Differences in the Shiga Toxin (Stx) 2a Phage Regulatory Switch Region Influence Stx2 Localization and Virulence of Stx-Producing Escherichia coli in Mice.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of foodborne illness globally, and infection with serotype O157:H7 is associated with increased risk of hospitalization and death in the U.S. The Stxs are encoded on a temperate bacteriophage (stx-phage), and phage induction leads to Stx expression; subtype Stx2a in particular is associated with more severe disease. Our earlier studies showed significant levels of RecA-independent Stx2 production by STEC O157:H7 strain JH2010 (stx2astx2c), even though activated RecA is the canonical trigger for stx-phage induction. This study aimed to further compare and contrast RecA-independent toxin production in Stx2-producing clinical isolates. Deletion of recA in JH2010 resulted in higher in vitro supernatant cytotoxicity compared to that from JH2016ΔrecA, and the addition of the chelator ethylenediaminetetraacetic acid (EDTA) and various metal cations to the growth medium exacerbated the difference in cytotoxicity exhibited by the two deletion strains. Both the wild-type and ΔrecA deletion strains exhibited differential cytotoxicity in the feces of infected, streptomycin (Str)-treated mice. Comparison of the stx2a-phage predicted protein sequences from JH2010 and JH2016 revealed low amino acid identity of key phage regulatory proteins that are involved in RecA-mediated stx-phage induction. Additionally, other STEC isolates containing JH2010-like and JH2016-like stx2a-phage sequences led to similar Stx2 localization, as demonstrated by JH2010ΔrecA and JH2016ΔrecA, respectively. Deletion of the stx2a-phage regulatory region in the wild-type strains prevented the differential localization of Stx2 into the culture supernatant, a finding that suggests that the stx2a-phage regulatory region is involved in the differential ΔrecA phenotypes exhibited by the two strains. We hypothesize that the amino acid differences between the JH2010 and JH2016 phage repressor proteins (CIs) lead to structural differences that are responsible for differential interaction with RecA. Overall, we discovered that non-homologous stx2a-phage regulatory proteins differentially influence RecA-independent, and possibly RecA-dependent, Stx2 production. These findings emphasize the importance of studying non-homologous regulatory elements among stx2-phages and their influence on Stx2 production and virulence of STEC isolates.

Shiga Toxin (Stx) Type 1a and Stx2a Translocate through a Three-Layer Intestinal Model.

Shiga toxins (Stxs) produced by ingested E. coli can induce hemolytic uremic syndrome after crossing the intact intestinal barrier, entering the bloodstream, and targeting endothelial cells in the kidney. The method(s) by which the toxins reach the bloodstream are not fully defined. Here, we used two polarized cell models to evaluate Stx translocation: (i) a single-layer primary colonic epithelial cell model and (ii) a three-cell-layer model with colonic epithelial cells, myofibroblasts, and colonic endothelial cells. We traced the movement of Stx types 1a and 2a across the barrier models by measuring the toxicity of apical and basolateral media on Vero cells. We found that Stx1a and Stx2a crossed both models in either direction. However, approximately 10-fold more Stx translocated in the three-layer model as compared to the single-layer model. Overall, the percentage of toxin that translocated was about 0.01% in the epithelial-cell-only model but up to 0.09% in the three-cell-layer model. In both models, approximately 3- to 4-fold more Stx2a translocated than Stx1a. Infection of the three-cell-layer model with Stx-producing Escherichia coli (STEC) strains showed that serotype O157:H7 STEC reduced barrier function in the model and that the damage was not dependent on the presence of the eae gene. Infection of the three-layer model with O26:H11 STEC strain TW08571 (Stx1a+ and Stx2a+), however, allowed translocation of modest amounts of Stx without reducing barrier function. Deletion of stx2a from TW08571 or the use of anti-Stx1 antibody prevented translocation of toxin. Our results suggest that single-cell models may underestimate the amount of Stx translocation and that the more biomimetic three-layer model is suited for Stx translocation inhibitor studies.

Fecal Microbiota Functional Gene Effects Related to Single-Dose Antibiotic Treatment of Travelers' Diarrhea.

BACKGROUND: Travelers' diarrhea (TD) is common among military personnel deployed to tropical and subtropical regions. It remains unclear how TD and subsequent antibiotic treatment impact the resident microflora within the gut, especially given increased prevalence of antibiotic resistance among enteric pathogens and acquisition of multidrug-resistant organisms. We examined functional properties of the fecal microflora in response to TD, along with subsequent antibiotic treatment. METHODS: Fecal samples from US and UK military service members deployed to Djibouti, Kenya, and Honduras who presented with acute watery diarrhea were collected. A sample was collected at acute presentation to the clinic (day 0, before antibiotics), as well as 7 and/or 21 days following a single dose of antibiotics (azithromycin [500 mg], levofloxacin [500 mg], or rifaximin [1650 mg], all with loperamide). Each stool sample underwent culture and TaqMan reverse transcription polymerase chain reaction analyses for pathogen and antibiotic resistance gene detection. Purified DNA from each sample was analyzed using the HumiChip3.1 functional gene array. RESULTS: In total, 108 day 1 samples, 50 day 7 samples, and 94 day 21 samples were available for analysis from 119 subjects. Geographic location and disease severity were associated with distinct functional compositions of fecal samples. There were no overt functional differences between pre- and postantibiotic treatment samples, nor was there increased acquisition of antibiotic resistance determinants for any of the antibiotic regimens. CONCLUSIONS: These results indicate that single-dose antibiotic regimens may not drastically alter the functional or antibiotic resistance composition of fecal microflora, which should inform clinical practice guidelines and antimicrobial stewardship. CLINICAL TRIALS REGISTRATION NUMBER: NCT01618591.

Switching Shiga Toxin (Stx) Type from Stx2d to Stx2a but Not Stx2c Alters Virulence of Stx-Producing Escherichia coli (STEC) Strain B2F1 in Streptomycin (Str)-Treated Mice.

Shiga toxin (Stx)-producing Escherichia coli (STEC) strain B2F1 produces Stx type 2d, a toxin that becomes more toxic towards Vero cells in the presence of intestinal mucus. STEC that make Stx2d are more pathogenic to streptomycin (Str)-treated mice than most STEC that produce Stx2a or Stx2c. However, purified Stx2d is only 2- or 7-fold more toxic by the intraperitoneal route than Stx2a or Stx2c, respectively. We hypothesized, therefore, that the toxicity differences among Stx2a, Stx2c, and Stx2d occur at the level of delivery from the intestine. To evaluate that hypothesis, we altered the toxin type produced by stx2d+ mouse virulent O91:H21 clinical isolate B2F1 to Stx2a or Stx2c. Because B2F1 encodes two copies of stx2d, we did these studies in a derivative of B2F1 in which stx2d1 was deleted. Although the strains were equivalently virulent to the Str-treated mice at the 1010 dose, the B2F1 strain that produced Stx2a was attenuated relative to the ones that produced Stx2d or Stx2c when administered at 103 CFU/mouse. We next compared the oral toxicities of purified Stx2a, Stx2c, and Stx2d. We found that purified Stx2d is more toxic than Stx2a or Stx2c upon oral administration at 4 µg/mouse. Taken together, these studies suggest that Stx2 toxins are most potent when delivered directly from the bacterium. Furthermore, because Stx2d and Stx2c have the identical amino acid composition in the toxin B subunit, our results indicate that the virulence difference between Stx2a and Stx2d and Stx2c resides in the B or binding subunit of the toxins.

Genetic and Virulence Profiles of Enteroaggregative Escherichia coli (EAEC) Isolated From Deployed Military Personnel (DMP) With Travelers' Diarrhea.

To discern if there was a particular genotype associated with clinical enteroaggregative Escherichia coli (EAEC) strains isolated from deployed military personnel (DMP) with travelers' diarrhea (TD), we characterized a collection of EAEC from DMP deployed to Afghanistan, Djibouti, Kenya, or Honduras. Although we did not identify a specific EAEC genotype associated with TD in DMP, we found that EAEC isolated at the first clinic visit were more likely to encode the dispersin gene aap than EAEC collected at follow-up visits. A majority of the EAEC isolates were typical EAEC that adhered to HEp-2 cells, formed biofilms, and harbored genes for aggregative adherence fimbriae (AAF), AggR, and serine protease autotransporters of Enterobacteriaceae (SPATEs). A separate subset of the EAEC had aggR and genes for SPATEs but encoded a gene highly homologous to that for CS22, a fimbriae more commonly found in enterotoxigenic E. coli. None of these CS22-encoding EAEC formed biofilms in vitro or adhered to HEp-2 cells. Whole genome sequence and single nucleotide polymorphism analyses demonstrated that most of the strains were genetically diverse, but that a few were closely related. Isolation of these related strains occurred within days to more than a year apart, a finding that suggests a persistent source and genomic stability. In an ampicillin-treated mouse model we found that an agg4A+ aar- isolate formed a biofilm in the intestine and caused reduced weight gain in mice, whereas a strain that did not form an in vivo biofilm caused no morbidity. Our diverse strain collection from DMP displays the heterogeneity of EAEC strains isolated from human patients, and our mouse model of infection indicated the genotype agg4A+ aar- and/or capacity to form biofilm in vivo may correlate to disease severity.

The Virulence of Escherichia coli O157:H7 Isolates in Mice Depends on Shiga Toxin Type 2a (Stx2a)-Induction and High Levels of Stx2a in Stool.

In this study we compared nine Shiga toxin (Stx)-producing Escherichia coli O157:H7 patient isolates for Stx levels, stx-phage insertion site(s), and pathogenicity in a streptomycin (Str)-treated mouse model. The strains encoded stx2a, stx1a and stx2a, or stx2a and stx2c. All of the strains elaborated 105-106 cytotoxic doses 50% (CD50) into the supernatant after growth in vitro as measured on Vero cells, and showed variable levels of increased toxin production after growth with sub-inhibitory levels of ciprofloxacin (Cip). The stx2a+stx2c+ isolates were 90-100% lethal for Str-treated BALB/c mice, though one isolate, JH2013, had a delayed time-to-death. The stx2a+ isolate was avirulent. Both an stx2a and a recA deletion mutant of one of the stx2a+stx2c+ strains, JH2010, exhibited at least a three-log decrease in cytotoxicity in vitro and both were avirulent in the mice. Stool from Str-treated mice infected with the highly virulent isolates were 10- to 100-fold more cytotoxic than feces from mice infected with the clinical isolate, JH2012, that made only Stx2a. Taken together these findings demonstrate that the stx2a-phage from JH2010 induces to higher levels in vivo than does the phage from JH2012. The stx1a+stx2a+ clinical isolates were avirulent and neutralization of Stx1 in stool from mice infected with those strains indicated that the toxin produced in vivo was primarily Stx1a. Treatment of mice infected with Stx1a+Stx2a+ isolates with Cip resulted in an increase in Stx2a production in vivo and lethality in the mice. Our data suggest that high levels of Stx2a in stool are predictive of virulence in mice.

The Role of the AggR Regulon in the Virulence of the Shiga Toxin-Producing Enteroaggregative Escherichia coli Epidemic O104:H4 Strain in Mice.

An O104:H4 Shiga toxin (Stx)-producing enteroaggregative Escherichia coli (EAEC) strain caused a large outbreak of bloody diarrhea and the hemolytic uremic syndrome in 2011. We previously developed an ampicillin (Amp)-treated C57BL/6 mouse model to measure morbidity (weight loss) and mortality of mice orally infected with the prototype Stx-EAEC strain C227-11. Here, we hypothesized that mice fed C227-11 cured of the pAA plasmid or deleted for individual genes on that plasmid would display reduced virulence compared to animals given the wild-type (wt) strain. C227-11 cured of the pAA plasmid or deleted for the known pAA-encoded virulence genes aggR, aggA, sepA, or aar were fed to Amp-treated C57BL/6 mice at doses of 1010-1011CFU. Infected animals were then either monitored for morbidity and lethality for 28 days or euthanized to determine intestinal pathology and colonization levels at selected times. The pAA-cured, aggR, and aggA mutants of strain C227-11 all showed reduced colonization at various intestinal sites. However, the aggR mutant was the only mutant attenuated for virulence as it showed both reduced morbidity and mortality. The aar mutant showed increased expression of the aggregative adherence fimbriae (AAF) and caused greater systemic effects in infected mice when compared to the C227-11 wt strain. However, unexpectedly, both the aggA and aar mutants displayed increased weight loss compared to wt. The sepA mutant did not exhibit altered morbidity or mortality in the Amp-treated mouse model compared to wt. Our data suggest that the increased morbidity due to the aar mutant could possibly be via an effect on expression of an as yet unknown virulence-associated factor under AggR control.

Shiga Toxin (Stx) Type 1a Reduces the Oral Toxicity of Stx Type 2a.

BACKGROUND: Shiga toxin (Stx) is the primary virulence factor of Stx-producing Escherichia coli (STEC). STEC can produce Stx1a and/or Stx2a, which are antigenically distinct. However, Stx2a-producing STEC are associated with more severe disease than strains producing both Stx1a and Stx2a. METHODS AND RESULTS: To address the hypothesis that the reason for the association of Stx2a with more severe disease is because Stx2a crosses the intestinal barrier with greater efficiency that Stx1a, we covalently labeled Stx1a and Stx2a with Alexa Fluor 750 and determined the ex vivo fluorescent intensity of murine systemic organs after oral intoxication. Surprisingly, both Stxs exhibited similar dissemination patterns and accumulated in the kidneys. We next cointoxicated mice to determine whether Stx1a could impede Stx2a. Cointoxication resulted in increased survival and an extended mean time to death, compared with intoxication with Stx2a only. The survival benefit was dose dependent, with the greatest effect observed when 5 times more Stx1a than Stx2a was delivered, and was amplified when Stx1a was delivered 3 hours prior to Stx2a. Cointoxication with an Stx1a active site toxoid also reduced Stx2a toxicity. CONCLUSIONS: These studies suggest that Stx1a reduces Stx2a-mediated toxicity, a finding that may explain why STEC that produce only Stx2a are associated with more severe disease than strains producing Stx1a and Stx2a.

Mapping of genetic loci that modulate differential colonization by Escherichia coli O157:H7 TUV86-2 in advanced recombinant inbred BXD mice.

BACKGROUND: Shiga toxin (Stx)-producing E. coli (STEC) are responsible for foodborne outbreaks that can result in severe human disease. During an outbreak, differential disease outcomes are observed after infection with the same STEC strain. One question of particular interest is why some infected people resolve infection after hemorrhagic colitis whereas others progress to the hemolytic uremic syndrome (HUS). Host age and infection dose have been implicated; however, these parameters do not appear to fully account for all of the observed variation in disease severity. Therefore, we hypothesized that additional host genetic factors may play a role in progression to HUS. METHODS AND RESULTS: To mimic the genetic diversity in the human response to infection by STEC, we measured the capacity of an O157:H7 outbreak isolate to colonize mouse strains from the advanced recombinant inbred (ARI) BXD panel. We first infected the BXD parental strains C57BL/6 J (B6) and DBA/2 J (D2) with either 86-24 (Stx2a+) or TUV86-2, an Stx2a-negative isogenic mutant. Colonization levels were determined in an intact commensal flora (ICF) infection model. We found a significant difference in colonization levels between the parental B6 and D2 strains after infection with TUV86-2 but not with 86-24. This observation suggested that a host factor that may be masked by Stx2a affects O157:H7 colonization in some genetic backgrounds. We then determined the TUV86-2 colonization levels of 24 BXD strains in the ICF model. We identified several quantitative trait loci (QTL) associated with variation in colonization by correlation analyses. We found a highly significant QTL on proximal chromosome 9 (12.5-26.7 Mb) that strongly predicts variation in colonization levels and accounts for 15-20 % of variance. Linkage, polymorphism and co-citation analyses of the mapped region revealed 36 candidate genes within the QTL, and we identified five genes that are most likely responsible for the differential colonization. CONCLUSIONS: The identification of the QTL on chromosome 9 supports our hypothesis that individual genetic makeup affects the level of colonization after infection with STEC O157:H7.

Virulence Potential of Activatable Shiga Toxin 2d-Producing Escherichia coli Isolates from Fresh Produce.

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are food- and waterborne pathogens that are often transmitted via beef products or fresh produce. STEC strains cause both sporadic infections and outbreaks, which may result in hemorrhagic colitis and hemolytic uremic syndrome. STEC strains may elaborate Stx1, Stx2, and/or subtypes of those toxins. Epidemiological evidence indicates that STEC that produce subtypes Stx2a, Stx2c, and/or Stx2d are more often associated with serious illness. The Stx2d subtype becomes more toxic to Vero cells after incubation with intestinal mucus or elastase, a process named "activation." Stx2d is not generally found in the E. coli serotypes most commonly connected to STEC outbreaks. However, STEC strains that are stx2d positive can be isolated from foods, an occurrence that gives rise to the question of whether those food isolates are potential human pathogens. In this study, we examined 14 STEC strains from fresh produce that were stx2d positive and found that they all produced the mucus-activatable Stx2d and that a subset of the strains tested were virulent in streptomycin-treated mice.

Reduced Toxicity of Shiga Toxin (Stx) Type 2c in Mice Compared to Stx2d Is Associated with Instability of Stx2c Holotoxin.

Shiga toxin (Stx) is an AB5 ribotoxin made by Stx-producing Escherichia coli (STEC). These organisms cause diarrhea, hemorrhagic colitis and the hemolytic uremic syndrome. STEC make two types of Stxs, Stx1 and/or Stx2. Stx2 has one prototype (a) and six subtypes (b-g), but only STEC that make Stx2a, and/or Stx2c, or Stx2d are associated with severe disease. However, Stx2c is about 10-fold less toxic than Stx2d in vivo despite only two amino acid differences in the A subunit at positions 291 and 297. We made mutations at these two sites to create intermediate toxins between Stx2c and Stx2d, and determined the 50% cytotoxic dose on Vero cells before and after heat treatment, and the 50% lethal dose in mice of the toxins. We found that serine 291 was associated with increased toxicity in vivo and that either amino acid change from that in Stx2c to that in Stx2d increased heat stability. We also assessed the secondary structure of Stx2c and Stx2d by circular dichroism (CD) spectroscopy. The CD studies suggest that Stx2c has a less-ordered secondary structure than Stx2d. We conclude that both amino acids at positions 291 and 297 in Stx2c contribute to its decreased stability and in vivo toxicity compared to Stx2d.

Shiga toxin 2a and Enteroaggregative Escherichia coli--a deadly combination.

In 2011, a Shiga toxin (Stx) type 2a-producing enteroaggregative E. coli (EAEC) strain of serotype O104:H4 caused a large lethal outbreak in Northern Europe. Until recently, the pathogenic mechanisms explaining the high virulence of the strain have remained unclear. Our laboratories have shown that EAEC genes encoded on the pAA virulence plasmid, particularly the AggR-regulated AAF/I fimbriae, enhance inflammation and enable the outbreak strain to both adhere to epithelial cells and translocate Stx2a across the intestinal epithelium, possibly explaining the high incidence of the life threatening post-diarrheal sequelae of hemolytic uremic syndrome. Epidemiologic evidence supports a model of EAEC pathogenesis comprising the concerted action of multiple virulence factors along with induction of inflammation. Here, we suggest a model for the pathogenesis of the O104:H4 outbreak strain that includes contributions from EAEC alone, but incorporating additional injury induced by Stx2a.

Protective efficacy and pharmacokinetics of human/mouse chimeric anti-Stx1 and anti-Stx2 antibodies in mice.

In the United States, Shiga toxin (Stx)-producing Escherichia coli (STEC) is the most frequent infectious cause of hemorrhagic colitis. Hemolytic uremic syndrome (HUS) is a serious sequela that may develop after STEC infection that can lead to renal failure and death in up to 10% of cases. STEC can produce one or more types of Stx, Stx1 and/or Stx2, and Stx1 and Stx2 are responsible for HUS-mediated kidney damage. We previously generated two monoclonal antibodies (MAbs) that neutralize the toxicity of Stx1 or Stx2. In this study, we evaluated the protective efficacy of human/mouse chimeric versions of those monoclonal antibodies, named cαStx1 and cαStx2. Mice given an otherwise lethal dose of Stx1 were protected from death when injected with cαStx1 either 1 h before or 1 h after toxin injection. Additionally, streptomycin-treated mice fed the mouse-lethal STEC strain B2F1 that produces the Stx2 variant Stx2d were protected when given a dose of 0.1 mg of cαStx2/kg of body weight administered up to 72 h post-oral bacterial challenge. Since many STEC strains produce both Stx1 and Stx2 and since either toxin may lead to the HUS, we also assessed the protective efficacy of the combined MAbs. We found that both antibodies were required to protect mice from the presence of both Stx1 and Stx2. Pharmacokinetic studies indicated that cαStx1 and cαStx2 had serum half-lives (t1/2) of about 50 and 145 h, respectively. We propose that cαStx1 and cαStx2, both of which have been tested for safety in humans, could be used therapeutically for prevention or treatment early in the development of HUS.

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.

Clinical isolates of Shiga toxin 1a-producing Shigella flexneri with an epidemiological link to recent travel to Hispañiola.

Shiga toxins (Stx) are cytotoxins involved in severe human intestinal disease. These toxins are commonly found in Shigella dysenteriae serotype 1 and Shiga-toxin-producing Escherichia coli; however, the toxin genes have been found in other Shigella species. We identified 26 Shigella flexneri serotype 2 strains isolated by public health laboratories in the United States during 2001-2013, which encode the Shiga toxin 1a gene (stx1a). These strains produced and released Stx1a as measured by cytotoxicity and neutralization assays using anti-Stx/Stx1a antiserum. The release of Stx1a into culture supernatants increased ≈100-fold after treatment with mitomycin C, suggesting that stx1a is carried by a bacteriophage. Infectious phage were found in culture supernatants and increased ≈1,000-fold with mitomycin C. Whole-genome sequencing of several isolates and PCR analyses of all strains confirmed that stx1a was carried by a lambdoid bacteriophage. Furthermore, all patients who reported foreign travel had recently been to Hispañiola, suggesting that emergence of these novel strains is associated with that region.

The presence of the pAA plasmid in the German O104:H4 Shiga toxin type 2a (Stx2a)-producing enteroaggregative Escherichia coli strain promotes the translocation of Stx2a across an epithelial cell monolayer.

BACKGROUND: A Shiga toxin type 2a (Stx2a)-producing enteroaggregative Escherichia coli (EAEC) strain of serotype O104:H4 caused a large outbreak in 2011 in northern Europe. Pathogenic mechanisms for this strain are unclear. We hypothesized that EAEC genes encoded on the pAA virulence plasmid promoted the translocation of Stx2a across the intestinal mucosa. METHODS: We investigated the potential contribution of pAA by using mutants of Stx-EAEC strain C227-11, either cured of the pAA plasmid or deleted for individual known pAA-encoded virulence genes (ie, aggR, aggA, and sepA). The resulting mutants were tested for their ability to induce interleukin 8 (IL-8) secretion and translocation of Stx2a across a polarized colonic epithelial (T84 cell) monolayer. RESULTS: We found that deletion of aggR or aggA significantly reduced bacterial adherence and (independently) translocation of Stx2a across the T84-cell monolayer. Moreover, deletion of aggR, aggA, sepA, or the Stx2a-encoding phage from C227-11 resulted in reduced secretion of IL-8 from the infected monolayer. CONCLUSIONS: Our data suggest that the AggR-regulated aggregative adherence fimbriae I enhance inflammation and enable the outbreak strain to both adhere to epithelial cells and translocate Stx2a across the intestinal epithelium.

Comparisons of native Shiga toxins (Stxs) type 1 and 2 with chimeric toxins indicate that the source of the binding subunit dictates degree of toxicity.

Shiga toxin (Stx)-producing E. coli (STEC) cause food-borne outbreaks of hemorrhagic colitis. The main virulence factor expressed by STEC, Stx, is an AB5 toxin that has two antigenically distinct forms, Stx1a and Stx2a. Although Stx1a and Stx2a bind to the same receptor, globotriaosylceramide (Gb3), Stx2a is more potent than Stx1a in mice, whereas Stx1a is more cytotoxic than Stx2a in cell culture. In this study, we used chimeric toxins to ask what the relative contribution of individual Stx subunits is to the differential toxicity of Stx1a and Stx2a in vitro and in vivo. Chimeric stx1/stx2 operons were generated by PCR such that the coding regions for the A2 and B subunits of one toxin were combined with the coding region for the A1 subunit of the heterologous toxin. The toxicities of purified Stx1a, Stx2a, and the chimeric Stxs were determined on Vero and HCT-8 cell lines, while polarized HCT-8 cell monolayers grown on permeable supports were used to follow toxin translocation. In all in vitro assays, the activity of the chimeric toxin correlated with that of the parental toxin from which the B subunit originated. The origin of the native B subunit also dictated the 50% lethal dose of toxin after intraperitoneal intoxication of mice; however, the chimeric Stxs exhibited reduced oral toxicity and pH stability compared to Stx1a and Stx2a. Taken together, these data support the hypothesis that the differential toxicity of the chimeric toxins for cells and mice is determined by the origin of the B subunit.

When a healthy diet turns deadly.

The health benefits of a high fiber diet (HFD) result in part from the action of metabolic end products made by gut commensals on the host epithelium. Butyrate is one such beneficial metabolite; however, butyrate paradoxically enhances the capacity of Escherichia coli-produced Shiga toxin type 2 (Stx2) to kill tissue culture cells. We recently showed that mice fed an HFD exhibited increased butyrate in gut contents and had an altered intestinal microbiota with reduced numbers of Escherichia species. Furthermore, mice fed an HFD and infected with Stx-producing E. coli (STEC) were colonized to a higher degree, lost more weight and succumbed to infection at greater rates compared with STEC-infected low fiber diet animals. The HFD animals showed higher levels of the Stx receptor globotriaocylceramide (Gb3) in both the gut and kidneys. We speculate that an HFD that leads to increased intestinal butyrate and Gb3 in the intestines and kidneys may explain the higher rate of the hemolytic uremic syndrome in females over males.

New Therapeutic Developments against Shiga Toxin-Producing Escherichia coli.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is an etiologic agent of bloody diarrhea. A serious sequela of disease, the hemolytic uremic syndrome (HUS) may arise in up to 25% of patients. The development of HUS after STEC infection is linked to the presence of Stx. STEC strains may produce one or more Stxs, and the Stxs come in two major immunological groups, Stx1 and Stx2. A multitude of possible therapeutics designed to inhibit the actions of the Stxs have been developed over the past 30 years. Such therapeutics are important because antibiotic treatment of STEC infections is contraindicated due to an increased potential for development of HUS. The reason for the increased risk of HUS after antibiotic treatment is likely because certain antibiotics induce expression of the Stxs, which are generally associated with lysogenic bacteriophages. There are a few potential therapeutics that either try to kill STEC without inducing Stx expression or target gene expression within STEC. However, the vast majority of the treatments under development are designed to limit Stx receptor generation or to prevent toxin binding, trafficking, processing, or activity within the cell. The potential therapies described in this review include some that have only been tested in vitro and several that demonstrate efficacy in animals. The therapeutics that are currently the furthest along in development (completed phase I and II trials) are monoclonal antibodies directed against Stx1 and Stx2.

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.