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 level of expression of recombinant EspF correlates with ability of E...
The level of expression of recombinant EspF correlates with ability of EPEC strains to induce a drop in TER in T84 monolayers. Bacteria were grown in the presence or absence of IPTG to induce EspF expression. Cultures were (a) concentrated and examined by immunoblot for EspF expression, or (b) used to infect T84 cells for 4 hours, and the change in TER was determined. Lane 1 in a and the open bar in b represent culture medium alone. Lane 2 and the filled bar represent wild-type EPEC strain E2348/69 in the absence of IPTG. Lanes 3–6 and the gray bars represent espF mutant strain UMD874 containing plasmid pBPM32 grown in the absence (lane 3) and the presence of varying concentrations of IPTG (lanes 4–6), as indicated. Data in b represent the mean (± SEM) of two experiments with triplicate samples. The difference between monolayers infected with UMD874 (pBPM32) in the absence of IPTG and those infected with UMD874 (pBPM32) in the presence of 0.1 mM IPTG was significant (P = 0.04).