Shiga Toxin–Producing \u3ci\u3eEscherichia coli\u3c/i\u3e in Montana: Bacterial Genotypes and Clinical Profiles

The diseases and virulence genes associated with Shiga toxin–producing Escherichia coli (STEC) are characterized incompletely. We analyzed, by polymerase chain reaction, 82 STEC isolates collected prospectively in Montana and profiled associated illnesses by patient chart review. All E. coli O157:H7 contained stx2-group genes, as well as eae, iha, espA, and ehxA; 84% contained stx1. Non-O157:H7 STEC less frequently contained stx1( P = .046 ), stx2 (P \u3c .001), iha (P \u3c .001), eae, and espA (P = .039 for both), were isolated less often from patients treated in emergency departments (P = .022), and tended to be associated less frequently with bloody diarrhea (P = .061). There were no significant associations between stx genotype and bloody diarrhea, but isolates containing stx2c or stx2d-activatable were recovered more often from patients who underwent diagnostic or therapeutic procedures (P = .033). Non-O157:H7 STEC are more heterogeneous and cause bloody diarrhea less frequently than do E. coli O157:H7. Bloody diarrhea cannot be attributed simply to the stx genotype of the infecting organism

A novel murine infection model for Shiga toxin-producing Escherichia coli

Enterohemorrhagic E. coli (EHEC) is an important subset of Shiga toxin-producing (Stx-producing) E. coli (STEC), pathogens that have been implicated in outbreaks of food-borne illness and can cause intestinal and systemic disease, including severe renal damage. Upon attachment to intestinal epithelium, EHEC generates attaching and effacing (AE) lesions characterized by intimate attachment and actin rearrangement upon host cell binding. Stx produced in the gut transverses the intestinal epithelium, causing vascular damage that leads to systemic disease. Models of EHEC infection in conventional mice do not manifest key features of disease, such as AE lesions, intestinal damage, and systemic illness. In order to develop an infection model that better reflects the pathogenesis of this subset of STEC, we constructed an Stx-producing strain of Citrobacter rodentium, a murine AE pathogen that otherwise lacks Stx. Mice infected with Stx-producing C. rodentium developed AE lesions on the intestinal epithelium and Stx-dependent intestinal inflammatory damage. Further, the mice experienced lethal infection characterized by histopathological and functional kidney damage. The development of a murine model that encompasses AE lesion formation and Stx-mediated tissue damage will provide a new platform upon which to identify EHEC alterations of host epithelium that contribute to systemic disease

Shiga toxin (Stx) type 2‐induced increase in O‐linked N‐acetyl glucosamine protein modification: a new therapeutic target?

Shiga toxin (Stx)‐producing Escherichia coli (STEC) causes bloody diarrhea, which may progress to the potentially fatal hemolytic uremic syndrome (HUS). Development of HUS after STEC infection is dependent on Stx, and is particularly linked to Stx type 2a, Stx2a (Melton‐Celsa, 2014; Scheutz, 2014). In this issue of EMBO Molecular Medicine, Lee et al report that O‐linked N‐acetyl glucosamine protein modification (O‐GlcNAcylation) is increased in host cells after Stx exposure and the subsequent endoplasmic reticulum (ER) stress response. The elevated O‐GlcNAcylation resulted in elevated inflammatory and apoptotic processes. Inhibition of O‐GlcNAcylation with OSMI‐1 protected cells from the Stx2a‐induced damage. In mice intoxicated with Stx2a, OSMI‐1 treatment reduced kidney damage and increased mouse survival

Activation of Shiga-Like Toxins by Mouse and Human Intestinal Mucus Correlates with Virulence of Enterohemorrhagic \u3ci\u3eEscherichia coli\u3c/i\u3e O91:H21 Isolates in Orally Infected, Streptomycin-Treated Mice

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 smallintestinal 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\u27-2-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, 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-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. This finding implies that the increased cytotoxicity of SLT-IIvhb detected after incubation with mucus is probably not due to cleavage of the A subunit into the A1 and A2 fragments. Taken together, these results indicate that mouse or human intestinal 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

Mutations in hns reduce the adherence of Shiga toxin-producing E. coli 091:H21 strain B2F1 to human colonic epithelial cells and increase the production of hemolysin

Shiga toxin-producing Escherichia coli (STEC) 091:H21 strain B2F1, an isolate from a patient with the hemolytic uremic syndrome (HUS), produces elastase-activatable Shiga toxin (Stx) type 2d and adheres well to human colonic epithelial T84 cells. This adherence phenotype occurs even though B2F1 does not contain the locus of enterocyte effacement (LEE) that encodes the primary adhesin for E. coli O157:H7. To attempt to identify genes involved in binding of B2F1 to T84 cells a bank of mini-Tn5phoACmr transposon mutants of this strain was generated. Several of these mutants exhibited a reduced adherence phenotype, but none of the insertions in these mutants were within putative adhesin genes. Rather, insertional mutations within hns resulted in the loss of adherence. Moreover, the hns mutant also displayed an increase in the production of hemolysin and alkaline phosphatase and a loss of motility with no change in Stx2d-activatable expression levels. When B2F1 was cured of the large plasmid that encodes the hemolysin, the resulting strain adhered well to T84 cells. However, an hns mutant of the plasmid-cured B2F1 strain exhibited a reduction in adherence to T84 cells. Taken together, these results indicate that H-NS regulates the expression of several genes and some potential virulence factors in the intimin-negative B2F1 STEC strain and that the large plasmid is not required for T84 cell colonization

Shiga Toxin Type 2dact Displays Increased Binding to Globotriaosylceramide in vitro and Increased Lethality in Mice after Activation by Elastase

Shiga toxin type 2dact (Stx2dact), an Stx2 variant originally identified from Escherichia coli O91:H21 strain B2F1, displays increased cytotoxicity after activation by elastase present in intestinal mucus. Activation is a result of cleavage of two amino acids from the C-terminal tail of the A2 subunit. In this study, we hypothesized that activation leads to increased binding of toxin to its receptor on host cells both in vitro and in vivo. To test this theory, Stx2dact was treated with elastase or buffer alone and then each toxin was assessed for binding to purified globotriaosylceramide (Gb3) in an enzyme-linked immunosorbent assay, or cells in culture by immunofluorescence, or flow cytometry. Elastase- and buffer-treated Stx2dact were also evaluated for binding to mouse kidney tissue and for relative lethality in mice. We found that activated Stx2dact had a greater capacity to bind purified Gb3, cells in culture, and mouse kidney tissue and was more toxic for mice than was non-activated Stx2dact. Thus, one possible mechanism for the augmented cytotoxicity of Stx2dact after activation is its increased capacity to bind target cells, which, in turn, may cause greater lethality of elastase-treated toxin for mice and enhanced virulence for humans of E. coli strains that express Stx2dact

Enterohemorrhagic\u3ci\u3eEscherichia coli\u3c/i\u3e O157:H7 Requires Intimin To Colonize the Gnotobiotic Pig Intestine and To Adhere to HEp-2 Cells

In a previous study, enterohemorrhagic Escherichia coli (EHEC) O157:H7 with a deletion and insertion in the eaeA gene encoding intimin was used to establish that intimin is required for the organism to attach to and efface microvilli in the piglet intestine (M. S. Donnenberg, S. Tzipori, M. L. McKee, A. D. O’Brien, J. Alroy, and J. B. Kaper, J. Clin. Invest. 92:1418–1424, 1993). However, in the same investigation, a role for intimin in EHEC adherence to HEp-2 cells could not be definitively demonstrated. To analyze the basis for this discrepancy, we constructed an in-frame deletion of eaeA and compared the adherence capacity of this mutant with that of the wild-type strain in vitro and in vivo. We observed a direct correlation between the requisite for intimin in EHEC O157:H7 colonization of the gnotobiotic piglet intestine and adherence of the bacterium to HEp-2 cells. The in vitro-in vivo correlation lends credence to the use of the HEp-2 cell adherence model for further study of the intimin protein

Comparative Pathogenicity of \u3ci\u3eEscherichia coli\u3c/i\u3e O157 and Intimin-Negative Non-O157 Shiga Toxin-Producing \u3ci\u3eE. coli\u3c/i\u3e Strains in Neonatal Pigs

We compared the pathogenicity of intimin-negative non-O157:H7 Shiga toxin (Stx)-producing Escherichia coli (STEC) O91:H21 and O104:H21 strains with the pathogenicity of intimin-positive O157:H7 and O157:H- strains in neonatal pigs. We also examined the role of Stx2d-activatable genes and the large hemolysinencoding plasmid of O91:H21 strain B2F1 in the pathogenesis of STEC disease in pigs. We found that all E. coli strains that made wild-type levels of Stx caused systemic illness and histological lesions in the brain and intestinal crypts, whereas none of the control Stx-negative E. coli strains evoked comparable central nervous system signs or intestinal lesions. By contrast, the absence of intimin, hemolysin, or motility had little impact on the overall pathogenesis of systemic disease during STEC infection. The most striking differences between pigs inoculated with non-O157 STEC strains and pigs inoculated with O157 STEC strains were the absence of attaching and effacing intestinal lesions in pigs inoculated with non-O157:H7 strains and the apparent association between the level of Stx2d-activatable toxin produced by an STEC strain and the severity of lesions

One of Two Copies of the Gene for the Activatable Shiga Toxin Type 2d in Escherichia coli O91:H21 Strain B2F1 Is Associated with an Inducible Bacteriophage

Shiga toxin (Stx) types 1 and 2 are encoded within intact or defective temperate bacteriophages in Stx-producing Escherichia coli (STEC), and expression of these toxins is linked to bacteriophage induction. Among Stx2 variants, only stx(2e) from one human STEC isolate has been reported to be carried within a toxin-converting phage. In this study, we examined the O91:H21 STEC isolate B2F1, which carries two functional alleles for the potent activatable Stx2 variant toxin, Stx2d, for the presence of Stx2d-converting bacteriophages. We first constructed mutants of B2F1 that produced one or the other Stx2d toxin and found that the mutant that produced only Stx2d1 made less toxin than the Stx2d2-producing mutant. Consistent with that result, the Stx2d1-producing mutant was attenuated in a streptomycin-treated mouse model of STEC infection. When the mutants were treated with mitomycin C to promote bacteriophage induction, Vero cell cytotoxicity was elevated only in extracts of the Stx2d1-producing mutant. Additionally, when mice were treated with ciprofloxacin, an antibiotic that induces the O157:H7 Stx2-converting phage, the animals were more susceptible to the Stx2d1-producing mutant. Moreover, an stx(2d1)-containing lysogen was isolated from plaques on strain DH5α that had been exposed to lysates of the mutant that produced Stx2d1 only, and supernatants from that lysogen transformed with a plasmid encoding RecA were cytotoxic when the lysogen was induced with mitomycin C. Finally, electron-microscopic examination of extracts from the Stx2d1-producing mutant showed hexagonal particles that resemble the prototypic Stx2-converting phage 933W. Together these observations provide strong evidence that expression of Stx2d1 is bacteriophage associated. We conclude that despite the sequence similarity of the stx(2d1)- and stx(2d2)-flanking regions in B2F1, Stx2d1 expression is repressed within the context of its toxin-converting phage while Stx2d2 expression is independent of phage induction