Translocated EspF protein from enteropathogenic Escherichia coli disrupts host intestinal barrier function
Barry P. McNamara, … , Michael S. Donnenberg, Gail Hecht
Barry P. McNamara, … , Michael S. Donnenberg, Gail Hecht
Published March 1, 2001
Citation Information: J Clin Invest. 2001;107(5):621-629. https://doi.org/10.1172/JCI11138.
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Article

Translocated EspF protein from enteropathogenic Escherichia coli disrupts host intestinal barrier function

Abstract

The mechanisms by which enteropathogenic Escherichia coli (EPEC), an important cause of diarrhea among infants in developing countries, induce symptoms are not defined. EPEC have a type III secretion system required for characteristic attaching and effacing changes that modify the cytoskeleton and apical surface of host cells. Infection of polarized intestinal epithelial cell monolayers by EPEC leads to a loss of transepithelial electrical resistance, which also requires the type III secretion system. We demonstrate here that EspF, a protein that is secreted by EPEC via the type III secretion system, is not required for quantitatively and qualitatively typical attaching and effacing lesion formation in intestinal epithelial cells. However, EspF is required in a dose-dependent fashion for the loss of transepithelial electrical resistance, for increased monolayer permeability, and for redistribution of the tight junction–associated protein occludin. Furthermore, the analysis of EPEC strains expressing EspF-adenylate cyclase fusion proteins indicates that EspF is translocated via the type III secretion system to the cytoplasm of host cells, a result confirmed by immunofluorescence microscopy. These studies suggest a novel role for EspF as an effector protein that disrupts intestinal barrier function without involvement in attaching and effacing lesion formation.

Authors

Barry P. McNamara, Athanasia Koutsouris, Colin B. O’Connell, Jean-Philippe Nougayréde, Michael S. Donnenberg, Gail Hecht

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The effect of EPEC strains on intestinal barrier function in T84 monolay...
The effect of EPEC strains on intestinal barrier function in T84 monolayers. (a) The TER of polarized T84 monolayers was measured using a simplified apparatus. Monolayers were left uninfected (open circles) or infected at an moi of 500 with wild-type EPEC strain E2348/69 (open squares); espF mutant strain UMD874 (open triangles); strain UMD876, which retains espF but contains a deletion of downstream sequences (filled circles); UMD874 containing plasmid pBPM19, which has the cloned espF gene (filled squares); or UMD874 with control plasmid pACYC184 (filled triangles). TER was measured over time and expressed as a percentage change from baseline values. Data shown represent the means (± SEM) of four or five experiments containing triplicate or quadruplicate samples. The differences between monolayers infected with E2348/69 and UMD874 were significant at 2 hours, 4 hours, and 6 hours (P = 0.006, 0.02, and < 0.001, respectively; Student’s t test). There was no significant difference between monolayers infected with E2348/69 and UMD876 or between those infected with E2348/69 and UMD874 (pBPM19). There was also no significant difference between uninfected monolayers and those infected with UMD874 or UMD874 (pACYC184). The mean ± SEM TER of uninfected monolayers at baseline was 1138 ± 67 ohms·cm2. (b) TER was measured in monolayers infected for 6 hours at the indicated concentrations with wild-type EPEC strain E2348/69 (open squares) or with espF mutant strain UMD874 (open triangles) or left uninfected (open circle). Data shown represent the means (± SEM) of three or four experiments containing triplicate samples. The differences between monolayers infected with E2348/69 and UMD874 were significant at moi of 250 (P = 0.04) and 500 (P < 0.001). (c) Mannitol flux was measured as described in Methods and expressed as a percentage of that recorded in uninfected monolayers. Monolayers were infected for 6 hours with wild-type EPEC strain E2348/69 (open squares) or espF mutant strain UMD874 (open triangles). Data shown represent the means (± SEM) from two experiments containing triplicate samples. The differences between monolayers infected with E2348/69 and UMD874 were significant at moi of 500 (P = 0.02) and 1,000 (P = 0.001).