Activation of Shiga-like toxins by mouse and human intestinal mucus correlates with virulence of enterohemorrhagic Escherichia coli O91:H21 isolates in orally infected, streptomycin-treated mice

• Published in: Infection and Immunity 1996-05, Vol.64 (5), p.1569-1576
• Main Authors: Melton-Celsa, A.R. (Uniformed Services University of the Health Sciences, Bethesda, MD.), Darnell, S.C, O'Brien, A.D
• Format: Article
• Language: English
• Subjects: Animals; Bacterial Toxins - chemistry; Bacterial Toxins - genetics; Bacterial Toxins - metabolism; Bacteriology; Biological and medical sciences; Cercopithecus aethiops; Enterotoxins - chemistry; Enterotoxins - genetics; Enterotoxins - metabolism; ESCHERICHIA COLI; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli - pathogenicity; Escherichia coli Infections - drug therapy; Escherichia coli Infections - metabolism; ESTREPTOMICINA; Fundamental and applied biological sciences. Psychology; Humans; In Vitro Techniques; Intestines - metabolism; Male; Mice; Microbiology; Models, Biological; Mucus - metabolism; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; Shiga Toxin 1; Shiga Toxin 2; Species Specificity; Streptomycin - therapeutic use; STREPTOMYCINE; TOXINAS; TOXINE; Vero Cells; Virulence
• ISSN: 0019-9567; 1098-5522
• DOI: 10.1128/iai.64.5.1569-1576.1996

The enterohemorrhagic Escherichia coli (EHEC) O91:H21 isolates B2F1 and H414-36/89 are virulent in an orally infected streptomycin- treated mouse model. Previous studies demonstrated that B2F1 and H414-36/89 grow to high levels in mucus isolated from the mouse small intestine and colon and that growth in small-intestinal mucus is related to virulence. We measured the levels of Shiga-like toxins (SLTs) SLT-IIvha and SLT-IIvhb produced by B2F1 after growth in Luria-Bertani (LB) broth supplemented with mouse intestinal mucus by assaying the cytotoxicity of culture supernatants on Vero cells. Culture supernatants from B2F1 grown 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 cells than supernatants from B2F1 grown in LB broth. This increased toxicity was not reflected by a concomitant increase in SLT antigen content. Furthermore, when culture supernatants from B2F1 or K-12 strains carrying plasmids encoding SLTs cloned from H414-36/89 or purified SLT-IIvhb 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 incubation with human colonic mucus. In contrast, culture supernatants from LB-grown EHEC isolates that produced SLT-I, SIT-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-IIvhb that had been treated with mouse intestinal mucus or trypsin were cleaved to A1 fragments by the mucus, but trypsin-mediated cleavage, unlike treatment with mouse intestinal mucus, did not result in increased Vero cell cytotoxic activity