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Identification of novel bacterial effectors that influence the EHEC interaction with intestinal mucosa using signature tagged mutagenesis

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.

Reported by

Institute for Animal Health
Compton LaboratoryCompton
RG16 0NNRG Newbury
United Kingdom
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