPEC-infected C57BL/6J mouse model of infection were utilized. SERT activity was measured as Na(+) and Cl(-) dependent (3)[H] 5-HT uptake. SERT expression was measured by real-time quantitative reverse-transcription polymerase chain reaction, Western blotting, and immunofluorescence studies. Infection of Caco-2 cells with EPEC for 30-120 minutes decreased apical SERT activity (P < .001) in a type 3 secretion system dependent manner and via involvement of protein tyrosine phosphatases. EPEC infection decreased V(max) of the transporter; whereas cell surface biotinylation studies revealed no alteration in the cellular or plasma membrane content of SERT in Caco-2 cells. EPEC infection of mice (24 hours) reduced SERT immunostaining with a corresponding decrease in SERT messenger RNA levels, 5-HT uptake, and mucosal 5-HT content in the small intestine. Our results demonstrate inhibition of SERT by EPEC and define the mechanisms underlying these effects. These data may aid in the development of a novel pharmacotherapy to modulate the serotonergic system in treatment of infectious diarrheal diseases.

The bacterial virulence factor NleA is required for the disruption of intestinal tight junctions by enteropathogenicEscherichia coli

Enteropathogenic Escherichia coli (EPEC) is a diarrhoeal pathogen that adheres to epithelial cells of the small intestine and uses a type III secretion system to inject effector proteins into host cells. EPEC infection leads to disruption of host intestinal tight junctions that are important for maintaining intestinal barrier function. This disruption is dependent on the bacterial type III secretion system, as well as the translocated effectors EspF and Map. Here we show that a third type III translocated bacterial effector protein, NleA, is also involved in tight junction disruption during EPEC infection. Using the drug Brefeldin A, we demonstrate that the effect of NleA on tight junction integrity is related to its inhibition of host cell protein trafficking through COPII-dependent pathways. These results suggest that NleA's striking effect on virulence is mediated, at least in part, via its role in disruption of intestinal barrier function.

Distribution of espM and espT among enteropathogenic and enterohaemorrhagic Escherichia coli.

Enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) translocate dozens of type III secretion system effectors, including the WxxxE effectors Map, EspM and EspT that activate Rho GTPases. While map, which is carried on the LEE pathogenicity island, is absolutely conserved among EPEC and EHEC strains, the prevalence of espM and espT is not known. Here we report the results of a large screen aimed at determining the prevalence of espM and espT among clinical EPEC and EHEC isolates. The results suggest that espM, detected in 51 % of the tested strains, is more commonly found in EPEC and EHEC serogroups that are linked to severe human infections. In contrast, espT was absent from all the EHEC isolates and was found in only 1.8 % of the tested EPEC strains. Further characterization of the virulence gene repertoire of the espT-positive strains led to the identification of a new ζ2 intimin variant. All the espT-positive strains but two contained the tccP gene. espT was first found in Citrobacter rodentium and later in silico in EPEC E110019, which is of particular interest as this strain was responsible for a particularly severe diarrhoeal outbreak in Finland in 1987 that affected 650 individuals in a school complex and an additional 137 associated household members. Comparing the protein sequences of EspT to that of E110019 showed a high level of conservation, with only three strains encoding EspT that differed in 6 amino acids. At present, it is not clear why espT is so rare, and what impact EspM and EspT have on EPEC and EHEC infection.

Enteropathogenic Escherichia coli inhibits intestinal vitamin B1 (thiamin) uptake: studies with human-derived intestinal epithelial Caco-2 cells.

Infection with the gram-negative enteropathogenic Escherichia coli (EPEC), a food-borne pathogen, represents a significant risk to human health. Whereas diarrhea is a major consequence of this infection, malnutrition also occurs especially in severe and prolonged cases, which may aggravate the health status of the infected hosts. Here we examined the effect of EPEC infection on the intestinal uptake of the water-soluble vitamin B1 (thiamin) using an established human intestinal epithelial Caco-2 cell model. The results showed that infecting Caco-2 cells with wild-type EPEC (but not with nonpathogenic E. coli, killed EPEC, or filtered supernatant) leads to a significant (P < 0.01) inhibition in thiamin uptake. Kinetic parameters of both the nanomolar (mediated by THTR-2) and the micromolar (mediated by THTR-1) saturable thiamin uptake processes were affected by EPEC infection. Cell surface expression of hTHTR-1 and -2 proteins, (determined by the biotinylation method) showed a significantly (P < 0.01) lower expression in EPEC-treated cells compared with controls. EPEC infection also affected the steady-state mRNA levels as well as promoter activity of the SLC19A2 and SLC19A3 genes. Infecting Caco-2 cells with EPEC mutants that harbor mutations in the escN gene (which encodes a putative ATPase for the EPEC type III secretion system, TTSS) or the espA, espB, or espD genes (which encode structural components of the TTSS) did not affect thiamin uptake. On the other hand, mutations in espF and espH genes (which encode effector proteins) exhibited partial inhibition in thiamin uptake. These results demonstrate for the first time that EPEC infection of human intestinal epithelial cells leads to inhibition in thiamin uptake via effects on physiological and molecular parameters of hTHTR-1 and -2. Furthermore, the inhibition appears to be dependent on a functional TTSS of EPEC.

Attaching and Effacing Escherichia coli Downregulate DNA Mismatch Repair Protein In Vitro and Are Associated with Colorectal Adenocarcinomas in Humans

BackgroundMucosa-associated Escherichia coli are frequently found in the colonic mucosa of patients with colorectal adenocarcinoma, but rarely in healthy controls. Chronic mucosal E. coli infection has therefore been linked to colonic tumourigenesis. E. coli strains carrying eae (encoding the bacterial adhesion protein intimin) attach intimately to the intestinal mucosa and are classed as attaching and effacing E. coli (AEEC). Enteropathogenic Escherichia coli (EPEC) are the most common form of AEEC identified in man. EPEC utilise a type III secretion system to translocate effector proteins into host cells and infection induces wide-ranging effects on the host cell proteome. We hypothesised that EPEC infection could influence molecular pathways involved in colorectal tumourigenesis.Methodology/Principal FindingsWhen co-cultured with human colorectal cell lines, EPEC dramatically downregulated the expression of key DNA mismatch repair proteins MSH2 and MLH1 in an attachment specific manner. Cytochrome c staining and TUNEL analysis confirmed that this effect was not a consequence of apoptosis/necrosis. Ex vivo human colonic mucosa was co-cultured with EPEC and probed by immunofluorescence to locate adherent bacteria. EPEC entered 10% of colonic crypts and adhered to crypt epithelial cells, often in the proliferative compartment. Adenocarcinoma and normal colonic mucosa from colorectal cancer patients (n = 20) was probed by immunofluorescence and PCR for AEEC. Mucosa-associated E. coli were found on 10/20 (50%) adenocarcinomas and 3/20 (15%) normal mucosa samples (P<0.05). AEEC were detected on 5/20 (25%) adenocarcinomas, but not normal mucosa samples (P<0.05).Significance/ConclusionsThe ability of EPEC to downregulate DNA mismatch repair proteins represents a novel gene-environment interaction that could increase the susceptibility of colonic epithelial cells to mutations and therefore promote colonic tumourigenesis. The potential role of AEEC in colorectal tumourigenesis warrants further investigation.

558 Outcomes After Retreatment with Cyclosporine a (CsA) in Hospitalized Ulcerative Colitis (UC) Patients: A 7-Year Review from a Single Institution

Background and Aims: Although expression of the di/tripeptide transporter PepT1 has been observed in colon under inflammatory conditions, the inducing factors and underlying mechanisms have not yet been investigated. Here, we addressed the role of pathogenic bacteria in the regulation of colonic PepT1 expression/function and the potential role of PepT1 in bacterial-epithelial interaction. Methods: Colonic HT29-Cl.19A cells were infected with enteropathogenic E. coli (EPEC). PepT1 promoter activity and PepT1 expression/activity were analyzed using the luciferase assay, RT-PCR, nuclear run-on assay, immunoblotting, immunofluorescence staining and uptake experiments. Cdx2-PepT1 promoter binding was assessed by gel-shift and chromatin immunoprecipitation assay. In Vitro experiments were validated by ex vivo and In Vivo infection of wild type and PepT1 over-expressing mice with Citrobacter rodentium. Interleukin (IL)-8 and keratinocyte-derived chemokine (KC) expression levels were quantified by real-time RT-PCR and ELISA. Results: EPEC transcriptionally induced PepT1 expression/activity in HT29-Cl.19A cells. Cdx2 over-expression in HT29-Cl.19A cells induced PepT1 expression and Cdx2 silencing markedly reduced EPECinduced PepT1 expression, indicating the importance of Cdx2 in PepT1 expression. Furthermore, PepT1 expression required intimate adherence of EPEC to host cells through lipid rafts (LRs). Importantly, PepT1 expressed upon EPEC infection is functionally localized in LRs, and PepT1 associated with LRs delayed EPEC-LR binding as monitored in real time by an electric cell-substrate impedance-sensing technique. Remarkably, PepT1 over-expression in HT29-Cl.19A cells reduced EPEC-triggered NF-κB and MAP kinase activation and IL-8 production. In agreement with In Vitro data, ex vivo and In Vivo experiments showed that C. rodentium increased PepT1 mRNA and protein expression levels in mouse colon. Furthermore, PepT1 over-expression in mouse colon reduced C. rodentium adherence and C. rodentium-induced KC production. Conclusions: We demonstrate that i) EPEC transcriptionally induces functional PepT1 expression in LRs of colonocytes by intimately attaching to host cell membranes through LRs, ii) the transcription factor Cdx2 is crucial for EPECinduced PepT1 expression, and iii) PepT1 associated with LRs is involved in bacterialepithelial interaction and intestinal inflammation. Our findings not only reveal a novel mechanism underlying the regulation of colonic epithelial PepT1 expression/function under pathological conditions, but also highlight the potential contribution of this transporter to host defense mechanisms in response to pathogenic attack.

557 Colectomy Rate in Hospitalized Ulcerative Colitis Patients Undergoing Therapy with Cyclosporine (CsA)

Background and Aims: Although expression of the di/tripeptide transporter PepT1 has been observed in colon under inflammatory conditions, the inducing factors and underlying mechanisms have not yet been investigated. Here, we addressed the role of pathogenic bacteria in the regulation of colonic PepT1 expression/function and the potential role of PepT1 in bacterial-epithelial interaction. Methods: Colonic HT29-Cl.19A cells were infected with enteropathogenic E. coli (EPEC). PepT1 promoter activity and PepT1 expression/activity were analyzed using the luciferase assay, RT-PCR, nuclear run-on assay, immunoblotting, immunofluorescence staining and uptake experiments. Cdx2-PepT1 promoter binding was assessed by gel-shift and chromatin immunoprecipitation assay. In Vitro experiments were validated by ex vivo and In Vivo infection of wild type and PepT1 over-expressing mice with Citrobacter rodentium. Interleukin (IL)-8 and keratinocyte-derived chemokine (KC) expression levels were quantified by real-time RT-PCR and ELISA. Results: EPEC transcriptionally induced PepT1 expression/activity in HT29-Cl.19A cells. Cdx2 over-expression in HT29-Cl.19A cells induced PepT1 expression and Cdx2 silencing markedly reduced EPECinduced PepT1 expression, indicating the importance of Cdx2 in PepT1 expression. Furthermore, PepT1 expression required intimate adherence of EPEC to host cells through lipid rafts (LRs). Importantly, PepT1 expressed upon EPEC infection is functionally localized in LRs, and PepT1 associated with LRs delayed EPEC-LR binding as monitored in real time by an electric cell-substrate impedance-sensing technique. Remarkably, PepT1 over-expression in HT29-Cl.19A cells reduced EPEC-triggered NF-κB and MAP kinase activation and IL-8 production. In agreement with In Vitro data, ex vivo and In Vivo experiments showed that C. rodentium increased PepT1 mRNA and protein expression levels in mouse colon. Furthermore, PepT1 over-expression in mouse colon reduced C. rodentium adherence and C. rodentium-induced KC production. Conclusions: We demonstrate that i) EPEC transcriptionally induces functional PepT1 expression in LRs of colonocytes by intimately attaching to host cell membranes through LRs, ii) the transcription factor Cdx2 is crucial for EPECinduced PepT1 expression, and iii) PepT1 associated with LRs is involved in bacterialepithelial interaction and intestinal inflammation. Our findings not only reveal a novel mechanism underlying the regulation of colonic epithelial PepT1 expression/function under pathological conditions, but also highlight the potential contribution of this transporter to host defense mechanisms in response to pathogenic attack.

Genetic Characterization of Antibiotic Resistance in EnteropathogenicEscherichia coliCarrying Extended-Spectrum β-Lactamases Recovered from Diarrhoeic Rabbits

A total of 52 Escherichia coli strains isolated from diarrhoeic rabbits were investigated for their enteropathogenic E. coli (EPEC) pathotype by PCR amplification of eae and bfp virulence genes. A total of 22 EPEC isolates were identified, serotyped and studied for antibiotic resistance and screened for the detection of extended-spectrum beta-lactamases (ESBLs). The EPEC isolates belonged to three serogroups (O26, O92 and O103). The most common serogroup (O103:K-:H2) was observed among 17 EPEC strains, the O92:K-serogroup in three isolates (the antibiotic sensitive ones) and the remaining O26:K-serogroup in two isolates (the ESBLs isolates). Resistances to ampicillin and tetracycline were the most frequent and detected followed by resistance to nalidixic acid, streptomycin, trimethoprim-sulphamethoxazole, cefoxitin, gentamicin and ciprofloxacin. All the isolates were sensitive for amikacin, ceftazidime, aztreonam, imipenem, chloramphenicol, tobramycin and amoxicillin + clavulanic acid. Two isolates recovered from two adult animals showed an intermediate susceptibility to cefotaxime, and a positive screening test for ESBL was demonstrated in both. The bla(TEM) gene was demonstrated in the majority of ampicillin-resistant isolates. The aac(3)-II or aac(3)-IV genes were detected in the four gentamicin-resistant isolates. In addition, the aadA gene was detected in 60% of streptomycin-resistant isolates. The tet(A) or tet(B) genes were identified in all tetracycline-resistant isolates. A total of nine EPEC isolates showed the phenotype SXT-resistant, and the sul1 and/or sul2 and/or sul3 genes were detected in all of them. Our findings showed that the molecular detection by the eae and bfp genes by PCR followed by serotyping is useful for monitoring trends in EPEC infections of rabbits allowing the identification of their possible reservoirs. The detection of genes involved in the resistance to antibiotics of different families in a relatively high proportion of faecal E. coli isolates of rabbits is of great interest and could be considered a serious public health problem.

Modelling of Infection by EnteropathogenicEscherichia coliStrains in Lineages 2 and 4 Ex Vivo and In Vivo by UsingCitrobacter rodentiumExpressing TccP

Enteropathogenic Escherichia coli (EPEC) strains colonize the human gut mucosa via attaching-and-effacing (A/E) lesion formation, while in vitro they employ diverse strategies to trigger actin polymerization. Strains belonging to the EPEC-1 lineage trigger strong actin polymerization via tyrosine phosphorylation of the type III secretion system (T3SS) effector Tir, recruitment of Nck, and activation of N-WASP. Strains belonging to EPEC-2 and EPEC-4 can trigger strong actin polymerization by dual mechanisms, since while employing the Tir-Nck pathway they can additionally activate N-WASP via the T3SS effectors TccP2 and TccP, respectively. It is currently not known if the ability to trigger actin polymerization by twin mechanisms increases in vivo virulence or fitness. Since mice are resistant to EPEC infection, in vivo studies are frequently done using the murine model pathogen Citrobacter rodentium, which shares with EPEC-1 strains the ability to induce A/E lesions and trigger strong actin polymerization via the Tir:Nck pathway. In order to model infections with EPEC-2 and EPEC-4, we constructed C. rodentium strains expressing TccP. Using a mouse intestinal in vitro organ culture model and oral gavage into C57BL/6 mice, we have shown that TccP can cooperate with Tir of C. rodentium. The recombinant strains induced typical A/E lesions ex vivo and in vivo. Expression of TccP did not alter C. rodentium colonization dynamics or pathology. In competition with the wild-type strain, expression of TccP in C. rodentium did not confer a competitive advantage.

Effect of Aqueous Extract of Aegle marmelos Fruit on Adherence and β-Lactam Resistance of Enteropathogenic Escherichia coli by Down Regulating Outer Membrane Protein C

Problem statement: Enteropathogenic Escherichia Coli (EPEC) continue to be a major health problem, leading to death due to diarrhea, predominantly in children below the age of five. Due to evolution of multi drug resistance in EPEC and side effects caused to host by antibiotics necessitated a search for alternative medicines from medicinal plants. One such medicinal plant used since ancient times to cure diarrhea is Aegle marmelos. This study was done to investigate the effect of aqueous extract of Aegle marmelos fruit (AEAM) on outer membrane protein C (OmpC) of EPEC, which plays a key role in adherence and antibiotic resistance. Approach: Fixation of minimum inhibitory concentration. In presence and absence of AEAM antibiotic susceptibility test was performed. Expression analysis of OmpC and OmpF was carried out by RT-PCR of EPEC in presence and absence of AEAM. Morphological changes of EPEC in presence and absence of AEAM were analyzed by TEM. In infant mouse ileal loop model, histological analysis, adherence of bacteria to ileal loops and Western blotting for caspase-3 and Hsp70 were done. Results: OmpC (~42kDa) a porin, played an important role in selective transport of nutrients and also acted as an adhesin, whereas OmpF (~38kDa) is also a porin which is non selective. Susceptibility of EPEC to β-lactam antibiotics in presence of AEAM can be attributed to down regulation of OmpC and upregulation of OmpF. The changes in Omp expression also triggered morphological changes in EPEC. Histology and western blot of Hsp70 and Caspase-3 in rat ileal loop confirmed the effect of AEAM on attenuating the virulence of EPEC by preventing its infection due to loss of adherence. Loss of adherence was due to morphological changes and down regulation of OmpC in EPEC. Conclusion: From this study, we concluded that the protection offered by AEAM against EPEC was due to down regulation of OmpC, leading to loss of adherence and up regulation of OmpF, which allowed the entry of β-lactam antibiotics into bacteria. Hence, AEAM, along with β-lactam antibiotics can be used in treatment of EPEC infections.

Theex vivoresponse of human intestinal mucosa to enteropathogenicEscherichia coliinfection

In vitro organ culture (IVOC) represents a gold standard model to study enteropathogenic E. coli (EPEC) infection of human intestinal mucosa. However, the optimal examination of the bacterial-host cell interaction requires a directional epithelial exposure, without serosal or cut surface stimulation. A polarized IVOC system (pIVOC) was developed in order to overcome such limitations: apical EPEC infection produced negligible bacterial leakage via biopsy edges, resulted in enhanced colonization compared with standard IVOC, and showed evidence of bacterial detachment, as in natural rabbit EPEC infections. Examination of mucosal innate immune responses in pIVOC showed both interleukin (IL)-8 mRNA and protein levels were significantly increased after apical EPEC infection. Increased IL-8 levels mainly depended on flagellin expression as fliC-negative EPEC did not elicit a significant IL-8 response despite increased mucosal colonization compared with wild-type EPEC. In addition, apical application of purified flagella significantly increased IL-8 protein levels over non-infected controls. Immunofluorescence staining of EPEC-infected small intestinal biopsies revealed apical and basolateral distribution of Toll-like receptor (TLR) 5 on epithelium, suggesting that EPEC can trigger mucosal IL-8 responses by apical flagellin/TLR5 interaction ex vivo and does not require access to the basolateral membrane as postulated in cell culture models.

Actin Pedestal Formation by Enteropathogenic Escherichia coli Is Regulated by IQGAP1, Calcium, and Calmodulin

During infection, enteropathogenic Escherichia coli (EPEC) injects effector proteins into the host cell to manipulate the actin cytoskeleton and promote formation of actin pedestals. IQGAP1 is a multidomain protein that participates in numerous cellular functions, including Rac1/Cdc42 and Ca(2+)/calmodulin signaling and actin polymerization. Here we report that IQGAP1, Ca(2+), and calmodulin modulate actin pedestal formation by EPEC. Infection with EPEC promotes both the interaction of IQGAP1 with calmodulin and the localization of IQGAP1 and calmodulin to actin pedestals while reducing the interaction of IQGAP1 with Rac1 and Cdc42. IQGAP1-null fibroblasts display a reduced polymerization of actin in response to EPEC. In addition, antagonism of calmodulin or chelation of intracellular Ca(2+) reduces EPEC-dependent actin polymerization. Furthermore, IQGAP1 specifically interacts with Tir in vitro and in cells. Together these data identify IQGAP1, Ca(2+), and calmodulin as a novel signaling complex regulating actin pedestal formation by EPEC.

Invasion of enteropathogenic Escherichia coli into host cells through epithelial tight junctions

Enteropathogenic Escherichia coli (EPEC) has been shown to disrupt the barrier function of host intestinal epithelial tissues through entering tight junctions. However, the mechanism by which this occurs remains poorly understood. In this study, we determined that EPEC invades host cells through tight junctions as it initiates infection. Immunofluorescence microscopy revealed redistribution of the tight-junction proteins occludin and ZO-1 from an intercellular to a cytoplasmic location after EPEC invasion. Flotillin-1 was recruited to sites of EPEC entry. EPEC entered host cells through tight-junction membrane microdomains. Tight-junction ultrastructure was disrupted following EPEC infection, accompanied by loss of barrier function. EPEC infection caused a time-dependent decrease in trans-epithelial electrical resistance. Subcellular fractionation using discontinuous sucrose density gradients demonstrated a decline in raft-associated occludin following exposure to EPEC. These results indicate the important role of host membrane tight-junction microdomains in EPEC invasion.

Immunization of mice withLactobacillus caseiexpressing intimin fragments produces antibodies able to inhibit the adhesion of enteropathogenicEscherichia colito cultivated epithelial cells

Enteropathogenic Escherichia coli (EPEC) are frequently isolated as a cause of infantile diarrhea in developing countries. Its pathogenicity is distinguished by histopathological alterations at the site of infection, known as attaching and effacing (A/E) lesions, in which bacterial virulence factors and host proteins participate. Intimin, a bacterial adhesin expressed by all EPEC described to date, is responsible for the intimate adherence of the bacteria to host cells and is essential for the formation of A/E lesions. Mucosal vaccination may represent an efficacious intervention to prevent EPEC infection and lower morbidity and mortality rates. Strategies for mucosal vaccinations that use lactic acid bacteria for the delivery of heterologous antigens rely on their safety profile and ability to stimulate the immune system. In the present work, we have constructed Lactobacillus casei strains expressing different fragments of intimin beta, a subtype that is frequently expressed by EPEC strains. Mucosal immunization of mice with L. casei expressing intimin fragments induced specific systemic and mucosal antibodies. These antibodies were able to recognize native intimin on the surface of EPEC and to inhibit in vitro EPEC binding to epithelial cells.

EspC translocation into epithelial cells by enteropathogenicEscherichia colirequires a concerted participation of type V and III secretion systems

EspC is a non-locus of enterocyte effacement (LEE)-encoded autotransporter protein secreted by enteropathogenic Escherichia coli (EPEC) that causes a cytopathic effect on epithelial cells, including cytoskeletal damage. EspC cytotoxicity depends on its internalization and functional serine protease motif. Here we show that during EPEC infection, EspC is secreted from the bacteria by the type V secretion system (T5SS) and then it is efficiently translocated into the epithelial cells through the type III secretion system (T3SS) translocon. By dissecting this mechanism, we found that EspC internalization during EPEC-host cell interaction occurs after 1 h, unlike purified EspC (8 h). LEE pathogenicity island is involved in specific EspC translocation as three espC-transformed attaching and effacing (AE) pathogens translocated EspC into the cells. A role for effectors and other factors involved in the intimate adherence encoded in LEE were discarded by using an exogenous EspC internalization model. In this model, an isogenic EPEC DeltaespC strain allows the efficient internalization of purified EspC. Moreover, isogenic mutants in T3SS were unable to translocate endogenous and exogenous EspC into epithelial cells, as EspC-EspA interaction is required. These data show for the first time the efficient delivery of an autotransporter protein inside the epithelial cells by EPEC, through cooperation between T5SS and T3SS.