INTRODUCTION

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Enteroaggregative Escherichia coli (EAggEC) has been associated with persistent diarrhea in young children (3, 5, 15, 25), especially in developing countries. Most EAggEC strains harbor a 65-MDa plasmid (called pAA), which is required for expression of aggregative adherence fimbriae (AAFs). These structures mediate the defining aggregative adherence (AA) phenotype to HEp-2 cells (14, 15, 21) as well as adherence to the colonic mucosa (7). The pAA plasmid is also required for the development of mucosal damage in in vitro models (10, 16).

Clinical, volunteer, and animal model studies suggest that EAggEC diarrhea may be due to a secretogenic enterotoxin (3, 13, 25). When tested in Ussing chambers, filtrates from EAggEC strain 17-2 produced an increase in potential difference (PD) and short-circuit current (Isc), attributed by Savarino et al. (20) to a heat-stable, plasmid-encoded enterotoxin of less than 10 kDa in molecular mass (EAST1). No data yet exist to support a role for EAST1 in EAggEC diarrhea.

Nataro et al. (13) reported that EAggEC strain 042 (serotype O44:H18) caused significant diarrhea in three of five adult volunteers, whereas strains 17-2, 34b, and JM221 (EAggEC of serotypes O3:H2, O?:H?, and O92:H33, respectively) did not induce enteric symptoms. Except for 042, each of these strains expressed AAF/I fimbriae, while EAST1 was produced by strains 042 and 17-2 but not by strain 34b or JM221 (13). Mathewson et al. (12) had previously reported that strain JM221 caused mild diarrhea in some adult volunteers, and Tzipori et al. (23) had shown that strains 17-2 and JM221 were able to cause diarrhea in gnotobiotic piglets. The basis for this strain heterogeneity has not been determined, and these data suggest the presence of unrecognized virulence factors.

A role for cytotoxins in EAggEC disease has been suggested by in vivo and in vitro models (10, 16, 24), which exhibit damage to intestinal epithelium. A candidate cytotoxin has been identified by Eslava et al. (8), as sera from children in a Mexican EAggEC outbreak consistently recognized two proteins of 108 and 116 kDa obtained from ammonium sulfate precipitates of EAggEC culture supernatants. Together these proteins elicited hemorrhagic and necrotic lesions in rat ileal loops (8). In this communication, we report that a fraction containing the 108- and 116-kDa proteins purified from EAggEC 049766, the strain implicated in the Mexican outbreak, is able to cause enterotoxic and cytotoxic effects on rat jejunal tissue mounted in Ussing chambers. We have identified the toxin moiety as the 108-kDa protein and have localized it to the pAA plasmid.

	MATERIALS AND METHODS

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Bacterial strains. EAggEC strain 049766, implicated in an outbreak of persistent diarrhea in Mexican infants, has been characterized as belonging to serotype O?:H10 and is capable of attaching with an aggregative pattern to HEp-2 cells (6, 15). EAggEC strain 065126 was isolated from a Mexican child with diarrhea. Strain 042 was isolated from a child with diarrhea in Lima, Peru, in 1983; this strain has been shown to cause diarrhea in adult volunteers (13). Strains 049766, 065126, and 042 hybridize with the EAggEC-specific (AA) probe (2). Plasmid-cured 042 has been described previously (16). E. coli strain K-12 was used as a control in Ussing chamber experiments; E. coli HB101 was used as a host for cloning experiments.

Preparation of protein fractions. Culture conditions and preparation of precipitates were performed as follows. EAggEC strain 049766 was grown overnight at 37°C in 200 ml of Luria broth (LB). After centrifugation at 12,000 × g for 10 min, supernatants were precipitated with 60% saturated ammonium sulfate for 18 h at 4°C, collected by centrifugation, dissolved with 0.07 M potassium phosphate buffer (pH 8.2), and dialyzed for 4 days against the same buffer. Protein concentration was determined by the method of Bradford (4). Purification of EAggEC-secreted proteins was obtained by further precipitation of protein suspensions in 0.07 M potassium phosphate buffer with 1.75 M K₂HPO₄, dialyzed at 4°C against Tris-EDTA buffer (0.05 M Tris-0.1 M EDTA [pH 8.0]) and eluted with the same buffer from a DEAE-cellulose column.

Protein electrophoresis and immunologic methods. Proteins present in precipitated culture supernatants from the DEAE chromatography fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by the method of Laemmli (11) under reducing conditions (boiling for 5 min in the presence of mercaptoethanol). Proteins separated by SDS-PAGE were transferred to nitrocellulose BA85 membranes (Schleicher & Schuell, Keene, N.H.) by the method of Towbin et al. (22). The membranes were incubated with rabbit antibodies generated in our laboratory against the 108- and 116-kDa proteins (diluted 1:100) or with sera from children in the Mexican EAggEC outbreak (diluted 1:25). Immunostaining was performed with peroxidase-labeled polyclonal antibodies against rabbit immunoglobulins (dilution 1:5,000) and developed with 4-chloronaphthol by standard methods (22). The antibodies against each of the 108- and 116-kDa proteins were elicited by excising proteins from polyacrylamide gels and injecting the gel slices subcutaneously into rabbits in two doses, 2 weeks apart. The antibody responses and specificity were determined by immunoblotting. The gamma fractions from the antisera were diluted 1:25 prior to use in Ussing chamber experiments.

Electrophysiologic measurements in rat jejunum. Ussing chamber experiments in Mexico City were performed as described previously (18, 19). Jejunal segments removed from adult male Sprague-Dawley rats under sodium pentobarbital anesthesia were placed in ice-cold Ringer's solution for mammals and gassed with an O₂-CO₂ (95%:5%) mixture. The excised segments were cut open along the mesenteric border, washed with cold Ringer's solution, divided into two fragments (experimental and control), and mounted between the circular openings (6-mm diameter, 0.28 cm²) of two adjacent Ussing hemichambers. Each hemichamber was filled with 10 ml of Ringer's solution and kept at 37°C under constant O₂-CO₂ bubbling. Transmural PD and Isc were recorded at 1-min intervals by means of a voltage clamp apparatus. Samples containing 1.5 to 25 µg of precipitate per ml were diluted with Ringer's solution at 37°C and added to the mucosal hemichamber of rat jejunum preparations after 10 min of equilibration, and both hemichambers were gassed with O₂-CO₂. Transmural resistance (R) values were obtained from PD and Isc values by using Ohm's law. Statistical analyses were performed with Student's t test on data recorded from at least four experiments.

	RESULTS

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Ussing chamber effects of 108- and 116-kDa supernatant proteins. Precipitation of EAggEC 049766 culture supernatants using 60% saturated (NH₄)SO₄ yielded several proteins, most prominent of which were 108- and 116-kDa species (Fig. 1, lane C) that were absent from precipitates of culture supernatants of an E. coli K-12 strain (lane A). Addition of the precipitates obtained from the 60% saturated (NH₄)₂SO₄ supernatant of 049766 (lane C) to the mucosal hemichamber of rat jejunum strips mounted in Ussing chambers evoked a significant increase of PD and Isc while producing a decrease of R (Fig. 2). Rises in PD and Isc began approximately 20 min after addition of culture supernatants. PD and Isc rose from 0.5 to 1.04 mV and from 5.1 to 16.7 µA/cm², respectively, while the R values decreased about 50% (P = 2.8 × 10⁸), from 102 to 58 cm². Maximum increases were attained approximately 95 min after inoculation. Precipitates from culture supernatants of an E. coli K-12 and from uninoculated broth had no effect on jejunal preparations from the same animals (Fig. 2). These data suggest that the supernatant of strain 049766 contains an enterotoxin.

View larger version (30K): [in this window] [in a new window]   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.

View larger version (27K): [in this window] [in a new window]   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.

Association of enterotoxic activity with the 108-kDa protein. The 108- and 116-kDa proteins were found to be immunogenic. Serum samples from children with diarrhea due to strain 049766 in the Mexican outbreak, reacted against the supernatant of the same strain (Fig. 3A, lane a) by Western immunoblotting, recognized either both the 108- and 116-kDa proteins (Fig. 3B, lane e) or only the 108-kDa species (Fig. 3B, lane f).

View larger version (33K): [in this window] [in a new window]   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.

View larger version (22K): [in this window] [in a new window]   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).

View larger version (24K): [in this window] [in a new window]   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.

Localization of the gene encoding the 108-kDa enterotoxin. Genetic analyses in our laboratories has focused on EAggEC strain 042 (7, 24). We decided to use these data to localize the 108-kDa toxin and to substantiate its enterotoxic effects. Concentrated supernatants of strain 042 were found to contain the 108- and 116-kDa proteins, detected by SDS-PAGE (Fig. 3A, lane c) and by immunoblotting with antibodies against 108- and 116-kDa proteins (Fig. 3B, lane c). As expected from previous experiments, these concentrated supernatants also induced increases of PD and Isc (Fig. 6); however, strain 042 cured of its 65-MDa virulence plasmid (pAA2) was found to be lacking the 108-kDa protein, and the fractionated supernatant of plasmid-cured 042 had no effect on jejunal preparations mounted in the Ussing chamber (Fig. 6). We next tested a series of clones derived from plasmid pAA2 and found that HB101(pJPN201), harboring a 13-kb insert which flanks the previously described AAF/II genes (7), expressed the 108-kDa protein by SDS-PAGE (Fig. 3A, lane d) and by Western blotting (Fig. 3B and C, lanes d). The 116-kDa protein was not encoded by pJPN201 (Fig. 3D, lane d). Again, as expected, concentrated fractionated supernatant of HB101(pJPN201) induced rises in jejunal PD and Isc in Ussing chambers (Fig. 6); the rises were neutralized by anti-108-kDa protein antibodies. These data confirm that the 108-kDa protein is indeed an enterotoxin.

View larger version (20K): [in this window] [in a new window]   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).

Histopathologic examination of rat mucosal tissue. Since the 108-kDa protein induced a decrease in electrical resistance, histopathologic analysis of full-thickness rat jejunal tissue was performed by light microscopy after Ussing chamber experiments. Control-treated rat jejunal sections appeared normal, with intact mucosa and minimal mucus secretion (Fig. 7A). However, specimens treated with the 108-kDa toxin derived either from 049766 or from 042 [in HB101(pJPN201)] demonstrated identical histopathologic abnormalities (Fig. 7B). The mucosal surface of toxin-treated specimens was covered with a thick mucus blanket. The epithelial layer demonstrated coagulation necrosis, with exfoliation of epithelial cells and occasional karyorrhexis of nuclei. Beneath the epithelium were observed increased numbers of mononuclear cells, and eosinophils and multifocal crypt abscesses were observed in several specimens. The submucosa exhibited edema and widening of the lymphatic channels.

View larger version (115K): [in this window] [in a new window]   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.

	DISCUSSION

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EAggEC is an emerging agent of pediatric diarrhea. Clinically, the disease presents as watery diarrhea, but the responsible enterotoxin has not yet been identified with certainty. Data do not yet exist to support a role for the ST-like toxin EAST1 in EAggEC diarrhea (21), and a 120-kDa protein that cross-reacted with hemolysin antibodies (1) has not been shown to have enterotoxic properties. Here we present data suggesting that a 108-kDa protein secreted by EAggEC strains is a heat-labile enterotoxin. This protein is recognized by sera from patients in an outbreak of EAggEC diarrhea.

The following data suggest that the 108-kDa protein is an enterotoxin: (i) fractions containing both the 108-kDa protein and a distinct 116-kDa protein produce rises in Isc, whereas a fraction from a strain producing only the 116-kDa protein does not; (ii) polyclonal antibodies raised against the 108-kDa protein abolish enterotoxic activity in a dose-related fashion, whereas anti-116-kDa protein antibodies have no effect; (iii) a 108-kDa protein-encoding subclone from the pAA plasmid induces increases in Isc; a pAA-cured EAggEC strain does not.

The 108-kDa toxin appears to induce not only enterotoxic effects but also tissue damage, inflammation, and mucus secretion; these effects correlated with a fall in R value. These data are consistent with other reports that EAggEC strains elaborate one or more cytotoxins and induce damage to the intestinal mucosa (10, 16, 24).

Thus, our data indicate that at least some EAggEC strains secrete a high-molecular-mass (ca. 108-kDa) protein which is encoded on the pAA virulence plasmid and has enterotoxic and perhaps cytotoxic activity on intestinal preparations. The enterotoxic effects were characterized by an increase of Isc and PD and a decrease in R, indicating induction of a net secretory state and damage to epithelial cells and/or their cellular junctions. This enterotoxin is immunogenic, as antibodies against the 108-kDa protein can be found in sera from children with EAggEC infection. The 108-kDa enterotoxin could play an important role in the diarrhea produced by EAggEC.

Our data allow us to hypothesize a model of EAggEC infection in which initial adherence is mediated by AAF fimbriae, followed by the induction of a net secretory state induced by the 108-kDa enterotoxin and also perhaps EAST1. This may be followed by the development of cytotoxicity on the mucosa also induced by the 108-kDa toxin or by an as yet unidentified factor. Studies to identify these factors are ongoing.