Since Shiga-toxigenic Escherichia coli (STEC) strains are not considered to be enteroinvasive, the mechanism(s) by which Shiga toxin (Stx) gains access to the circulation and to target tissues expressing its target receptor Gb3 is crucial to the disease process. There is increasing evidence that by facilitating translocation of Stx across the intestinal epithelium and by transporting bound toxin to remote sites such as the renal endothelium, polymorphonuclear leucocytes (PMNs) play a key role in the pathogenesis of serious STEC disease. Plasma levels of PMN-attracting CXC chemokines such as IL-8 also appear to correlate in humans with the severity of disease. Thus, the capacity of STEC strains to elicit CXC chemokine responses in intestinal epithelial cells may be a crucial step in pathogenesis. In order to determine which STEC factor(s) are responsible for the induction of CXC chemokine responses by intestinal epithelial (HCT-8) cells, a real-time reverse transcription PCR assay was developed to quantitatively measure relative expression of chemokine mRNA for IL-8, ENA-78, GCP-2, MGSA, MIP-2α and MIP-2β. Similarly, a commercially available sandwich ELISA was used to measure levels of IL-8 protein secreted by HCT-8 cells in response to infection with STEC. When HCT-8 cells were infected with the wellcharacterised locus of enterocyte effacement (LEE)-negative O113:H21 strain 98NK2 or the LEE-positive STEC strain EDL933, there were significant differences in the levels of chemokine mRNA and IL-8 protein expression. In particular, the LEE-negative strain 98NK2 induced significantly higher and earlier levels of chemokine mRNAs, including IL-8, MIP-2α and MIP-2β at 1 and 4 h, and ENA-78 at 4 h. However, EDL933 elicited no significant upregulation of any of the chemokine mRNAs at 1 h, and only modest increases in IL-8, MIP-2α and MIP-2β by 4 h, post-infection. These results were confirmed by IL-8 ELISA which showed that 98NK2 elicited significant levels of IL-8 protein by 2 h post-infection, and remained high until 4 h post-infection. In comparison, EDL933 did not elicit significant IL-8 induction over that of control cells, even at 4 h post-infection. When a range of STEC isolates from clinical samples were tested for their capacity to induce chemokine production in HCT-8 cells, highly significant differences were observed between the strains. Infection of HCT-8 cells with a range of LEE-negative STEC strains isolated from patients with severe STEC disease resulted in significantly higher and earlier upregulation of IL-8 and MIP-2α mRNA than that elicited by several LEE-positive STEC strains. Similarly, levels of IL-8 protein in LEE-negative STEC-infected HCT-8 culture supernatants were significantly higher than in LEE-positive STEC-infected culture supernatants. Only one LEE-positive strain, an O26 strain 95ZG1, was capable of inducing chemokine responses comparable to that induced by infection with the LEE-negative STEC strains. These results were also shown not to be attributable to differences in the adherence, initial doses or growth of the strains during the assay, or to a loss of viability of the HCT-8 cells. These results, therefore, suggest that there may be interesting differences in the ability of STEC strains to induce chemokine production in intestinal epithelial cells. The factor(s) that contribute to chemokine induction by epithelial cells in response to STEC were then examined. The difference in responses could not be attributed to the expression or non-expression of LEE genes, the presence or absence of an STEC megaplasmid or to differences in O serogroup. Although purified Stx1 and Stx2 were able to induce IL-8 and MIP-2α mRNA, and IL-8 protein, the levels of chemokine induction in response to wild-type STEC did not correlate with the type or amount of Stx produced by these strains in vitro. Similarly, deletion of the single stx2 gene from 98NK2 had no significant effect on chemokine induction compared to wild-type 98NK2-infected HCT-8 cells. Interestingly, several of the LEE-negative STEC strains eliciting the strongest chemokine responses belonged to flagellar serotype H21. Incubation of HCT-8 cells with purified H21 flagella elicited IL-8 and MIP-2α mRNA responses similar to those seen in the presence of the most potent LEE-negative STEC strains. Deletion of the fliC gene largely abolished the capacity of 98NK2 to elicit IL-8 and MIP-2α mRNA and IL-8 protein responses in HCT-8 cells. Similarly, deletion of both stx2 and fliC from 98NK2 elicited a response similar to that observed with deletion of fliC alone. Flagella were then purified from the high chemokine-inducing STEC strains 95HE4 (O91:H7) and 95ZG1 (O26:H11). Purified H7 and H11 flagella were similarly able to induce both IL-8 and MIP-2α mRNA, and IL-8 protein, in HCT-8 cells at levels similar to their respective wild-type strains. Deletion of fliC from two other STEC strains, 97MW1 (O113:H21) and 86-24 (O157:H7), confirmed that flagellin was responsible for the majority of chemokine responses in these wild-type strains. However, an inability of EDL933 to induce these responses was unexpected and later found to be due to a lack of expression of H7 flagella by this strain. Purified H21 FliC (His6-FliC) alone was able to induce chemokine production (including IL-8, MIP-2α and MIP-2β at 1 and 4 h, and ENA-78 at 4 h) by HCT-8 cells at similar levels to that observed for 98NK2. Taken together, these data suggest that although Stx is capable of inducing CXC chemokine responses, the elevated responses observed in cells infected with certain STEC strains are largely attributable to the production of flagellin. Purified His6-H21 flagellin was also able to induce p38 MAPK activation in vitro and IL-8 and MIP-2α mRNA were superinduced in the presence of both Stx2 and H21 flagellin. Blockade of the p38 pathway with SB203580 resulted in a down-regulation of IL-8 protein levels (by up to 61%) in response to H21 flagellin, but not IL-8 mRNA, suggesting that this inhibition may occur post-transcriptionally. Blocking the ERK and JNK pathways similarly decreased IL-8 secretion in response to H21 flagellin, suggesting that all three MAPK pathways are involved in this response. Indeed, concurrent inhibition of all three pathways resulted in virtually complete inhibition of IL-8 protein production (98%). Transfected HeLa and MDCK cells stably expressing TLR5 activated p38 in the presence of purified H21 flagellin, whereas dominant-negative (DN) TLR5-expressing cells did not, supporting previous studies that show that flagellin acts via TLR5. These data suggest that TLR5 and the p38, ERK and JNK MAPK pathways all play an important role in the response of intestinal epithelial cells to H21 flagellin from STEC, and that the combined effects of Stx and flagellin on host intestinal epithelial cells may result in an augmented inflammatory response. A role for flagellin in virulence was then investigated. BALB/c mice were orally inoculated with wild-type 98NK2 or 98NK2ΔfliC. Of the 16 mice challenged with the wildtype strain 98NK2, 9 (56%) died during the experiment (median survival time 7.6 days). However, only 3 of 16 mice (19%) challenged with 98NK2ΔfliC died (median survival time > 14 days). The difference in survival rate was statistically significant. No significant differences in the level of intestinal colonisation of 98NK2 or 98NK2ΔfliC were observed. Thus, flagellin directly contributes to the virulence of STEC in streptomycin-treated mice. Since the streptomycin-treated mouse is a model for systemic Stx-mediated pathology, these results suggest that the pro-inflammatory effects of flagellin play an important role in the pathogenesis of Stx-mediated STEC disease in vivo. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2004.

Normal endothelial cell function is controlled in part by a tightly regulated balance between angiopoietin-1 and -2 (Ang-1 and Ang-2). Angiopoietin dysregulation (decreased Ang-1 and increased Ang-2) leads to an activated endothelium that is contractile, adhesive, and prothrombotic. Since an activated endothelial phenotype is seen in invasive group A streptococcal infection, E. coli O157:H7-induced hemolytic-uremic syndrome (HUS), and sepsis, we hypothesized that angiopoietin dysregulation might also be present in these syndromes, and to that end, measured angiopoietin levels in several well-characterized patient cohorts. Decreased Ang-1 and/or increased Ang-2 were found in all three syndromes, and were predictive of clinical outcome in HUS and sepsis. The prognostic utility of Ang-2 in sepsis was further enhanced by combination with biomarkers of inflammation. Angiopoietin dysregulation may therefore represent a shared final common pathway to endothelial activation as well as a clinically useful prognostic biomarker in streptococcal toxic shock, HUS, and sepsis.

Normal endothelial cell function is controlled in part by a tightly regulated balance between angiopoietin-1 and -2 (Ang-1 and Ang-2). Angiopoietin dysregulation (decreased Ang-1 and increased Ang-2) leads to an activated endothelium that is contractile, adhesive, and prothrombotic. Since an activated endothelial phenotype is seen in invasive group A streptococcal infection, E. coli O157:H7-induced hemolytic-uremic syndrome (HUS), and sepsis, we hypothesized that angiopoietin dysregulation might also be present in these syndromes, and to that end, measured angiopoietin levels in several well-characterized patient cohorts. Decreased Ang-1 and/or increased Ang-2 were found in all three syndromes, and were predictive of clinical outcome in HUS and sepsis. The prognostic utility of Ang-2 in sepsis was further enhanced by combination with biomarkers of inflammation. Angiopoietin dysregulation may therefore represent a shared final common pathway to endothelial activation as well as a clinically useful prognostic biomarker in streptococcal toxic shock, HUS, and sepsis.

Shiga toxin-producing E. coli (STEC) cause diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). All STEC produce one or both of two Shiga toxins, Stx1 and Stx2. STEC strains that produce Stx2 are more strongly associated with HUS than strains that produce Stx1 or both Stx1 and Stx2. Epidemiologic evidence indicates a recent increase in the rate of HUS among STEC outbreaks. The increasing rate of HUS could be explained by a shift in the toxin profiles of STEC strains. The purpose of this study was to examine trends in toxin profiles of human STEC O157 isolates from 1996 to 2008 and to assess whether an increase in the number of Stx2-only-producing strains could be correlated with a recent increase in HUS cases. Data from three independent datasets, collected from PulseNet, eFORS and NARMS, were used. Additionally, trends such as seasonal variations, geographical variations, gender differences, and age differences were examined for each toxin profile. Results from this study show a shift in the toxin profile of human STEC O157 strains in the United States, in that the proportion of Stx2-only producing strains has increased dramatically since 1996.

Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Science, 2003. / "February 2003." Addendum and corrigenda inserted at back. Includes bibliographical references (leaves 249-272).

Thesis (Ph. D.)--University of Nevada, Reno, 2008. / Includes bibliographical references. Online version available on the World Wide Web.

A Síndrome Hemolítica Urêmica (SHU) é a principal doença associada à infecção com linhagens de Escherichia coli produtoras de toxina de Shiga (Stx), doença para qual não há uma vacina ou tratamento específico. A toxina Stx é formada por uma subunidade A enzimaticamente ativa e uma B pentamérica responsável pela ligação da toxina na célula hospedeira. Neste trabalho propomos o uso de Bacillus subtilis, uma bactéria não patogênica e formadora de esporos, como veículo vacinal para a expressão de formas atóxicas da Stx2, sob o controle de um promotor induzível por estresse (PgsiB). Camundongos BALB/c imunizados com células vegetativas ou esporos das linhagens vacinais de B. subtilis, por diferentes vias, induziram baixos níveis de anticorpos anti-Stx em soro (IgG) e fezes (IgA). Avaliamos também o potencial imunogênico da Stx gerada em linhagens recombinates de E. coli, mas os anticorpos gerados não foram capazes de neutralizar a toxina nativa. Os resultados indicam que formas alternativas de expressão e/ou o uso de adjuvantes são necessárias para gerar formulações vacinais eficazes contra a SHU. / The Hemolytic Uremic Syndrome (HUS) is the main disease associated with infections with Shiga toxin (Stx) - producing Escherichia coli strain and no effective vaccine or treatment exist. The Stx toxin consist of an enzymatically active A subunit and a pentameric B subunit responsible toxin binding to host cells. In this work we propose the use of Bacillus subtilis, a harmless spore form bacteria as a vaccine vehicle for the expression atoxic forms of Stx2, under the control of stress inducible (PgsiB) promoter. BALB/c mice immunized with vegetative cells and spores of the B. subtilis vaccine strain using different immunization routes elicited low specific antibody levels at serum (IgG) or fecal extracts (IgA). We also investigated the immunogenic potencial of StxB purified from recombinant E. coli strain, but the induced anti-StxB antibodies did not neutralize the native toxin. The results indicate that alternative expression system or the incorporation of the adjuvants are required for the generation of vaccine formulation active against HUS.

Atypical hemolytic uremic syndrome (aHUS) is a rare renal disorder characterized by thrombotic microangiopathy, thrombocytopenia, and acute kidney injury. Its pathogenesis has been attributed to a ‘triggering' event that leads to dysregulation of the complement cascade at the level of the endothelial cell surface. Consistent with this understanding of the disease, mutations in complement genes have been definitively implicated in aHUS. However, the existence of other genetic contributors is supported by two observations. First, in ~50% of cases, disease-causing variants are not identified in complement genes, and second, disease penetrance is typically incomplete and highly variable. To test this hypothesis, we identified pathways established to have crosstalk with the complement cascade, focusing initially on the coagulation pathway. Using targeted genomic enrichment and massively parallel sequencing we screened 36 European-American patients with sporadic aHUS patients for genetic variants in 85 complement and coagulation genes, identifying deleterious rare variants in several coagulation genes. The most frequently mutated coagulation gene in our study cohort was PLG, which encodes a zymogen of plasmin and plays key role in fibrinolysis. These results implicate the coagulation pathway in the pathogenesis of aHUS. Based on this outcome, we developed a clinical genetic testing panel to screen disease-related genes in a group of ultra-rare complement-mediated diseases that includes, in addition to aHUS, thrombotic thrombocytopenic purpura (TTP), C3 glomerulonephritis (C3GN) and dense deposit disease (DDD) patients. Data from 193 patients validate the usage of this panel in clinical practice and also provide confirmatory insight into the pathogeneses of these diseases. Specifically, we found that in aHUS and TTP patients, variants were frequently identified in complement regulator genes, while in C3GN and DDD patients, variants were additionally found in C3 convertase genes. To understand variability in disease penetrance, we completed targeted genetic screening in two aHUS families grossly discordant for disease penetrance, identifying in one family a co-segregating Factor X-deficiency variant (F10 p.Glu142Lys) that abrogated the effect of the complement mutation. Functional studies of the F10 p.Glu142Lys variant show that it decreases Factor X activity predicting to a hypo-coagulable state and further illustrating the importance of complement-coagulation crosstalk in exacerbating, but also mitigating the aHUS phenotype. In our final studies, we have sought to complete a comprehensive analysis for other potentially related pathways by using bioinformatics to identify candidate pathways coupled with whole exome sequencing. Preliminary data from 43 aHUS patients and 300 controls suggest that pathways for dermatan and heparan sulfate synthesis, which are relevant to the formation of the extra-cellular matrix and cell surface adhesion, may be implicated in the aHUS.

BACKGROUND: Over the past 5 years, 634 cases of Shigatoxin E. coli (STEC) infection were reported to Tennessee Health Department 1. At our local children’s hospital, 4-5 children are hospitalized with STEC infection each year. Some of these children had no history of ingesting food items that could have placed them at risk to develop STEC infection; however, there are other ways that humans could get infected, such as exposure to contaminated water from cattle farms 2. GOALS: To determine if bodies of water in the city are contaminated with STEC. METHODS: Fifty (50) ml of water samples were collected from selected areas of Johnson City, TN. Samples were inoculated to Sorbitol McConkey Agar (SMA) plates under sterile techniques & incubated at 36C for 18 hours under aerobic conditions. RESULTS: Table 1 E. coli Strains Isolated from Water Samples Colony Types Founders Park Sinking Creek Carroll Creek Cherokee Creek Colorless (STEC) 14 (3.5) 24 (6) 32 (8) 35 (8.75) Pink (Non-STEC ) 8 (2) 3 (0.75) 7 (1.75) 4(1) DISCUSSION/ CONCLUSION: All sampled sites were positive for STEC. STEC is a normal flora of the gastrointestinal tract of cattle. Around city neighborhoods are pastures, as cattle farming is a major livelihood in Northeastern, TN. It is highly possible that water runoff from these pastures contaminates the waters around the city. Public health measures should be undertaken to inform the community that these waters are contaminated with STEC to prevent STEC infection. References: Reportable Conditions. TN Epi-news, TN Health Dept Issue 3, Volume 9, 2016 Escherichia coli O157:H7 Infections in Children Associated with Raw Milk & Raw Colostrum From Cows—California, 2006. MMWR Weekly, 57(23); 625-628, June 23, 2008.

Enterohemorrhagic E. coli (EHEC) infections are a leading cause of foodborne illness in the United States. Shiga-like toxins are produced that can cause hemorrhagic colitis and can lead to dangerous complications, such as acute kidney injury and hemolytic uremic syndrome (HUS). There are currently no specific treatments for HUS, and therefore more research into EHEC and HUS needs to be done. Our study focuses on Shiga-like toxin induction of endoplasmic reticulum (ER) stress in in vitro and in vivo systems, using human monocyte-like THP-1 cells and a non-human primate model of HUS. We used qPCR to determine the levels of ER stress marker expression induced by both Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) challenges. We also looked at ER stress marker expression in non-human primates that survived a lethal Stx2 challenge after being given a Stx2 binding tetravalent peptide. We expected to see increased ER stress marker expression in THP-1 cells challenged with both Shiga-like toxins and in animals that received lethal doses of the toxins. Although results were inconclusive for THP-1 cell experiments, our preliminary non-human primate data suggest that the timing of ER stress marker production is important, and Shiga-like toxins may suppress the unfolded protein response (UPR) in some baboon tissues. We also show that the therapeutic peptide TVP may reverse this UPR suppression and relieve ER stress leading to animal survival. Our study, along with the current literature, shows that Shiga-like toxin induced ER stress is a promising area for future study.