In vitro effects of a high-molecular-weight heat-labile enterotoxin from enteroaggregative Escherichia coli

Abstract

The pathogenic mechanisms of enteroaggregative Escherichia coli (EAggEC) infection are not fully elucidated. In this work we show that an ammonium sulfate precipitate of culture supernatant of EAggEC strain 049766 increased the potential difference (PD) and the short-circuit current (Isc) in rat jejunal preparations mounted in Ussing chambers. The precipitate contained two major proteins of 108 and 116 kDa, which were partially copurified by chromatography in DEAE-cellulose. This chromatographic fraction (peak I) increased jejunal PD and Isc in a dose-dependent manner, accompanied by a decrease in tissue electrical resistance. These effects were inhibited by incubation of peak I at 75 degreesC for 15 min or for 1 h with proteinase K at 37 degreesC. Rabbit polyclonal antibodies against peak I containing both the 108- and 116-kDa proteins inhibited the enterotoxic effect. Specific polyclonal antibodies raised against the 108-kDa but not against the 116-kDa protein inhibited the enterotoxic effect, suggesting that the 108-kDa protein is the active toxic species. Moreover, another EAggEC strain (065126) producing the 116-kDa protein but not the 108-kDa protein had no effect on rat jejunal mucosa in the Ussing chamber. The >100-kDa fraction derived from prototype EAggEC strain 042, which also expressed both 108- and 116-kDa proteins, also produced an enterotoxic effect on rat jejunal preparations in Ussing chambers; however, the same strain cured of its 65-MDa adherence plasmid did not. A subclone derived from the 65-MDa plasmid expressing the 108-kDa toxin (and not the 116-kDa protein) elicited rises in Isc. Tissue exposed to any preparation containing the 108-kDa toxin exhibited similar histopathologic changes, characterized by increased mucus release, exfoliation of cells, and development of crypt abscesses. Our data suggest that some EAggEC strains produce a ca. 108-kDa enterotoxin/cytotoxin which is encoded on the large virulence plasmid.

FIG. 1

SDS-PAGE characterization of protein fractions (40 μg per lane) from EAggEC strain 049766. E. coli K-12 was used as a control. The crude 60% (NH₄)₂SO₄-precipitated supernatant (lane C) of 049766 produced several proteins, including those at 108 and 116 kDa. The high-molecular-weight fraction was partially purified by reprecipitation with 1.75 M K₂HPO₄ (lane B) and chromatography through DEAE-cellulose (peak I, lane D). Concentrated supernatant of E. coli K-12 (lane A) did not reveal secreted proteins in the range of 108 to 116 kDa.

FIG. 2

Time course of PD, Isc, and R values of rat jejunum preparations exposed to 60% (NH₄)₂SO₄-precipitated supernatants from cultures of EAggEC strain 049766 or E. coli K-12. Twenty-five micrograms of protein was used from concentrated supernatants or 25 μl of uninoculated LB. The symbols represent the mean values of experiments performed on four different animals.

FIG. 3

SDS-PAGE (A) and Western immunoblotting (B to D) of >100-kDa fractions from supernatants of strains 049766 (lanes a), 065126 (lanes b), 042 (lanes c), and HB101(pJPN201) (lanes d). In panel B, Western blots in lanes a to d are reacted with anti-peak I antibodies, and those in lanes e and f are reacted with antibodies from two different patients in the 049766 outbreak. Blots in panel C are reacted with anti-108-kDa protein antibodies, and those in panel D are reacted with anti-116-kDa protein antibodies. Lower-molecular-weight bands in all lanes most likely represent breakdown products of the high-molecular-weight species, since they are generally absent in blots lacking reactivity in the region from 108 to 116 kDa. MW, molecular weight markers.

FIG. 4

Inhibition of enterotoxicity by antibodies against the peak I fraction. Twenty-five-microgram aliquots of peak I proteins were preincubated for 20 min with rabbit serum directed against the identical fraction and then added to the mucosal hemichambers of rat jejunum preparations (n = 4).

FIG. 5

Inhibition experiments in Ussing chambers with antibodies against either 108- or 116-kDa protein. Bars 1 to 3, PD and Isc increments induced by the peak I fraction of strain 049766 alone or after preincubation with monospecific antibodies against either the 108- or 116-kDa protein (n = 7). Bar 4 represents the rises induced by strain 065126, which lacks the 108-kDa species.

FIG. 6

Enterotoxic activity of >100-kDa fractionated supernatants containing the 108-kDa protein. One-hundred micrograms of concentrated supernatant protein was added to the mucosal hemichambers of rat jejunum preparations (n = 4) (see text).

FIG. 7

Morphologic effects of 108-kDa protein on rat jejunal mucosa. The rat jejunal preparations were removed from Ussing chambers, fixed with 4% formalin, and embedded in paraffin. The sections were stained with hematoxylin and eosin. (A) Untreated control preparation. (B) Preparation treated with 108-kDa protein from HB101(pJPN201). Note the mucus blanket with cell debris on the luminal side (asterisk), damage of the epithelial layer (arrowhead), and crypt abscesses (arrow) in the treated section.