Angela R. Melton-Celsa

Multicenter Evaluation of a Sequence-Based Protocol for Subtyping Shiga Toxins and Standardizing Stx Nomenclature

ABSTRACT When Shiga toxin-producing Escherichia coli (STEC) strains emerged as agents of human disease, two types of toxin were identified: Shiga toxin type 1 (Stx1) (almost identical to Shiga toxin produced by Shigella dysenteriae type 1) and the immunologically distinct type 2 (Stx2). Subsequently, numerous STEC strains have been characterized that express toxins with variations in amino acid sequence, some of which confer unique biological properties. These variants were grouped within the Stx1 or Stx2 type and often assigned names to indicate that they were not identical in sequence or phenotype to the main Stx1 or Stx2 type. A lack of specificity or consistency in toxin nomenclature has led to much confusion in the characterization of STEC strains. Because serious outcomes of infection have been attributed to certain Stx subtypes and less so with others, we sought to better define the toxin subtypes within the main Stx1 and Stx2 types. We compared the levels of relatedness of 285 valid sequence variants of Stx1 and Stx2 and identified common sequences characteristic of each of three Stx/Stx1 and seven Stx2 subtypes. A novel, simple PCR subtyping method was developed, independently tested on a battery of 48 prototypic STEC strains, and improved at six clinical and research centers to test the reproducibility, sensitivity, and specificity of the PCR. Using a consistent schema for nomenclature of the Stx toxins and stx genes by phylogenetic sequence-based relatedness of the holotoxin proteins, we developed a typing approach that should obviate the need to bioassay each newly described toxin and that predicts important biological characteristics.

Shiga Toxin–Producing Escherichia coli 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<.001), iha (P<.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 stx(2d-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.

Pathogenesis of Shiga-Toxin Producing Escherichia coli

Shiga toxin (Stx)-producing Escherichia coli (STEC) are food-borne pathogens that cause hemorrhagic colitis and a serious sequela, the hemolytic uremic syndrome (HUS). The largest outbreaks of STEC are due to a single E. coli serotype, O157:H7, although non-O157 serotypes also cause the same diseases. Two immunologically distinct Stxs are found in E. coli, Stx1 and Stx2. The Stxs are AB₅ toxins that halt protein synthesis in the host cell, a process that may lead to an apoptotic cell death. Stx-mediated damage to renal glomerular endothelial cells is hypothesized as the precipitating event for HUS. A subset of STEC referred to as the enterohemorrhagic E. coli has the capacity to intimately attach to and efface intestinal epithelial cells, a pathology called the A/E lesion. The A/E lesion is mediated by the adhesin intimin, its bacterially encoded receptor, Tir, and effectors secreted through a type III secretion system. The proteins needed for the A/E lesion are encoded within a large pathogenicity island called the locus of enterocyte effacement or LEE. There are several animal models for STEC infection, but no one model fully represents the spectrum of STEC illness. Currently there is no cure for STEC infection, and therapies are based mainly on alleviating symptoms. However, chimeric or humanized monoclonal antibodies have been developed that neutralize the Stxs, and those therapies may be able to prevent the development of HUS in an STEC-infected patient.

Mouse Model of Hemolytic-Uremic Syndrome Caused by Endotoxin-Free Shiga Toxin 2 (Stx2) and Protection from Lethal Outcome by Anti-Stx2 Antibody

ABSTRACT Hemolytic-uremic syndrome (HUS) results from infection by Shiga toxin (Stx)-producing Escherichia coli and is the most common cause of acute renal failure in children. We have developed a mouse model of HUS by administering endotoxin-free Stx2 in multiple doses over 7 to 8 days. At sacrifice, moribund animals demonstrated signs of HUS: increased blood urea nitrogen and serum creatinine levels, proteinuria, deposition of fibrin(ogen), glomerular endothelial damage, hemolysis, leukocytopenia, and neutrophilia. Increased expression of proinflammatory chemokines and cytokines in the sera of Stx2-treated mice indicated a systemic inflammatory response. Currently, specific therapeutics for HUS are lacking, and therapy for patients is primarily supportive. Mice that received 11E10, a monoclonal anti-Stx2 antibody, 4 days after starting injections of Stx2 recovered fully, displaying normal renal function and normal levels of neutrophils and lymphocytes. In addition, these mice showed decreased fibrin(ogen) deposition and expression of proinflammatory mediators compared to those of Stx2-treated mice in the absence of antibody. These results indicate that, when performed during progression of HUS, passive immunization of mice with anti-Stx2 antibody prevented the lethal effects of Stx2.

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

ABSTRACT 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 stx2e 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 stx2d1-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 stx2d1- and stx2d2-flanking regions in B2F1, Stx2d1 expression is repressed within the context of its toxin-converting phage while Stx2d2 expression is independent of phage induction.

Shiga Toxin (Stx) Classification, Structure, and Function.

Shiga toxin (Stx) is one of the most potent bacterial toxins known. Stx is found in Shigella dysenteriae 1 and in some serogroups of Escherichia coli (called Stx1 in E. coli). In addition to or instead of Stx1, some E. coli strains produce a second type of Stx, Stx2, that has the same mode of action as Stx/Stx1 but is antigenically distinct. Because subtypes of each toxin have been identified, the prototype toxin for each group is now designated Stx1a or Stx2a. The Stxs consist of two major subunits, an A subunit that joins noncovalently to a pentamer of five identical B subunits. The A subunit of the toxin injures the eukaryotic ribosome and halts protein synthesis in target cells. The function of the B pentamer is to bind to the cellular receptor, globotriaosylceramide, Gb3, found primarily on endothelial cells. The Stxs traffic in a retrograde manner within the cell, such that the A subunit of the toxin reaches the cytosol only after the toxin moves from the endosome to the Golgi and then to the endoplasmic reticulum. In humans infected with Stx-producing E. coli, the most serious manifestation of the disease, hemolytic-uremic syndrome, is more often associated with strains that produce Stx2a rather than Stx1a, and that relative toxicity is replicated in mice and baboons. Stx1a and Stx2a also exhibit differences in cytotoxicity to various cell types, bind dissimilarly to receptor analogs or mimics, induce differential chemokine responses, and have several distinctive structural characteristics.

Phase 1 Safety and Pharmacokinetic Study of Chimeric Murine-Human Monoclonal Antibody cαStx2 Administered Intravenously to Healthy Adult Volunteers

ABSTRACT Hemolytic-uremic syndrome (HUS) is a serious complication of infection by Shiga toxin-producing Escherichia coli. Shiga toxin type 2 (Stx2) is responsible for the renal toxicity that can follow intestinal infection and hemorrhagic colitis due to E. coli. A chimeric mouse-human antibody, designated cαStx2, that has neutralizing activity in a mouse model was produced and tested in healthy adult volunteers. In this phase I dose escalation study, cαStx2 was generally well tolerated. Pharmacokinetic studies indicated that clearance was stable over the dose range of 1.0 to 10 mg/kg of body weight (0.249 ± 0.023 ml/kg/h) but was higher for the 0.1-mg/kg dose (0.540 ± 0.078 ml/kg/h), suggesting saturable elimination. A similar nonlinear trend was observed for the volume of distribution, where average values ranged from 0.064 ± 0.015 liter/kg for the 1.0- to 10-mg/kg doses and 0.043 ± 0.005 for the 0.01-mg/kg dose. The relatively small volume of distribution suggests that the antibody is limited to the vascular (plasma) compartment. The mean half-life was 165 ± 66 h, with lowest values observed for the 0.1-mg/kg dose (56.2 ± 9.7 h) and the highest values reported for the 10.0-mg/kg dose (206.4 ± 12.4 h). Future studies are needed to confirm the safety of this cαStx2, and innovative clinical trials will be required to measure its efficacy in preventing or treating HUS.

Activation of Shiga toxin type 2d (Stx2d) by elastase involves cleavage of the C-terminal two amino acids of the A2 peptide in the context of the appropriate B pentamer

Shiga toxins (Stx) are potent ribosome‐inactivating toxins that are produced by Shigella dysenteriae type 1 or certain strains of Escherichia coli. These toxins are composed of one A subunit that can be nicked and reduced to an enzymatically active A1(≈27 kDa) and an A2 peptide (≈4 kDa) as well as a pentamer of B subunits (≈7 kDa/monomer) that binds the eukaryotic cell. Purified Shiga toxin type 2d is activated 10‐ to 1000‐fold for Vero cell toxicity by preincubation with mouse or human intestinal mucus or purified mouse elastase, whereas Stx2, Stx2c, Stx2e and Stx1 are not activatable. E. coli strains that produce the activatable Stx2d are more virulent in a streptomycin (str)‐treated mouse model of infection [lethal dose 50% (LD50) = 101] than are E. coli strains that produce any other type of Stx (LD50= 1010). To identify the element(s) of Stx2d that are required for mucus‐mediated activation, toxin genes were constructed such that the expressed mutant toxins consisted of hybrids of Stx2d and Stx1, Stx2 or Stx2e, contained deletions of up to six amino acids from the C‐terminus of the A2 of Stx2d or were altered in one or both of the two amino acids of the A2 of Stx2d that represent the only amino acid differences between the activatable Stx2d and the non‐activatable Stx2c. Analysis of these mutant toxins revealed that the A2 portion of Stx2d is required for toxin activation and that activation is abrogated if the Stx1 or Stx2e B subunit is substituted for the Stx2d B polypeptide. Furthermore, mass spectrometry performed on buffer‐ or elastase‐treated Stx2d indicated that the A2 peptide of the activated Stx2d was two amino acids smaller than the A2 peptide from buffer‐treated Stx2d. This finding, together with the toxin hybrid results, suggests that activation involves B pentamer‐dependent cleavage by elastase of the C‐terminal two amino acids from the Stx2d A2 peptide.

Human Intestinal Tissue and Cultured Colonic Cells Contain Globotriaosylceramide Synthase mRNA and the Alternate Shiga Toxin Receptor Globotetraosylceramide

ABSTRACT Escherichia coli O157:H7 and other Shiga toxin (Stx)-producing E. coli (STEC) bacteria are not enteroinvasive but can cause hemorrhagic colitis. In some STEC-infected individuals, a life-threatening sequela of infection called the hemolytic uremic syndrome may develop that can lead to kidney failure. This syndrome is linked to the production of Stx by the infecting organism. For Stx to reach the kidney, the toxin must first penetrate the colonic epithelial barrier. However, the Stx receptor, globotriaosylceramide (Gb3), has been thought to be absent from human intestinal epithelial cells. Thus, the mechanisms by which the toxin associates with and traverses through the intestine en route to the kidneys have been puzzling aspects of STEC pathogenesis. In this study, we initially determined that both types of Stx made by STEC, Stx1 and Stx2, do in fact bind to colonic epithelia in fresh tissue sections and to a colonic epithelial cell line (HCT-8). We also discovered that globotetraosylceramide (Gb4), a lower-affinity toxin receptor derived from Gb3, is readily detectable on the surfaces of human colonic tissue sections and HCT-8 cells. Furthermore, we found that Gb3 is present on a fraction of HCT-8 cells, where it presumably functions to bind and internalize Stx1 and Stx2. In addition, we established by quantitative real-time PCR (qRT-PCR) that both fresh colonic epithelial sections and HCT-8 cells express Gb3 synthase mRNA. Taken together, our data suggest that Gb3 may be present in small quantities in human colonic epithelia, where it may compete for Stx binding with the more abundantly expressed glycosphingolipid Gb4.

Dietary choice affects Shiga toxin-producing Escherichia coli (STEC) O157:H7 colonization and disease

Significance We demonstrated that dietary fiber content affects susceptibility to Shiga toxin (Stx)-producing Escherichia coli (STEC) infection in mice. We showed that high fiber diet (HFD)-fed mice had elevated levels of butyrate, a beneficial gut metabolite that paradoxically enhances the cell-killing capacity of Stx. We also found that the amount of gut bacteria in HFD-fed mice increased whereas the percent of commensal Escherichia species (spp) decreased compared with animals fed a low fiber diet (LFD). These changes led to higher E. coli O157:H7 colonization levels, more weight loss, and greater rates of death in HFD-fed than in LFD-fed STEC-infected animals. The likelihood that a single individual infected with the Shiga toxin (Stx)-producing, food-borne pathogen Escherichia coli O157:H7 will develop a life-threatening sequela called the hemolytic uremic syndrome is unpredictable. We reasoned that conditions that enhance Stx binding and uptake within the gut after E. coli O157:H7 infection should result in greater disease severity. Because the receptor for Stx, globotriaosylceramide, is up-regulated in the presence of butyrate in vitro, we asked whether a high fiber diet (HFD) that reportedly enhances butyrate production by normal gut flora can influence the outcome of an E. coli O157 infection in mice. To address that question, groups of BALB/c mice were fed high (10%) or low (2%) fiber diets and infected with E. coli O157:H7 strain 86-24 (Stx2+). Mice fed an HFD exhibited a 10- to 100-fold increase in colonization, lost 15% more body weight, exhibited signs of morbidity, and had 25% greater mortality relative to the low fiber diet (LFD)-fed group. Additionally, sections of intestinal tissue from HFD-fed mice bound more Stx1 and expressed more globotriaosylceramide than did such sections from LFD-fed mice. Furthermore, the gut microbiota of HFD-fed mice compared with LFD-fed mice contained reduced levels of native Escherichia species, organisms that might protect the gut from colonization by incoming E. coli O157:H7. Taken together, these results suggest that susceptibility to infection and subsequent disease after ingestion of E. coli O157:H7 may depend, at least in part, on individual diet and/or the capacity of the commensal flora to produce butyrate.