Role of EspFu/Tccp in the establishment of the attaching and effacing lesion caused by atypical enteropathogenic Escherichia coli displaying different interactions with epithelial cells invitro
Abstract number: P1967
Rocha S., Abe C., Bando S., Sperandio V., Elias W.
Enteropathogenic Escherichia coli (EPEC) has been classified as typical and atypical (aEPEC), based on the presence or absence of the EAF plasmid, respectively. EPEC cause the attaching-effacing (A/E) lesion on the intestinal mucosa, which is triggered by proteins encoded by the pathogenicity island named LEE. However, some aEPEC strains are unable to cause A/E in vitro, despite the presence of LEE.
Objective: Structural and functional analysis of the LEE region of A/E-negative aEPEC strains.
Methods: Four aEPEC strains were studied: O55:H7 (displaying the localized-like adherence pattern/LAL), O55:H7 (displaying the diffuse adherence pattern/DA), O125ac:H6 (displaying the aggregative adherence/AA) and O88:HNM (nonadherent/NA). Adherence and capacity to cause A/E (FAS assay) were investigated in HeLa, HEp-2, Caco, T84 and HT29 cells, and Tir phosphorylation in HEp-2 cells. The espFu/tccp gene was searched by PCR. Transcription of LEE operons was measured by real time PCR (qRT-PCR). Expression of intimin, Tir, EspA, EspB and EspD was detected by immunoblotting. A EspFu-expressing plasmid (pKC471) was used to transform the A/E-negative aEPEC strains.
Results: The adherence patterns observed in HEp-2 and HeLa cells were maintained in all cell lines of intestinal origin. The capacity to cause A/E, to phosphorylate Tir and the presence of espFu/tccP was detected only in the LAL-expressing strain. Transcriptional profiles of LEE as measured by qRT-PCR were analysed in comparison to the atypical EPEC strain BA320 (LAL/FAS+). LEE 15 transcription levels were decreased in the AA, DA and NA strains in both culture conditions (DMEM and HeLa cells), except for LEE 4 (espA) which showed higher transcription level in the DA strain in DMEM. All four aEPEC strains studied expressed intimin, Tir, EspA, EspB and EspD. The DA and NA strains harbouring espFu/tccp were able to cause A/E in HeLa and Hep-2 cells. The presence of espFu/tccp was not efficient to induce the A/E lesion in vitro by the AA strain.
Conclusions: Despite the incapacity to cause A/E, the transcription and expression of LEE 15 demonstrated that LEE is functional in these strains. The incapacity to cause A/E in vitro by the DA and NA aEPEC strains is due to the absence of espFu/tccp. The complete mechanism involved in the establishment of A/E by the AA aEPEC of serotype O125ac:H6 remains unknown.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
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