ACTIVATION OF SHIGA-LIKE TOXINS BY MOUSE AND HUMAN INTESTINAL MUCUS CORRELATES WITH VIRULENCE OF ENTEROHEMORRHAGIC ESCHERICHIA-COLI O91-H21ISOLATES IN ORALLY INFECTED, STREPTOMYCIN-TREATED MICE

The enterohemorrhagic Escherichia coli (EHEC) 091:H21 isolates B2F1 an d H414-36/89 are virulent in an orally infected streptomycin-treated m ouse model. Previous studies demonstrated that B2F1 and H414-36/89 gro w to high levels in mucus isolated from the mouse small intestine and colon and that growth in small-intestinal mucus is related to virulenc e. We measured the levels of Shiga-like toxins (SLTs) SLT-IIvha and SL T-IIvhb produced by B2F1 after growth in Luria-Bertani (LB) broth supp lemented with mouse intestinal mucus by assaying the cytotoxicity of c ulture supernatants on Vero cells. Culture supernatants from B2F1 grow n in mouse intestinal mucus, but not EHEC strains that produce SLT-II or SLT-IIc, were approximately 35- to 350-fold more toxic for Vero cel ls than supernatants from B2F1 grown in LB broth. This increased toxic ity was not reflected by a concomitant increase in SLT antigen content . Furthermore, when culture supernatants from B2F1 or K-12 strains car rying plasmids encoding SLTs cloned from H414-36/89 or purified SLT-II vhb from B2F1 were incubated with mouse intestinal mucus, the samples exhibited greater cytotoxicity than when they were incubated with N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer alone. These toxin preparations also showed increased cytotoxicity after incu bation with human colonic mucus. In contrast, culture supernatants fro m LB-grown EHEC isolates that produced SLT-I, SLT-II, SLT-IIc, or SLT- IIe did not show increased cytotoxicity after incubation with mouse or human intestinal mucus. The A subunits of purified SLT-II and SLT-IIv hb that had been treated with mouse intestinal mucus or trypsin were c leaved to A(1) fragments by the mucus, but trypsin-mediated cleavage, unlike treatment with mouse intestinal mucus, did not result in increa sed Vero cell cytotoxic activity. This finding implies that the increa sed cytotoxicity of SLT-IIvhb detected after incubation with mucus is probably not due to cleavage of the A subunit into the A(1) and A(2) f ragments. Taken together, these results indicate that mouse or human i ntestinal mucus directly activates SLT-II-related toxins from B2F1 and H414-36/89 and suggest that toxin activation may explain the low 50% lethal doses of B2F1 and H414-36/89 in streptomycin-treated mice.