Involvement of the enteroaggregative Escherichia coli plasmid-encoded toxin in causing human intestinal damage

Abstract

Enteroaggregative Escherichia coli (EAEC) strains have been shown to adhere to human intestinal tissue in an in vitro organ culture (IVOC) model, and certain strains manifest mucosal toxicity. We have recently described the EAEC plasmid-encoded toxin (Pet), a member of a specific serine protease subclass of the autotransporter proteins. When injected into rat ileal loops, Pet both elicited fluid accumulation and had cytotoxic effects on the mucosa. Furthermore, the Pet protein caused rises in short circuit current from rat jejunal tissue mounted in a Ussing chamber and rounding of intestinal epithelial cells in culture. We therefore hypothesized that the mucosal pathology induced by EAEC strains in the IVOC model was related to expression of the Pet protein. Here, we have examined the effects of EAEC strain 042 and its isogenic pet mutant in the IVOC model. 042-infected colonic explants exhibited dilation of crypt openings, increased cell rounding, development of prominent intercrypt crevices, and absence of apical mucus plugs. Colonic tissue incubated with the pet mutant exhibited significantly fewer mucosal abnormalities both subjectively and as quantitated morphometrically by measurement of crypt aperture diameter. Mucosal effects were restored upon complementation of the pet mutation in trans. Interestingly, we found that the ability of 042 to damage T84 cells was not dependent upon Pet. The data suggest that the Pet toxin is active on the human intestinal mucosa but that EAEC may have other mechanisms of eliciting mucosal damage.

FIG. 1

SDS-PAGE analysis of pet mutants. Concentrated culture supernatants from strains 042 (lane 1), JIF1 (lane 2), and JIF2 (lane 3), which have the presence, absence, and complemented expression of Pet, respectively, are shown. The positions of Pet and the 116-kDa secreted protein are indicated.

FIG. 2

Scanning electron photomicrographs of in vitro-cultured human colonic tissues infected with the EAEC strain 042 (A), the pet mutant strain JIF1 (B), and the complemented strain JIF2 (C) for 8 h, and of uninoculated colonic tissue (D). All of these tissue sections derived from the same intestinal biopsy specimen. For the tissues shown in panels A and C the surface of the colon is markedly abnormal, as manifested by increased crypt apertures (white arrowheads), prominent mucosal crevices (white arrow), goblet cell pitting (black arrowhead), and rounding of epithelial cells (black arrow). Bars, 50 μm.

FIG. 3

Scattergram illustrating crypt aperture diameters induced by 042 and mutant strains in the IVOC model. An average of 19 openings (range, 11 to 31 openings) were measured per sample, in blinded fashion. Means per sample are illustrated by separate data points, and overall means are indicated by the horizontal bars. P values are noted in the text. Cont, control.

FIG. 4

Transmission electron photomicrographs of T84 cell monolayers infected for 6 h. (A) EAEC strain 042. The appearance of the cells is comparable to that in images previously published by Nataro et al. (25); i.e., the monolayer demonstrates adherent bacteria, loss of microvilli, ballooning of the apical cytoplasm, and subnuclear vacuolization. (B) The pet mutant strain JIF1. The effects induced by the pet mutant strain are similar to those observed for the wild-type strain (042). (C) Pet-treated T84 cells. The appearance of the cells is similar to that of uninfected T84 cells.

FIG. 4

Transmission electron photomicrographs of T84 cell monolayers infected for 6 h. (A) EAEC strain 042. The appearance of the cells is comparable to that in images previously published by Nataro et al. (25); i.e., the monolayer demonstrates adherent bacteria, loss of microvilli, ballooning of the apical cytoplasm, and subnuclear vacuolization. (B) The pet mutant strain JIF1. The effects induced by the pet mutant strain are similar to those observed for the wild-type strain (042). (C) Pet-treated T84 cells. The appearance of the cells is similar to that of uninfected T84 cells.

FIG. 5

Enterotoxic activity of concentrated culture supernatants derived from 042 and the pet mutant strain JIF1. Supernatants from overnight cultures were size fractionated (saving the fraction with mass >50 kDa), and equivalent loadings were added to each Ussing chamber, into which had been mounted full-thickness rat jejunal tissue. Data points are the means of at least three experiments; error bars represent standard errors of the means. The supernatants from 042 and JIF1 generated significant rises in PD and I_sc compared with those of negative controls (P < 0.05, Student’s t test).