Attaching and effacing escherichia coli infections : pathogenesis, host response and epidemiology.

Exploitable results

The distribution of different intimin types between AEEC isolated from different hosts suggests that the type of intimin may influence the host specificity and the tissue tropism. The goal of this work package was to test the hypothesis that intimin subtype is responsible for the AEEC host specificity. Major finding during the project: E. coli strains of serotype O103:H2 isolated from rabbits with diarrhea (EPEC) or from humans with HUS (EHEC) are clonal. These strains produce an identical Tir but the rabbit AEEC strain produces a b1 intimin and elicits a weak accumulation of F-actin (FAS-response) in human HeLa cells whereas the human AEEC strain produces an e intimin and is strongly FAS-positive. In addition, human AEEC is not pathogenic for rabbits whereas rabbit AEEC induced diarrhea and mortality. Trans complementation of eae-b1 null mutant with a plasmid expressing intimin e and vice versa, were carried out. In infected human HeLa cells, the accumulation of F-actin was fully restored whatever the construction used for complementation (eae-b1 or eae-e suggesting that the intensity of FAS-response was linked to the copy number of intimin gene. Since such results were obtained from a plasmid source, we carry out the allelic exchange of b1-intimin in the rabbit EPEC with the e-intimin derived from the human EHEC, and vice-versa, to allow expression of a changed intimin type from the chromosome with the native promoter and with its own transcriptional regulation elements. A rabbit EPEC strain producing a hybrid b1/e intimin was successfully constructed. This hybrid has the N-terminus of the b1 intimin and the variable C-terminal binding domain of intimin e. In infected human HeLa cells, the accumulation of F-actin (FAS) was compared between the wild type human EHEC strain producing e intimin, the wild type rabbit EPEC strain producing b1 intimin and the rabbit EPEC strain producing the hybrid b1/e intimin. The human EHEC strain produced the strongest FAS response whereas the rabbit strain producing the b1 intimin or the hybrid b1/e intimin produced a less intense FAS response. The response was similar between the rabbit strains producing either the b1 intimin or the hybrid b1/e intimin. These results suggest that on HeLa cells the intensity of the FAS response was not due to the binding domain of the intimins but more likely due to the regulation of the LEE since the intensity of FAS-response was linked to the copy number of intimin gene. However, we cannot rule out the hypothesis that additional factor(s) unrelated to intimin exist in the O103:H2 genome that influence the FAS and consequently intestinal tissue tropism. Using EHEC strain of serotype O103:H2 expressing intimin e to infect human biopsies, partner CR2 showed tropism towards the follicle-associated epithelium of human intestinal Peyer's patch mucosa. This result supports the contribution of intimin to tissue specificity. We have characterized in this model the rabbit EPEC strain expressing a b1-intimin and the rabbit EPEC strain expressing the hybrid b1/e intimin. The study has shown that intimin e strains O103:H2 have a similar human intestinal tropism to intimin g expressing strains, and also show adherence to FAE of isolated lymphoid follicles of the duodenum.

Objectives: - To examine the putative role of EPEC for the evolution of EHEC by acquisition of phages encoding Stx; - To establish an in vivo phage transduction model for wild-type E. coli (EPEC) strains. Major finding during the project: Establishing an in vivo transduction model. In porcine ligated intestinal loops we repeatedly were able to lysogenise a porcine EPEC strain 1390 of O45 with stx2::cat phage induced from an O157 donor strain. On the other hand the human prototype strain E2348/69 was not lysogenised with stx2::cat phage under the same conditions. Chromosomal integration site of F3538(stx2::cat). F3538(stx2::cat) occupies a new so far un-described site for Stx-phages in the chromosome of E. coli O157:H7 strain 3538 and tranductants. Stability of F3538(stx2::cat) in the lysogens. F3538(stx2::cat) could not be mobilised from the transductant neither in vitro nor in vivo. In summary, it was demonstrated that EHEC can be developed from EPEC by acquisition of stx phage (stx2) in vivo using a porcine EPEC strain in a porcine ligated intestinal loop system.

Escherichia coli produces polysaccharide capsules that, based on their mechanisms of synthesis and assembly, have been classified into four groups. The group 4 capsule polysaccharide is frequently identical to that of the cognate LPS O side-chain and has, therefore, also been termed the O-antigen capsule. The genes involved in the assembly of the group 1, 2 and 3 capsules have been described, but those required for the assembly of group 4 capsule remained obscure. We found that enteropathogenic E. coli (EPEC) produces group 4 capsule and we identified an operon containing seven genes: ymcD, ymcC, ymcB, ymcA, yccZ, etp and etk, which are required for formation of the capsule. The encoded proteins appear to constitute a polysaccharide secretion system. The group 4 capsule operon is absent from the genomes of enteroaggregative E. coli and uropathogenic E. coli. E. coli K12 contains the group 4 capsule operon, but does not express it, because of the presence of IS1 at its promoter region. In contrast, EPEC, enterohemorrhagic E. coli (EHEC), and Shigella possess an intact group 4 capsule operon. We recently found that all the tested EPEC serotypes, including O111, O119, O142, and O55, and the closely related entrohemorrhagic E. coli serotype O157, form group 4 capsule and/or express Etk. This correlation suggests that group 4 capsule play a role in the virulence of these closely related pathogens.

EPEC, a major cause of severe disease with diarrhoea in infants, is also involved in weaned rabbit colibacillosis. EPEC O103 is frequent in rabbit-fattening units of Western Europe. It causes high mortality and growth retardation, leading to substantial economic losses. WP10 had two objectives: - To analyse immune responses associated with protection in the rabbit AEEC model. Very few data are available on the responses in this type of infection, whichever the species involved; - To find a vaccination train that may be used against AEEC infections in rabbit breeding units. This WP benefited of the work proposed in WP08 (new attenuated strains that could be used as live vaccine), WP09 (physiopathology) and WP19 (stability of LEE). Work in the rabbit model may be representative to develop vaccines against AEEC in other animals. Major finding during the project: Vaccine candidate strain. We constructed (by allelic exchange of an EPEC O103 strain mutated in espB and tir) two essential virulence genes. Upon live oral administration to weaned rabbits, the E22DTirEspB mutant strain efficiently colonized the intestinal tract without any adverse consequences. The rabbits were challenged with the highly pathogenic parental strain E22. The mutant provided complete protection to rabbits and total resistance to intestinal colonization by E22. In addition, E22DTirEspB strain induced a specific humoral response against the bacterial adhesion AF/R2. The Abs prevents bacterial attachment to epithelial cells in vitro. These results open the way for the development of an efficient vaccine strategy against rabbit EPEC infections. Immune response of the rabbits. Using ELISA and Western-blot, we have been able to show the production of specific antibodies against Eae/Intimin, Afr2G, and EspA. However, it was very difficult to estimate by ELISPOT the frequency of specific antibody producing cell due to heavy non-specific responses.

The first step in the pathogenesis of most intestinal bacterial pathogens is the adherence to the cytoplasmic membrane of enterocytes via specific bacterial structures and proteins (= primary colonisation factors). This step is also often the basis of the host specificity of the bacterial pathogens. The identification of such primary colonisation factors could greatly help in the comparison of bovine and human strains identical by other properties. The general objectives of WP14 were to identify such adhesions present in bovine EHEC strains, to confirm their role in intestinal colonisation and in enteropathogenicity in calves and to look for the expression of these new adhesin(s) in human EHEC. Major finding during the project: Ex vivo and in vitro adherence assays. - Intestinal explants experiments with bovine EPEC/EHEC strains. Intestinal explants experiments were performed using mucosal tissues from the colon obtained from 18-month-old cattle at the slaughterhouse. Explants were inoculated with two O26 EHEC strains and with an in vivo pathogenic O118 EHEC strain as positive control. This part of the work-package suffered a lot of problems regarding the short survival time of the intestinal explants (less than 5 hours), despite testing several variations in the culture conditions and parameters of the explants. No further work was therefore performed with explants and efforts were re-directed towards the development of an adherence assay on cells in culture. - Adherence assay on MDBK (Madin Darby Bovine Kidney) cells with bovine EPEC/EHEC strains. The tests were performed with 64 strains of bovine origin belonging to the O5, O26, O103, O111, O118, O145 and O157 serogroups. With the exception of the O145 serogroup, the other strains presented a phenotype with various intensity of adherence: localised-like (LAL), diffuse (DA) and aggregative (AA) adherence patterns were observed irrespectively of the serotype or the pathotype of the strain. A quantitative assay on the MDBK cell line was also derived, by comparing adherent bovine EPEC/EHEC strains and negative control strains. In spite of some variations during the assay and of a significant level of non-specific adherence by the negative control strains, a one-log difference in the numbers of adherent bacteria was consistently observed between EPEC/EHEC and negative control strains. This assay is therefore suitable to test mutant strains. - Adherence assay on primary intestinal cell cultures with bovine EPEC/EHEC strains. The adherence assay with the 64 strains of bovine origin belonging to the O5, O26, O103, O111, O118, O145 and O157 serogroups was also adapted to primary culture of bovine jejunocytes and colonocytes. The adherence patterns of most strains were similar to the ones on MDBK cells: localised-like (LAL), diffuse (DA) and aggregative (AA) adherence patterns. Production and testing of mutants: i) Presence of adhesin-encoding genes in bovine EPEC/EHEC strains. Following sequence alignments and gene probe derivation, the collection of bovine EPEC/EHEC strains was screened for the presence of putative adhesin-encoding genes (afa8, f17, afr2, clp, bfp, lpfA, lifA/efa1, iha). The positive hybridisation results can be summarised as follows: bfp-like gene was detected in human EPEC strains, but not in the other strains; lpfA-like gene was detected in all O157 and O145 EHEC, but not in the other serogroups; whereas lifA/efa1-like gene was detected in most non-O157 non-O145 EHEC; iha-like gene was present in several EPEC/EHEC too, irrespectively of the serotypes; and clpE-like gene (but not clpG) was present in O26 EPEC strains only. - Production of mutants in putative adhesin-encoding genes of bovine EPEC/EHEC strains. In order to evaluate the relative implication of the putative adhesins encoded by the lifA/efa1, clpE and iha gene-like sequences in the adherence on MDBK cells of bovine EPEC/EHEC stains, a mutagenesis strategy by allelic exchange was followed. Two mutants in the lifA/efa1 and clpE genes were obtained from an O26 EPEC strain which show a rise (one log difference) in their adherence properties on MDBK cells and primary enterocyte cultures, but a decrease of adherence in the porcine and rabbit ligated loop assays (see also WP11). They are being further studied for the exact localisation and genetic consequence of the mutations. - Experimental challenge of calves with mutants in putative adhesin-encoding genes of bovine EPEC/EHEC strains. Six calves were experimentally orally infected with deletion mutant strains of lifA/efa1, clpE or cif genes (see WP05 for cif gene), and two calves per strains. Preliminary results indicate a less clinically severe enteritis in those calves in comparison with those infected with the wild O26 strains.

The specific objectives are: - To analyse the immune response, especially cytokine, during pig AEEC infections - To construct a DNA vaccine specific for AEEC infection - To analyse the immune response of this DNA vaccine. Cytokine production during AEEC infection: Although A/E lesions have been experimentally reproduced in newborn piglets, such lesions have been much more difficult to induce in older conventional pigs. We used two different strategies to analyse the immune response during AEEC infection in pigs. The first strategy was to use a ligated loop model in pig. Ligated intestinal loops were prepared in 4 weeks old pigs and inoculated with 108 CFU of bacteria. Pigs were evaluated for colonisation and AE lesion using histopatholgy, immuno-histochemistry and cytokine expression level. In each pig there were two loops inoculated with the same strains: AEEC 1390 or D5, (both O45, Intb) or the negative control (123). The results indicated that the colonisation and AE-lesions were 80-90% reproducible. We also analyse the local immune response on the different ileal samples using a RT-PCR technique or a DNA array specific for the pig immune system. This array contains 62 genes including 20 cytokines, 10 chemokines and 12 immunological receptors. We demonstrated that inflammatory cytokine genes are induces by both pathogenic (D5 and 1390) and non-pathogenic (123) strain of E. coli. By contrast the expression of IL-8 is only induced by AEEC strains. The second strategy was to examine the development of A/E lesions in weaned pigs treated with dexamethasone, an immunosuppressant agent. We demonstrated that dexamethasone, given orally for 7 days at an immunosuppressive dose, enhanced the development of A/E lesions weaned pigs experimentally challenged with the 1390 strain. We successfully reproduce typical intimate adherence leading to A/E lesions. The local intestinal cytokine response following challenge of weaned pigs with the 1390 strain was also investigated. A significant upregulation of IL-1, IL-6, IL-8 and IL-12p40 mRNA were observed in the ileum of control weaned pigs challenged with the 1390 strain and showing few A/E lesions, but not in pigs having received dexamethasone prior to experimental challenge and showing more extensive A/E lesions. Construction of DNA vaccines: DNA vaccines are able to induce antigen-specific cellular and humoral immune responses. It is also possible to manipulate the immune response generated through the co-delivery of DNA encoding for cytokines. Wa constructed DNA plasmids encoding for two bacterial antigens (i.e. Eae, and Tir) which are considered as good candidates for protective immunity as well as DNA plasmids encoding for 3 porcine cytokines (GM-CSF, IL-4 and IL-6). The cytokines, already cloned and sequenced in pigs, presented adjuvant properties. GM-CSF is secreted by multiple cells and is able to recruit professional antigen-presenting cells. IL-4 and IL-6 have been implicated in the development of mucosal immunity and in the induction of IgA. Cytokine and antigen specific DNA fragments were then inserted in the pcDNA3.1/V5-His-TOPO expression vector which links in frame a V5 epitope and a 6 histidine (His6) tag at the N-terminal end of the proteins. Constructs were then verified by a sequence analysis of the entire insert. The gene expression in eukariotic cells was then evaluated by western blot analysis using supernatant of transfected in COS-7 cells. Immune response upon DNA vaccination: Two animal experiments were performed in order to analyse the immunogenicity of the DNA vaccine constructions. The first experiment included 4 groups of 3 piglets. Each group was vaccinated 3 times at two weeks intervals with a different plasmid combination (Eae+Tir; Eae+Tir+GM-CSF; Eae+Tir+IL-4; Eae+Tir+IL-6). The different plasmids were injected subcutaneously at the base of the ear and electroporation was used to improve transfection efficacy. In the second experiment, 24 pigs divided in 4 groups were followed up for 7 weeks (between 4-11 weeks of age). The animals were vaccinated 3 times at two weeks intervals with different constructions (unvaccinated control; vaccinated with Eae+Tir; vaccinated with Eae+Tir+IL-4; vaccinated with Eae+Tir+IL-6) In both experiments, blood samples were taken before the vaccination and bimonthly thereafter. The animals were followed for two month, their humoral immune response was analysed by western blot and ELISA. The first experiment indicates that some animals have antibody directed to intimin before immunisation and that vaccination increased the percentage of positive animals and/or their titre. However, due to the limited number of animal per group and the variability of their response, it was impossible to compare the adjuvant of the three cytokine tested. In the second experiment we failed to see any effect of the vaccination.

The objective of this WP was to quantify and analyse in vivo and in vitro, the stability of the LEE in AEEC strains of serotype O103:H2. The specific aim is to introduce a tetAR(B) cassette marker in the LEE and take the advantage of a simple one-step selection for Tc-sensitive revertants. We further characterized the stability of the LEE in a representative collection of AEEC with LEE inserted in different tRNA. Major finding during the project: In contrast to other E. coli pathogenicity islands, the LEE was supposed to be stable. But preliminary observations obtained in our laboratory suggested that the LEE was very likely unstable when AEEC strains of serotype O103:H2 were used to infect orally rabbits. Therefore, our objective was to confirm this result and to quantify in vivo and in vitro the stability of the LEE in two AEEC strains of serotype O103:H2, the rabbit EPEC strain E22 and the human EHEC strain PMK5. These strains produce b1 and e intimin subtypes respectively. A major limitation to study the stability of the LEE of AEEC strains was the difficulty to detect deletions of the PAI. Therefore, we have developed a strategy based on the island probing, a method used to identify the Shigella flexneri she PAI. We have been able to isolate several Tc-sensitive derivatives of E22 in which the LEE was excised. In contrast, Tc-sensitive revertant clones of PMK5 were never obtained. In EPEC strain, the frequency of excision of the LEE was about 1.10-6 (corresponding to the number of Tc-sensitive clones relative to the total population plated onto LB medium containing kanamycin). We found no the effects of culture conditions (temperature, osmolarity, etc.) on excision of the LEE in vitro. We have then orally infected 10 rabbits with E22 tir::tetAR, lacZ::km. The inoculated strain was recovered from the rabbits by plating dilutions of the faeces onto MacConkey medium with Kanamycin. Instability of the LEE was estimated by plating the Km+ Lac - derivatives onto fusaric acid and kanamycin medium in order to select Tc-sensitive revertants. The method is based on the finding that Tc-resistant bacteria are hypersensitive to lipophilic chelating agents such as fusaric acid. Using this method, we have been able to confirm that the LEE was also unstable. The frequency of the appearance of Tc-sensitive revertants was estimated to less than 10-6. Of note we have been able to isolate these revertants in only two rabbits out of ten.

The main serotype implicated in human disease caused by EHEC strains has been O157 but other serotypes have also been implicated in causing human and/or animal diseases. The main objective of this WP is to develop diagnostic tests for the detection of non-O157 EHEC in particular and AEEC in general. We will develop two different types of test: - The first type of diagnostic test will be based on immuno-magnetic separation (IMS) for selective enrichment of AEEC strains. Theses IMS tests will use polyclonal (already developed by partner CR2) and monoclonal (to be developed by partner CR10) antibodies directed against intimin; - The second type of test will be sandwich ELISA tests for characterisation of AEEC strains. The ELISA will be based on intimin Mabs produced against the different intimin types as well as Mabs directed toward surface adhesin (see WP16). Major finding during the project The primary aim of WP21 was to develop new reagents for isolation and detection of VTEC. Intimin and LPS are well-characterised surface VTEC antigens. Accordingly, mono-specific antibodies against these major antigens were generated and evaluated in this project. Intimin - several attempts were made to generate monoclonal antibodies using different intimin domains as an antigen. Intimin shows antigenic polymorphism. The carboxy terminus domain, comprising the receptor-binding activity, is variable, yet expose on the bacterial cell surface. The amino terminus is conserved among the different VTEC strains, yet masked by the LPS. Monoclonal antiserum made using the carboxy terminal 280 amino acids of intimin a and intimin b, although reacted with the antigen did not cross-react with the native intimin on the bacterial cell surface. This rendered the antibodies unsuitable for immuno magnetic separation (IMS). Polyclonal antibodies made against the conserved region of intimin bound recognised native intimin, but the binding avidity was too low for IMS. The conclusion from this investigation was that intimin is unlikely to provide a good target for capturing VTEC from biological material and foodstuff or for ELISA. LPS- is a strong antigen that is constitutively expressed by VTEC. However, VTEC bacteria can expressed one of a number of distinct LPS antigens; accordingly, a number of serogroup-specific antibodies are needed to be used in parallel IMS for each sample. Nevertheless, as the conserved intimin domain was proven unsuitable for a pan VTEC detection/isolation system, we evaluated the use of LPS-specific antisera. Antibodies directed against O26, O103 and O111 and O157 were evaluated. Using the antisera in reconstituted biological material and in field studies, LPS antibodies were proven to be highly specific and sensitive in IMS. Conclusion - at present, a bank of serogroup-specific LPS antibodies are the recommended method for IMS of VTEC.

Objectives: - To establish a large collectionof AEEC strains, available to all the partners, characterized by serotype and profile of virulence markers, in particular by typing of their stx and eae genes. - To characterize possible new intimin types and investigate on their distribution among AEEC strains . - To identify the different pathogroups of AEEC on the basis of the types and combination of virulence genes. - To investigate the host specificity of AEEC and possible epidemiological links between the presence of virulence factors and severe human disease. Major finding during the project: - Establishment of a culture collection. It included a total of 50 O serogroups and 300 strains, isolated from healthy and diseased humans and from different animals species. Consensus PCR protocols for detection of the main AEEC virulence genes (all variants of stx and eae, EHEC-hlyA, bundle forming pili, flagellar (fliC) genes) have been harmonised. - Characterization of new intimin types. Partner CR12 identified four eae variant genes in isolates of human origin. Three of the newly discovered genes were designated as eae-eta, jota, and kappa. The fourth one was identical to the recently described eae-zeta. A PCR scheme for amplification and typing of E. coli eae gene was developed and an eae-nomenclature system based on the Greek alphabet has been proposed. - Distribution of intimin genes and other LEE locus-related genes among AEEC Partners CR10, CR11, and CR12 carried out investigations on the distribution of the main intimin types among AEEC. Four major clones corresponding to the major intimin types a,b,g, and e have been characterized. The three intimin a, g, and e clones include all the major serogroups of AEEC associated with human disease while the intimin b clone appears to be the most ubiquitous type, in that it has been found in both EPEC and EHEC isolates from several animal species: humans, cattle, pigs, rabbits, dogs, and birds. Moreover, it includes important diarrheagenic clones such as human EHEC O26 and EPEC O26:H11, O111:H2, and O128:H2. Partner CR10 has investigated the presence of four putative PAIs described in the chromosome of EHEC O157 among AEEC. One of these PAIs, termed O#122, was found to be strongly associated with AEEC in particular those firmly associated with human disease. It contains the putative virulence gene efa1/lifA, which was reported to enhance the adhesion. Partner CR10 investigated on the presence of the virulence genes, efa1/lifA and toxB, in a panel of 60 EHEC and 68 EPEC strains isolated from different sources and belonging to different serogroups. The toxB gene was present only in EHEC O157 and most of the O26 isolates, while, efa1/lifA seemed to be associated with the non-O157 EHEC strains analysed. As for the EPEC strains investigated, efa1/lifA and toxB were present in 41,5% and in 10.3% of isolates respectively. A polymorphism in the sequence of the toxB gene was observed between EHEC O157 and EHEC O26 by restriction with HindIII followed by hybridisation with a toxB gene probe. Identification of the LEE insertion site. We showed that the LEE is located in selC or in pheU tRNA loci in most of the AEEC clones. Using an original PCR strategy, we showed that the LEE is inserted in pheV in 8 out of the 14 strains which were negative for both SelC and pheU. These included the intimin e-positive EHEC and some b-positive EPEC. Determination of AEEC pathogroups. Partner CR11 completed the investigation on a group of STEC strains collected from 677 human patients in Germany over a three years period. Sixty-two% of the STEC strains were eae-positive and 37.4% were eae-negative. eae positive STEC were significantly associated with distinct serotypes of strains and with the presence of an stx1 and/or stx2 gene, whereas stx1c, stx2d and stx2e variant genes were only found in eae-negative STEC. EHEC-Hly, a marker of the EHEC-virulence plasmid, was present in 96.2% of eae-positive and in 65.2% of eae-negative STEC. Fourteen new serotypes of eae-positive STEC which were previously not described to occur in humans were detected. A collaborative study on STEC in sheep showed that STEC isolated from healthy sheep in Norway belonged to a few dominating serotypes and did not carry an eae-gene indicating that sheep are not a major source of eae-positive STEC and EHEC strains.

"Attaching and Effacing" (A/E) activity of AEEC requires a bacterial outer membrane protein called intimin that mediates intimate adhesion between the bacteria and the host epithelium potential interaction. The aim of this work package is to identify host-cell intimin receptors using a yeast two-hybrid system. Major finding during the project: While remaining extracellular EHEC and EPEC establish direct links with the cytoskeleton of the target epithelial cell leading to formation of actin rich pedestals underneath attached bacteria. The translocated adaptor protein Tir forms the transmembrane bridge between the cytoskeleton and the bacterium; the extracellular domain of Tir functions as a receptor for the bacterial adhesin intimin, while the intracellular amino and carboxy termini interact with a number of focal adhesion and other cytoskeletal proteins. Although binding of intimin to Tir is an essential step in colonisation and disease, large body of evidence point to the possibility that intimin also binds to a receptor encoded by the host cell. In order to identify the putative host intimin receptor, we employed the yeas two hybrid system, an established tool used to screen for protein:protein interactions. To this end, we have cloned eae (encoding intimin) in the yeast two-hybrid bait plasmid and used an epithelial cDNA library that was cloned in the yeast two-hybrid prey plasmid. We used Tir, cloned into the bait plasmid as a positive control. Unfortunately, we did not identify any putative host cell intimin receptor using intimin as the bait clone. However, using Tir as bait we identified cytokeratin 18 as a novel Tir partner protein. In order to determine if the observed Tir:CK18 protein interaction is relevant to infection, cytoplasmic extracts of infected HeLa cells were immuno-precipitated (IP) with mouse anti-CK18. Specific co-IP of Tir with the CK18 antiserum observed. In a reciprocal experiment Tir antiserum was used to IP soluble cytoplasmic and membrane extracts of WT-infected cells. Probing the IP content with antibodies specific for cytoskeletal components revealed that in addition to CK18, CK8 was co-IP in both soluble cytoplasmic and membrane extracts of WT- infected HeLa cells but not with the tir mutant. We examined if CK18 is recruited to the pedestals. HeLa and intestinal epithelial INT407 cells were infected with WT bacteria and triple stained with either anti-bacterial/CK18/Arp3 antibodies or with anti-bacterial/CK18/Tir antibodies and examined by confocal microscopy. In both cell lines concentrated CK18 was found to be co-localised with Tir and Arp3 beneath adherent bacteria. CK8 is a partner protein of CK18 in IF expressed by epithelial cells. Moreover, CK8 was co-IP with anti-Tir following infection. Consistent with these, immuno-staining revealed that CK8 is also recruited to the pedestals. In order to determine the functional significance of recruitment of CK18 to the pedestal, we used the small interfering RNAs, which provide a sequence-specific, post-transcriptional, gene silencing mechanism. HeLa cells were transfected with a CK18-specific RNA duplex, infected with WT EPEC then tripled stained using anti-bacterial/CK18/Arp3 antibodies; Arp3 staining was used as a marker for pedestal formation. Staining for CK18 revealed a transfection efficiency of less than 50%; silencing was confirmed by Western blotting. In transfected cells exhibiting reduced or no CK18 staining, pedestals were observed in 29 +/- 1.5% of the adherent bacteria whereas 63 +/- 3.6% of adherent bacteria were associated with Arp3 in un-transfected cells. This study is the first to implicate intermediate filaments in microfilament reorganisation following EPEC infection.

Certain AEEC strains inhibit host cell proliferation and trigger irreversible alterations of the cytoskeleton, characterised by the recruitment of vinculin and the assembly of actin stress fibres. This phenotype called CythoPathic Effect (CPE), is dependent on a functional type III secretion machinery but is Tir and intimin independent (Tir and intimin mutants are FAS negative but CPE positive). We used two approaches to identify the bacterial effector(s) responsible for CPE: - Construction of a mutant strain that show an enhanced secretion of all protein secreted through the type III secretion system and characterisation of new secretion products. - Development of random mutagenesis and screening for failure to induce CPE. Major finding during the project: We have identified a new TTSS translocated effector molecule responsible of the CPE that we called Cif (Cycle inhibiting factor), which blocks cell cycle G2/M transition, and induce the formation of stress fibres through the recruitment of focal adhesions. Cif is the first effector not encoded by the LEE but by a lambdoid prophage present in EPEC and EHEC. A cif mutant causes localized effacement of microvilli and intimately attaches to the host cell surface but is defective in the ability to block mitosis. When expressed in TTSS competent LEE positive pathogens, Cif is injected into the infected epithelial cells. These cells arrested at the G2/M phase displayed accumulation of inactive phosphorylated Cdk1. In conclusion, Cif is a new member of a growing family of bacterial cyclomodulins, which subvert the host eukaryotic cell cycle. The use of mutant with enhanced secretion was less efficient than the detection of CPE effectors by random mutagenesis. Therefore, we developped a new strategy to analyze and identify new TTSS effectors using an originalreporter system based on a translational fusion of the effector proteins with mature TEM-1 b-lactamase. Translocation was detected directly in living host cells by using the fluorescent b-lactamase substrate CCF2/AM. The TEM-1 system is not based on a cellular process but on the differential entry of the TEM-1 substrate in bacterial and eukaryotic cells and the use of the CCF2/AM fluorescent substrate enables the translocation analysis in living cells, without disruption of the host cell. In addition, this new reporter can be fused to the end of certain effectors without affecting their activity, which means that double activity tests can be carried out with efficiently translocated proteins, making the data more reliable. Because fewer than 100 molecules of TEM-1 can be readily detected within a cell, the system was sensitive enough to detect translocation of a weakly produced chromosomal-encoded Cif-TEM fusion. In the context of a growing importance of TTSS in bacterial pathogenicity, TEM-1 fusions used in combination with CCF2/AM fluorescent substrate is a new powerful tool for identifying undiscovered bacterial encoded molecules that are delivered into host cells.

The objectives of this work package were to understand the physiopathology of AEEC infections using rabbit, a relevant animal host highly susceptible to EPEC. We aimed at defining the importance of the strong inflammatory response in the development of the disease and at characterising bacterial effectors responsible for this inflammation. Major finding during the project: Rabbit EPEC infection induced dramatic villus atrophy in the distal ileum combined with a hypertrophy of the glandular region. Epithelial cells were irregular and dysplastic and strong exfoliation occurred. The brush border was not visible. Similar lesions were observed in the proximal colon. In contrast, 5 days after inoculation of a Tir/EspB mutant, there were no architectural intestinal modifications in the ileum or the colon. Villi were preserved and no inflammation was detectable. Epithelial cells were regular and the brush border was visible and comparable to that of the ileum of control rabbits. A new collaboration with a Mexican team allowed us to analyse the expression of proinflammatory cytokines from enterocytes and lymphocytes by EPEC-Infected rabbits and to study the role of EspA and intimin. Orally inoculated rabbits suffered from weight loss, mucosa inflammation, developed watery diarrhea, and died (day 7). At day 6 postinoculation, animals were analyzed for induction of proinflammatory cytokines in enterocytes. The role of lymphocyte-dependent immunity was determined through expression of proinflammatory cytokines by lymphocytes from Peyer's patches and spleen. EspA and intimin mutants were used to explore the role of the A/E lesion on the expression of these cytokines. Infected rabbit enterocytes increased IL-1b, IL-6, IL-8 and TNF-a mRNAs expression (by RT-PCR) but only slightly the anti-inflammatory IL-10. In contrast, intimin mutant-infected rabbits were unable to induce this proinflammatory cytokine profile but did induce a remarkable increase of IL-10. Whereas, EspA mutant increased the expression of IL-8 and TNF-a but only slightly IL-10. Peyer's patches lymphocytes (PP) also produced proinflammatory cytokines, which were dependent on EspA (except TNF-a) and intimin, while IL-10 was induced by EspA and intimin mutants. In contrast, spleen lymphocytes (systemic compartment) were unable to induce IL-1b and TNF-a. These data show the importance of the proinflammatory cytokines secreted by enterocytes and those expressed locally by PP lymphocytes, which can activate effector mechanisms at the epithelium. Furthermore, this cytokine profile depends on intimin, including IL-6 and IL-1b, which may be involved in the diarrhoea produced by EPEC.

The objectives of this section were: - To raise specific antibodies against AEEC virulence factors in hens (egg yolks are a rich source of immunoglubulins that can easily be incorporated into animal or human diets) - To examine the ability of these antibodies to prevent the development of intestinal colonisation and diarrhoea. It has clearly been demonstrated in the literature that the intimin Eae and the secreted proteins EspA and EspB are virulence factors that play an important role in the pathogenesis of various AEEC infections. These different virulence factors were thus considered as good candidates for protective immunity. Primer pairs specific for each of the virulence factors were chosen to amplify by PCR the eae, espA and espB genes from the genomic DNA of enteropathogenic E. coli porcine strain 1390. The different DNA amplified fragments were inserted in the pQE30 expression vector which links a His6 tag at the N-terminal end of the proteins. After transformation in the M15(pREP4) strain, the fusion proteins His-Eae, His-EspA and His-EspB were detected by Western blot analysis revealed with anti-histidine antibodies and with antibodies specifically directed against each of the virulence factors. All fusion proteins were then produced on a large scale and purified on a nickel affinity column in sufficient quantities for immunization of chickens and for use as antigens in the ELISA. Chickens were immunized with each of the purified proteins. Eggs were collected and egg yolks containing antibodies (IgY) were mixed with PBS, chloroform was added and the overall mixture vortexed. After centrifugation, the water phase containing IgY was conserved. Western blot analysis showed that purified IgY recognized homologous fusion proteins. The titer of the specific IgY for each protein, using homologous purified proteins as antigens in an ELISA, was 1/4800, 1/4000 and 1/5600 for anti-Eae, anti-EspA and anti-EspB IgY, respectively. AEEC strains originating from the rabbit, pig, dog, bovine, and human (including non-O157 EHEC and EPEC), and producing Eae of the a, b, d, or e subtypes, induced A/E lesions equally well on newborn pig ileal explants. On the other hand, O157:H7 AEEC strains producing Eae of the g subtype adhered to a much lesser extent to the ileal epithelium. However, replacement of the Eae of the g subtype by the Eae of the a-subtype in an O157:H7 strain resulted in induction of A/E lesions to a similar extent as observed for the homologous porcine AEEC strain. These results suggest that Eae of the g subtype of O157:H7 strains recognizes receptors on porcine ileal epithelial cells less well than the Eae of the other known subtypes. Nevertheless, our results confirm that pig ileal explants are an appropriate model for the examination of the effect of specific antibodies on the formation of A/E lesions by AEEC of diverse origin. Antibodies specific for the mature and carboxy terminal of Eae, Tir and, in the case of the homologous porcine strain 1390, Paa, significantly blocked the development of A/E lesions in ileal explants, both for the homologous strain 1390 and all of the tested AEEC strains from the calf, and human, the latter including both EPEC and O157:H7 EHEC strains. The anti-EspA, anti-EspB, and anti-EspD antibodies did not affect the development of A/E lesions by any of the tested AEEC strains, with the exception of a human EPEC strain, for which the anti-EspA antibodies did significantly block the development of A/E lesions. Anti-Paa antibodies did not block the development of A/E lesions due to this human EPEC strain that produced Eae but not Paa. Hence, the anti-Eae mature, anti-Tir, and anti-Paa antibodies, and possibly the anti-EspA antibodies, were considered as potential candidates for the blocking of development of A/E lesions in vivo.

The main objective of WP11 is to develop an infection model for AEEC infection in weaned pigs. Major finding during the project: The main objective of WP11 was to develop an infection model for AEEC infection in weaned pigs. In this line one of the deliverable (D23) was to develop an oral AEEC model. The efforts in the first 4 experiments were concentrated on 5-6 weeks old pigs, inoculated once with TSB grown 1010 CFU of the standard Canadian 1390 (O45) AEEC bacteria/pig (all from farm A). The strain was provided by CR6 (Dr. J. Fairbrother). In all subsequent experiments we used 4 weeks old pigs, from farms B, C, and D and inoculated them twice on subsequent two days. All together 159 pigs were used in 10 different experiments. In 5 of these experiments different predisposing factors were also used (Fumonisin mycotoxin, TGE-virus, F18+ETEC bacteria and high energy or high protein diet). In none of these experiments were repeatable attaching effacing lesion in a consequent number of pigs when ilea and caeca were tested by fluorescence microscopy (IF), and HE sections, at 3-5 days post infection. The predisposing effect of the high protein diet (per se) could reproducibly proven by the concomitant use of ETEC infections. To increase the chance of selecting more virulent strains, there were also 3 pools (with 4 AEEC strains in each) formed using Canadian and Hungarian AEEC strains. Slight AE lesions were only detected in 3 of all together 12 pigs inoculated that way. Based on these results it was concluded that 4 weeks old commercial pigs are basically quite resistant to the oral infection by porcine AEEC strains. The other objective (D24) within the above main objective was to establish an alternative virulence model in weaned pigs. For these experiments we have used all together 48 pigs that provided over 200 loops to be investigated for the pathogenicity and pathophysiology of the AEEC infection in these animals by IF and HE sections. The ligated intestinal loops were prepared in 4 weeks old pigs (as described in previous report). Loops were inoculated with approx. 5 x108 CFU of AEEC bacteria (or negative control). Pigs were kept in post surgical anaesthesia for 16-18 hours (as described before) We have used the Canadian O45 standard strain (86-1390) in every pig as a positive control strain, together with a standard negative (nonpathogenic) E. coli control strain. The other test organisms were Hungarian 9 different AEEC strains of different O-types and intimin types (described below, and characterized in detail in WP17). These strains were isolated from the upper intestine or from the rectal flora, and a human O157:H7 EHEC, as well as a bovine AEEC (O126 strain from J.Mainil) for their adhesive pathophysiology. For all these types of AEEC bacteria the ligated loop model in 4-week-old weaned pigs proved to be a useful model and detected differences between EHEC and porcine AEEC, as well as within porcine AEEC. Using this weaned pig loop model we could also complete the objective to describe potential new porcine types of AEEC (D25). These were reported within WP17 and they are the O4/O123, O28, O76, O108, O149, O157, (most of them are of b-type of intimin but some are of g, and some are untypable). This model was also succesfully used to study intestinal cellular immunity as a response to AEEC infection (Trizol-fixed intestinal wall and Peyer's patch samples were sent to CR7 for amplifications by RT-PCR to measure intestinal cytokine response. See WP13). DNA vaccine test was performed on 24 pigs for 7 weeks (between 4-11 weeks of age), using the DNA fragments AE, Tir, IL-4 and IL-6 in different combinations as well as controls (See WP13).

Among other properties, EPEC/EHEC differ from other pathogenic and non-pathogenic Escherichia coli by their surface antigens. Recognition of those could help in the specific diagnosis and description of specific virulence factors, including putative colonisation factors. The general objectives of WP16 were to raise MAbs to specific surface structures involved in the development of lesions by EPEC/EHEC strains and to identify the genetic basis of any newly recognised specific antigen. The factors targeted include those that are well defined, such as intimin, but also as yet undefined surface antigens. Major finding during the project: - Genetic basis of the MAb 2F3 recognized antigen. Prior to the project, a MAb has been raised to an undefined surface antigen of an O26 EHEC strain (Mab 2F3). Continued diagnostic work with an sELISA developed with this MAB has provided evidence that it is largely specific to EPEC/EHEC O26 strains (see result 21: development of immuno-capture and ELISA test diagnostic). A genomic DNA library from the O26 bovine EHEC strain 4276 was constructed using cosmid vectors (derivatives from cosmid pHC79) of different sizes and different cloning capacity. The constructs were packaged into l bacteriophages, which were subsequently able to infect an E. coli. More then 2000 clones were obtained. The entire library was screened by sELISA with the 2F3 MAb. One positive clone harboured a genomic locus of 30 kb, containing the entire O-antigen gene cluster and the second half of the colanic acid gene cluster. Six independent mutants obtained by in vitro insertional mutagenesis assay (Tn5 transposition system) had the transposon inserted into the O26 antigen gene cluster. The O26 O-antigen gene cluster from the EHEC strain 4276 was also cloned into an E. coli DH5a strain using long range PCR assay. Several clones expressing the 2F3 antigen and the O26 O antigen were obtained. The O26 O antigen gene cluster from an EHEC strain is therefore sufficient to synthesise the epitope recognised by the2F3 Mab. Finally Western blot assay on LPS extracts with the 2F3 MAb gave a positive result. The sequencing of one O26 antigen gene cluster from a 2F3-negative E. coli is complete. Comparison to the published sequence of an O26 antigen gene cluster from a 2F3-positive E. coli failed to highlight any significant difference. Derivation of other monoclonal antibodies (Mab): - Derivation of monoclonal antibodies (Mab) to the intimin adhesin. Prior to and following the start of the project, MAbs were raised to the intimin adhesin of the EPEC/EHEC: to a conserved intimin region and to specific regions of the b-intimin and g2-intimin. They have been examined for immunohistological staining of gut tissue (see below) collected from calves experimentally infected with O26 EPEC/EHEC strains (see result 14: identification of colonisation factors of bovine EHEC strains). - Derivation of monoclonal antibodies (Mab) to additional surface antigens. Outer membrane protein (OMP) and lipopolysaccharide (LPS) extracts from EPEC/EHEC strains of the O5, O111, O103, O118 and O157 serogroups were used to immunise mice. Hybridoma fusions were carried out on mice that demonstrated a better serum titre to the equivalent EPEC/EHEC whole cells than to corresponding non-EPEC/EHEC strains. Three MAbs with specificity to O111 LPS were obtained, one of which, was shown to be specific for the O111 EPEC/EHEC strains, was successfully applied in a sELISA to produce an EPEC/EHEC specific assay (see result 21: developement of immuno-capture and ELISA test diagnostic). - Identification of O26 specific surface antigens. In a different approach, the strains from the O26 strain cosmid library were examined for expression of O26 specific surface antigens using polyclonal sera raised to whole cell and surface antigen fractions of O26. Five of the clones reacted to polyclonal sera (Pabs). Unfortunately antisera raised with all five did not show O26 specific activity, since cross-reaction to other E. coli strains, EPEC/EHEC and non-EPEC/EHEC was observed by immunoblot and ELISA. Immunocytochemistry: Immunocytochemical work has been conducted with samples obtained from the experimental infections already carried out (see result 14: identification of colonisation factors of bovine EHEC strains). The b-intimin MAb 4H4 demonstrated clear, specific staining in the infected tissue. In addition, Pabs to EspA, EspB, EspD and Tir (obtained from CR2), all demonstrated specific staining in infected tissue. Attempts to apply the antibodies available to immunogold staining for electron microcopy examination have been successfully achieved with MAb 2F3 (O26 LPS) and 4H4 (b intimin), but staining with PAbs to EspA, Esp B, EspD and Tir showed little evidence of specificity.

The aim of this WP is to identify new virulence genes induced during the contact of AEEC strains with the epithelium. A technique known as IVET (in vivo expression technology) will be used to positively select for bacterial genes expressed upon infection of epithelial cells. This system is aimed at identifying bacterial genes that are preferentially expressed in the host during infection and are poorly transcribed under laboratory conditions. IVET will allow us the identification of promoters (and subsequently the genes) that are activated in vivo through the use of a promoter-less reporter gene located downstream of randomly inserted bacterial genomic sequences. Major finding during the project: The aim of this project was to use a whole-genome micro-array (instead of IVET) to screen for alteration in EHEC gene expression at a time when bacteria are attached to plasma membranes. The array was composed of 5318 50-mer oligonucleotides. Only genes with a p-value equal to or smaller than 0.05 were analysed further. Genes showing a iY2-fold difference in the expression ratio, between the two conditions, were classified as regulated genes. Expression profiles. Expression of 401 genes was different between RBC-associated and DMEM-grown EHEC; with 296 lower and 105 higher in the RBC-associated bacteria. Stress responses. The mRNA levels of rpoE and rseB, were more abundant in RBC-associated EHEC. RpoE controls cell envelope stress response and is transcribed when misfolded or unfolded proteins accumulate in the periplasm or outer membrane or when cells enter stationary growth phase. ResB is a regulator of RpoE and the higher level of expression in attached bacteria is therefore consistent with the elevated mRNA level of rpoE. Elevated levels of mRNA of groES and groEL (expressed in response to a variety of stress conditions in the cytoplasm) were found in RBC-associated EHEC. Moreover, the mRNA levels of both heat shock chaperone proteins DnaK and DnaJ were also elevated in the RBC-bound bacteria, although the p value was not as significant (p = 0.15). The mechanism controlling expression of these chaperones under the experimental conditions tested is not yet defined. mRNA of genes involved in translation. With the exception of rpsV encoding the 30S ribosomal subunit protein S22, the majority of mRNA encoding ribosomal proteins was lower in the attached bacteria. These results suggest a reduction of de novo protein synthesis in attached bacteria. This is supported by the fact that the level of tig mRNA, encoding the ribosome-bound trigger factor chaperone that maintains newly translated proteins in an open conformation, is also reduced in attached bacteria. mRNA expression profiles of the LEE genes. Of major interest was the expression profile of mRNA of the LEE genes. In general, expression levels of most of the LEE mRNA were significantly lower in RBC-associated bacteria. In particular, the levels of mRNA encoding the translocator proteins EspA, EspB and EspD were over 20 fold lower and the mRNA encoding intimin was 69 fold lower. In contrast to the almost uniform lower abundancy of LEE mRNA in RBC-associated EHEC, mRNA levels of four genes; espG, rorf3, orf15 and escN encoding the effector EspG, two proteins of unknown function and the TTSS ATPase respectively, were unchanged, although only orf15 was also statistically significant. Regulators. yhiE and yhiF were recently reported to be negative regulators of LEE4 gene expression and null function mutations in these genes resulted in enhanced binding of EHEC to CaCo2 cells . Moreover, over expression of yhiX (gadX) reduced the production of virulence proteins in EPEC. Consistent with this, the mRNA levels of yhiE, yhiF and yhiX were higher in RBC-associated EHEC than in DMEM-grown EHEC. These transcriptional regulators influence expression of genes implicated in the maintenance of pH homeostasis. Therefore, yhiE (gadE) in EHEC, like gadX in EPEC, may regulate expression of genes required for acid resistance and virulence. SdiA was also reported to have a negative effect on LEE gene expression. Consistent with this, the mRNA level of sdiA was higher in RBC-associated EHEC. Identification of new virulence determinants EspJ and TccP: Similarly to down regulation of the LEE-encoded proteins, expression of two other phage encoded genes (termed espJ and tccP) that encode type III effectors were also down in attached bacteria. By adding a carboxy terminal His tag to EspJ and TccP we have shown that both proteins are secreted and translocated by the EHEC TTSS. Mutations in espJ and tccP have shown that while the former is not requited for A/E lesion formation, the latter is essential for actin reorganisation by EHEC. TccP is only the second (after Tir) EHEC type III effector that was implicated in A/E lesion formation.

The specific objectives of this work package are: 1) investigating molecular aspects of the LEE thermoregulation, 2) determination of the pattern of expression of LEE genes during the infection of organ culture and 3) testing the role of IHF and Ler during infection of organ culture. Major finding during the project: Concerted regulation of the different virulence genes is essential for successful colonization of the host by pathogens. The genes encoding the TTSS and some of the effector proteins are located within a unique pathogenicity island, the locus of enterocyte effacement (LEE). Work in several groups as well as work in our laboratory indicate that the 41 LEE genes are organized in 11 transcriptional units including: LEE1, LEE2, LEE3, LEE4, LEE5, LEE6 (rorf1,rorf2), LEE7 (orf10, orf11), rorf3, map, cesF, escD, and map. Moreover, we found that some of these transcriptional units contain internal promoters as well. Our long-term goal is to decipher the complex regulatory network coordinating the expression of the genes encoding the TTSS and the effector proteins. We discovered that the first gene in the LEE1 operon encode a regulator termed Ler. Ler positively regulates the transcription of all the LEE transcriptional units except LEE1 (Friedberg et. al., 1999 and unpublished data). Therefore, the decision of whether to activate the LEE1 promoter is critical to the initiation of a regulatory cascade leading to the expression of other LEE operons. Entry of the pathogen into the host is associated with a shift in temperature from below 30 degrees Celsius to 37 degrees Celsius. We showed that EPEC exploits this temperature shift as a signal for activating the expression of its virulence genes. We further investigated the molecular mechanism of this thermoregulation. We found that the thermostat, which senses differences in temperature, is the LEE1 promoter (Umanski et. al., 2002). This property of the LEE1 promoter is dependent on H-NS, a nucleoid-associated protein. We also discovered that H-NS represses the expression of the LEE1 operons at 27 degrees Celsius, but not at 37 degrees Celsius, leading to thermo-regulated expression of all the Ler-regulated operons. In addition, the expression of LEE2, LEE3, LEE4 and LEE5 is repressed by H-NS at both 27 degrees C and 37 degrees Celsius. Upon shifting the culture temperature from 27 degrees Celsius to 37 degrees Celsius, Ler is synthesized, it in turn activating the expression of LEE2, LEE3, LEE4 and LEE5 by releasing the H-NS-mediated repression (Umanski et al., 2002). While H-NS represses the LEE1 operon, Integration Host Factor (IHF), another nucleoid-associated protein, is a positive regulator of the LEE1 promoter. We found that LEE1 expression is strictly dependent on binding IHF upstream to the LEE1 promoter (Friedberg et al., 1999, Yona-Nadler et al., 2003). Interestingly, IHF is also required for repression of the flhDC operon in EPEC. FlhDC are positive regulators of the flagellum encoding genes (numbering about 40). Thus, IHF represses flagellum synthesis (Yona-Nadler et al., 2003). Moreover, we showed that a unique EPEC factor is required for IHF-mediated flagellum repression (Yona-Nadler et al., 2003). We also investigated the mechanism that determines the Ler steady state concentration. We found that Ler acts as a specific auto-repressor of LEE1 transcription (Berdichevski et al., submitted). We further show that Ler specifically binds upstream of the LEE1 operon in vivo and in vitro. Comparison of the Ler affinities to different DNA regions suggest that the auto-regulation mechanism limits the steady state level of Ler to concentrations that are just sufficient for activation of the LEE2 and LEE3 promoters and probably other LEE promoters. This mechanism may reflect the need of EPEC to balance between maximizing colonization efficiency by increasing the expression of the virulence genes and minimizing the immune response of the host by limiting their expression. In addition, we found that the auto-regulation mechanism reduces the cell-to-cell variability in the levels of LEE1 expression (Berdichevski et al., submitted). These findings point to a new negative regulatory circuit that suppresses the noise, and optimises the expression levels of Ler and other LEE1 genes.

The objective of WP15 was to identify genes that influence the interaction of EHEC with bovine intestinal mucosae by screening signature-tagged transposon mutants for their ability to colonise calves. Wild-type and defined mutant strains would then be fed to calves and faecal excretion and clinical parameters scored. Major findings during the project: Creation and screening of the mutant bank: - Selection of a bovine virulent EHEC strain. Pairs of 4 and 14 day-old Friesian calves were separately infected with EHEC strains of serotype O5, O26, O103, O111, O157 and a porcine commensal E. coli. All EHEC strains were shed in high numbers for >7 days. An O157:H7 EHEC strain colonised calves in high numbers but without symptoms. An O5:H- EHEC strain induced transient bloody diarrhoea, but proved difficult to genetically manipulate. Finally, an O26:H- EHEC strain (193) was chosen, which induced transient diarrhoea in 4 day-old calves and was observed to adhere extensively to the colonic epithelium by microscopy. - Creation of the mutant bank. A bank of 3000 mini-Tn5Km2 tagged transposon mutants of EHEC O26:H- strain 193 was created following separate matings between strain 193 and 95 E. coli K-12 donor strains each containing a uniquely tagged transposon on a suicide plasmid. Insertion mutants were selected and arrayed into 96¡Vwell microtitre plates such that each mutant could be distinguished from the others by a unique sequence tag. - Screening of the mutant bank. Six plates of 95 mutants were separately fed to 4 day-old calves and output pools recovered from the faeces and colonic epithelium at 5 d.p.i. The composition of the output pools was then compared with that of the inocula by PCR for the unique tags followed by hybridisation. Analysis of non-colonising mutants: - Identification of EHEC genes influencing intestinal colonisation. Of 570 mutants screened 19 were completely absent in both output pools, indicating that they have defects in intestinal colonisation. A further 65 mutants were poorly represented in one or both of the output pools. The site of Tn insertion was determined in 62 colonisation-defective mutants and 59 different genes were identified. This was accomplished by subcloning the mutated region into pBluescript with selection for the Tn-encoded resistance and sequencing with a Tn-specific primer. The results indicate that the locus of enterocyte effacement (LEE) plays a major role in intestinal colonisation of calves by strain 193. In addition colonisation is facilitated by the cytotoxins PssA, EhxA and Efa1, as well as putative Type III secreted proteins unlinked to the LEE, a putative fimbrial operon and the EvgAS two-component sensory system. The elaboration of Type I fimbriae by EHEC O26:H- is apparently disadvantageous for persistence within the bovine intestine, since a constitutively fimbriated fimE mutant was isolated in the screen. - Analysis of defined mutants in vitro. All mutants in whom the Tn-insertion site was determined were examined for their ability to adhere to HeLa cells and elicit pedestal formation. In addition, the expression and secretion of LEE-encoded proteins by each mutant was assessed by Western blotting using EspD-specific antiserum. As expected, mutations located within the LEE greatly reduced secretion of effector proteins, adherence and actin nucleation. Mutation of the other genes identified by STM did not impair type III secretion or adherence, indicating that they may influence EHEC carriage in a LEE-independent manner. - Analysis of defined mutants in calves. The role of 3 genes identified by STM was assessed in calves. Mutants with insertions affecting the LEE (escN) and Type I fimbriation (fimE) were given orally to calves and the course of faecal excretion compared to that of the wild-type for up to 3 weeks (at least 2 calves/mutant). The induction of enteritis was scored and bacterial adherence to intestinal mucosae assessed by microscopy and direct enumeration. The escN mutant was severely impaired in intestinal colonisation, validating the STM approach as a means to identify intestinal colonisation factors. The fimE mutant behaved similarly to the wild-type, but was later found to have converted to the non-fimbriated state in vivo. A non-polar deletion of pssA was constructed by �ÜRed mutagenesis. This mutant was given orally to three 4 day-old calves and was not significantly attenuated compared to the wild type, although the pssA::Tn mutant was. Polar effects of the Tn-insertion in the original STM mutant could explain this discrepancy. The results have provided extremely valuable insights into EHEC pathogenesis in both animals and humans.

Objectives - Establishment of a strain collection of 150 AEEC strains; - Molecular characterization of AEEC from human, animal, and foods; - Comparison of PFGE with MLST for characterization of AEEC; - Assessment of the potential risk of animal AEEC to infect humans. Major finding during the project: Strain collection. One of the major requirements for the success of this project was the establishment of a strain collection of important AEEC strains of non-O157 serotypes. We have collected AEEC strains expressing defined O-antigens from a number of various sources and countries. The strain collection consists of 150 strains of sero-groups O49, O84, O103, O145, O128, and O156, derived from humans, animals, and food ASTS/MLST. We have worked out the complete conditions for the molecular typing with MLST/ASTS. To avoid confusion in the literature we have reclassified our work as MLST. We used the adk, arcA, fumC, icd, mtlD, mdh, and pgi houskeeping genes as well as the eae and escD genes from the LEE locus to amplify fragment from the strains and now we have a complete data set of 950 housekeeping and 256 LEE sequences. These data are under analysis and compared with the data of partner CR11. Phylogenetic trees were constructed and phylogenetic relationships were compared with PFGE patterns and virulence patterns (partner CR11). We have analysed the whole data set of E. coli O103 strains of our strain collection. The paper on O145 strains is in preparation. From these analyses it became clear that there exists typical animal adapted strains that are not found as human pathogens and only some analyzed strains have been found in both humans and animals. We have been able to better characterize serotype O103 and to show that EPEC and STEC in the O103 group are genetically related but represent two distinct clones with different animal reservoirs. PFGE: PFGE has been performed on all 150 strains and data were computerized, analysed and compared with MLST/ASTS for their suitability to define (sub)clones of pathogenic AEEC and to investigate the origin of EPEC and STEC in strains belonging to the same serogroup. Virulence gene typing. PCR-based virulence gene typing was performed on all strains using primers specific for stx, efa, cif, paa, ehlyA, etpD, katP, iutA, espP, cnf, cdt-I, bfpA, lpfA. The data from these experiments have been intergated into the PFGE/MLST data to give a complete strain characterization. Intimin genes. In a cooperation project with partner CR11, CR10, and CR5 we have identified and characterized new intimin genes. We determined the sequence of three novel variant intimin genes detected in attaching and effacing Escherichia coli isolates of human origin. We compared these sequences with those of published intimin-a, -b, -g, -e, and __q__ alleles. Sequence analysis of these ten intimin alleles confirmed the great genetic diversity of the intimin gene family in E. coli. Phylogenetic analysis indicates that recombination events have played a role in the evolutionary history of intimin genes. We have recommended an eae-nomenclature system based on the Greek alphabet and provide an updated PCR scheme for amplification and typing of E. coli eae. Intimin genes in porcine EPEC. By cooperation with partner CR10 we have been worked out the serotyping and intimin typing of a group of AEEC strains from healthy and diseased pigs from Hungaria. The results indicate that different groups of AEEC are present in healthy and diarrheic pigs. E. coli from healthy infants. Twenty-seven AEEC strains have been isolated from fecal samples of 14 children from Melbourne and from 12 children from Berlin. The 27 AEEC strains were classified as enterohemorrhagic E. coli (EHEC), typical enteropathogenic E. coli (EPEC) and atypical EPEC (25 strains negative for Shiga-toxins and EAF-plasmids). The AEEC were divided into 18 different serotypes, O-untypable and O-rough strains. Typing of their intimin genes revealed the presence intimin a in 6 strains, b in 11 strains, g in 7 strains, z in 2 strains and h in one strain. Analysis of HEp-2 cell adherence showed diffused (DA) or localized like adherence (LLA) patterns in 26 AEEC strains, local adherence (LA) was only found with the EAF-positive strain. Ten AEEC strains showed an attaching and effacing (AE) property with the fluorescent actin staining (FAS)-test. Our findings indicate that atypical EPEC could play a double role as natural immunizing strains to intimin in humans and as a reservoir for new emerging human pathogenic EPEC strains.

Weaned rabbit is a relevant non-human host for study in vivo EPEC and to a lesser extent EHEC pathogenesis. Several rabbits EPEC serotypes, such as O103:H2 and O26:H11, induced severe and lethal diarrhoea upon oral inoculations with as few as 104 CFU. The objective of this WP is to compare a wild-type pathogenic E. coli strain with different isogenic mutants in experimental rabbit infections. This approach was first used to analyse newly described putative virulence factor such as EspF, Paa, and Orf31 and was then extended to other putative contributor genes that were identified in WP05, 06, 07, 14 and 15. Major finding during the project: We have constructed null mutants in new identified genes as for example cif (see WP05) or in genes involved in the secretion as for example escN and in the regulation of the LEE as for example hha. We have then constructed null mutants in cdt, fliC and in orf16 from the LEE. Total DNA preparations from strains resulting from this procedure has been restricted and probed to confirm the allelic exchange. All the mutant resulted from a double crossing-over except. Western-blots and FAS or CPE assays have been performed to verify the loss of the putative virulence trait. The mutant strain have been then complemented with the wild type allele of the gene to establish that no other genes have been affected during the allelic exchange. We then have compared the ability of wild type and defined mutant strains to induce enteropathogenic responses in rabbits. However, to reduce the number of infected (and eventually killed) rabbits, we mainly used the rabbit ileal loop model to analyse the enteropathogenic response. Except when we muted the TTSS, tir or eae (intimin), none of the tested mutants has a significantly reduced virulence in the rabbit model.

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