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Role of Intimin in host specificity and tissue tropism

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.

Informazioni correlate

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INRA-ENVT Microbiologie
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