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. , 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. ). 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 × 10−8), from 102 to 58 Ωcm2. 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. ). These data suggest that the supernatant of strain 049766 contains an enterotoxin.
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 (more ...)
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 (more ...)
To identify the protein conferring the enterotoxic effect, 049766 precipitated supernatant preparations were enriched by a second precipitation with 1.75 M K2HPO4 (Fig. , lane B) and then were separated by DEAE-cellulose chromatography (lane D). The first peak obtained from the DEAE-cellulose column (hereafter designated peak I) produced a highly enriched fraction containing both the 108- and 116-kDa EAggEC proteins.
The peak I supernatant fraction induced increases in rat jejunal PD and Isc, and affected R, similarly to the crude 049766 precipitates. The effect of peak I proteins on Isc values was dose dependent (1.5 μg of protein induced mean increases in Isc of 2.07 μA/cm2, while 25 μg induced mean Isc rises of 13.06 μA/cm2), starting 20 min after the inoculation (Table ). In addition, the same mass of protein (25 μg) from the peak I fraction induced a greater increase of PD and Isc than the crude precipitate (0.71 mV and 14.32 μA/cm2 [peak I] versus 0.44 mV and 10.6 μA/cm2 [crude]). These data strongly suggest that the 108- and/or the 116-kDa proteins exhibit dose-dependent enterotoxic properties. Interestingly, however, the crude precipitate produced a significantly greater change in resistance (ΔR = 43.2 Ωcm2 versus 25.06 Ωcm2), suggesting that another factor(s) may also contribute to mucosal damage.
TABLE 1 Increase in PD and Isc after addition of various doses of partially purified 108- and 116-kDa EAggEC-secreted proteins in rat jejunum strips mounted in Ussingchambersa
Heat-treated peak I proteins (75°C for 15 min) lost enterotoxic activity (Table ). Preincubation with proteinase K also inhibited the effects of peak I proteins on jejunal PD and Isc (Table ). These data are consistent with the presence of a high-molecular-weight heat-labile protein enterotoxin.
TABLE 2 Jejunal PD and Isc values after addition of the 108- and 116-kDa EAggEC proteins preheated or preincubated with proteinaseKa 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. A, lane a) by Western immunoblotting, recognized either both the 108- and 116-kDa proteins (Fig. B, lane e) or only the 108-kDa species (Fig. B, lane f).
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 (more ...)
We took advantage of the immunogenicity of these proteins to identify the toxic species. Rabbits immunized with peak I proteins from strain 049766 produced antibodies against both the 108- and 116-kDa proteins (Fig. B, lane a). Monospecific polyclonal antibodies against either the 108-kDa (Fig. C, lane a) or 116-kDa (Fig. D, lane a) protein were prepared by excising the proteins from polyacrylamide gels and injecting the proteins into different rabbits. Each of these antibody preparations was then tested for the ability to inhibit the enterotoxicity of fractionated EAggEC supernatants in the Ussing chamber. As expected, anti-peak I antibodies neutralized PD, Isc, and R changes in Ussing chambers (Fig. ). No rises in Isc or decreases in R were detected (Fig. A and C). Preincubation of the peak I fraction with monospecific antibodies against the 108-kDa but not against the 116-kDa protein neutralized the effects of the preparation on jejunal PD and Isc (Fig. ). These data suggest that the 108-kDa protein is the enterotoxic species found in the peak I fraction.
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 (more ...)
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 (more ...)
We used our 108- and 116-kDa protein-specific polyclonal antibodies to screen our collection for strains that might express only the 108- or 116-kDa protein to further support our hypothesis that the 108-kDa protein was the active species. By Western immunoblotting strain 065126 was found to express the 116-kDa (Fig. D, lane b) but not the 108-kDa (Fig. C, lane b) protein. As predicted, the >100-kDa fraction of 065126 (Fig. A, lane b) did not induce changes in jejunal PD and Isc (Fig. ) and was not significantly different from the preparation treated with LB medium (P = 0.1).
Localization of the gene encoding the 108-kDa enterotoxin.
Genetic analyses in our laboratories has focused on EAggEC strain 042 (7
). 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. A, lane c) and by immunoblotting with antibodies against 108- and 116-kDa proteins (Fig. B, lane c). As expected from previous experiments, these concentrated supernatants also induced increases of PD and Isc (Fig. ); 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. ). 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. A, lane d) and by Western blotting (Fig. B and C, lanes d). The 116-kDa protein was not encoded by pJPN201 (Fig. D, lane d). Again, as expected, concentrated fractionated supernatant of HB101(pJPN201) induced rises in jejunal PD and Isc in Ussing chambers (Fig. ); the rises were neutralized by anti-108-kDa protein antibodies. These data confirm that the 108-kDa protein is indeed an enterotoxin.
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. A). However, specimens treated with the 108-kDa toxin derived either from 049766 or from 042 [in HB101(pJPN201)] demonstrated identical histopathologic abnormalities (Fig. B). 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.
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 (more ...)