No description available.

Shiga toxin-producing E. coli (STEC) are globally relevant bacterial pathogens responsible for epidemic outbreaks of hemorrhagic diarrhea with variable progression to potentially fatal systemic Shiga toxicosis. Predictive clinical biomarkers and targeted therapeutic interventions for systemic Shiga toxicosis in diagnosed STEC patients are not available, and the impact of Shiga toxin production on STEC colonization and survival remain unclear. Improved murine models of STEC infection are needed to address knowledge gaps surrounding the gastrointestinal effects of Shiga toxins, as previously published models utilize ablation of host defense responses or microbiota depletion to facilitate colonization and are poorly suited for study of the effects of Shiga toxins on host responses. Dextran sulfate sodium (DSS) colitis in rodents has been associated with outgrowths of commensal E. coli in the literature, suggesting that DSS colitis could open a gastrointestinal niche usable by pathogenic STEC. This DSS colitis-based approach successfully induced susceptibility to robust colonization by two clinical isolate STEC strains in standard C57BL/6 mice. Studies using a Shiga-like toxin 2 (STX2)-producing clinical isolate STEC strain and its paired isogenic STX2 deletion strain (STEC(ΔSTX2)) revealed that STX2 was associated with delayed gastrointestinal clearance of STEC and concurrent reduction in colonic interleukin 23 (IL-23) axis transcripts known to be critical for pathogen clearance in other gastrointestinal pathogen models. In vivo reductions in IL-23 axis transcripts in the DSS+STEC model were supported by decreased IL-23 protein secretion by human macrophage-like cells during Shiga intoxication in vitro. Increased morbidity during STX2-producing STEC infection was associated with renal injury consistent with murine systemic Shiga toxicosis characterized by elevations in renal transcripts of molecular injury markers and histologically apparent renal tubular injury in a subset of mice. The dissertation research establishes a novel model of DSS colitis-facilitated murine STEC infection that recapitulates progression to systemic Shiga toxicosis in a subset of infected mice and demonstrates a clear STEC survival benefit associated with STX2 production. Shiga toxin-induced suppression of IL-23 axis signaling is a novel finding facilitated by the DSS+STEC model, demonstrating its utility for future delineation of the impacts of Shiga toxins on gastrointestinal host responses to STEC.

Shiga producing E. coli (STEC) O157 is a zoonotic pathogen. In humans STEC O157 causes bloody diarrhoea and potentially fatal renal failure. Cattle are the major reservoir, where bacteria are limited to the intestinal tract and do not cause clinical signs of disease. Previous studies indicate that shiga toxins produced by STEC O157, suppress STEC-specific cellular immune responses in vivo. This study aimed to initially examine the humoral immune response in cattle following natural challenge and the effects of a toxoid vaccination on this humoral STEC specific-immune response. We determined a statistically significant suppression in Tir specific IgA in STEC O157 positive cattle compared to O157 negative cattle but not in super shedding cattle. Following toxoid vaccination we determined a significant increase in flagellin specific IgG1 antibody levels in toxoid vaccinated animals despite lower numbers of positive faecal samples compared to placebo vaccinated controls. These results suggest that shiga toxins produced by STEC O157 are actively suppressing the STEC specific immune response in natural colonisation. To clarify this suppression further calves were orally challenged with STEC O157 (either a PT21/28 Stx2c+, PT32 Stx2c+ or PT21/28 Stx2a+Stx2c+ strain) and their STEC specific immune responses monitored. STEC specific systemic antibody responses were variable and weak in some cases. STEC specific local antibody responses were only significantly increased following challenge with the PT21/28 Stx2a+Stx2c+ challenge. Transcripts for genes associated with immune responses, and in particular B cell activation, at the terminal rectum were analysed by reverse transcriptase quantitative PCR. Suppression of IL2RA transcripts was observed in calves challenged with PT21/28 Stx2a+Stx2c+ compared to control calves but not with the other two STEC O157 strains tested. This study also aimed to determine the effects of cattle colonisation with STEC O157 on the immune response to a non-bacterial T-cell dependent antigen, ovalbumin (OVA). Cattle were orally challenged with either a PT21/28 Stx2c+, PT32 Stx2c+ or PT21/28 Stx2a+Stx2c+ strain or unchallenged. Calves were subcutaneously immunised with OVA five days post challenge, on two separate occasions with a two week interval. Lymphocytes from lymph nodes local to the immunisation site demonstrated significantly increased OVA-specific proliferation and OVA-specific activation of CD4+ and CD8+ cells in calves that were challenged with the PT21/28 Stx2c+ strain (but not with the other two challenge strains), compared to unchallenged controls. These results indicate that colonisation with STEC O157 can alter local adaptive immune responses to non-bacterial antigens in a strain dependent manner, unexpectedly enhancing the immune response rather than suppressing it. Circulating T cell responses were unaffected. In conclusion this study provides some further evidence of adaption of the host immune response by STEC O157, which is strain dependent, and variable. It seems unlikely from the data in this study that STEC O157 colonisation is having a major impact on the responses of cattle to other vaccines or infections in the field.

Enterohemorrhagic E. coli (EHEC) infection is a leading cause of acute kidney failure in otherwise healthy children, and a leading cause of foodborne illness with an outsized economic impact from outbreaks. EHEC secrete two Shiga-like toxins (Stx1 and Stx2) which are AB5 holotoxins that inhibit protein synthesis in cells expressing the toxin receptor Gb3. Infection with EHEC typically begins with a diarrheal prodrome that can progress in 5-15% of cases to hemolytic uremic syndrome (HUS), a clinical diagnosis characterized by thrombocytopenia, hemolytic anemia, and thrombotic microangiopathy. Historically, strains of EHEC expressing Stx2 have been associated with more severe disease. We hypothesized that tissue injury due to the toxins leads to the release of damage-associated molecular patterns (DAMPs), which act through inflammatory receptors to promote the endothelial dysfunction that drives this disease alongside the inciting Shiga toxins. Here we demonstrate that two well-characterized DAMPs, extracellular histones and HMGB1, are produced in two different mouse models when Stx2 is present; one model represents challenge with the toxin alone, and the second model introduces toxin through secretion with a lysogenized bacterium, C. rodentium, mimicking EHEC colonization. We investigate whether Stx1, Stx2, or histones affect the endothelial expression of well-characterized members of the protein C pathway, namely the endothelial protein C receptor (ECPR), protease-activated receptor 1 (PAR1), and thrombomodulin (TM), on human aortic (HAEC) and human renal glomerular endothelial cells (HRGEC). We show that Stx and/or histones reduce endothelial expression of these anti-coagulant molecules and histones dramatically increase expression of pro-thrombotic PAR-1. These changes lead to physiologically important decreases in activated protein C (APC), a critical anti-coagulant and cytoprotective molecule. Finally, we demonstrate that histones exacerbate thrombin's barrier-disruptive effects on the endothelium, and prevent APC's protective effects. These data provide novel mechanistic insight into the endothelial dysfunction that characterizes HUS and also provide a new perspective on systemic consequences of the bacterial Shiga toxins that might drive organ injury in susceptible patients.

Orientador: Tomomasa Yano / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T11:52:53Z (GMT). No. of bitstreams: 1 Bernedo-Navarro_RobertAlvin_D.pdf: 2646572 bytes, checksum: 02b50bc28d5be6cddb78abfe621a047c (MD5) Previous issue date: 2013 / Resumo: Escherichia coli produtora de toxina de Shiga (STEC) é um grupo de importantes patógenos para humanos. Essas bactérias são relacionadas a várias doenças, como por exemplo, Síndrome Urêmica Hemolítica e produzem potentes toxinas denominadas toxinas de Shiga. Essas toxinas, tanto Stx1 quanto Stx2, compartilham um receptor celular comum, a globotriaosilceramida (Gb3) e exibem a mesma atividade biológica intracelular. O desenvolvimento de novos agentes neutralizantes dos danos induzidos por Stx pode representar uma estratégia promissora para o tratamento das doenças causadas por STEC em humanos. No presente estudo, nós desenvolvemos peptídeos sintéticos que exibem atividade neutralizante contra a citotoxicidade induzida por Stx tanto in vitro quanto in vivo e, além disso, que se ligam eficientemente ao receptor Gb3. O peptídeo P12-26 compete eficientemente com Stx2 para a ligação ao Gb3 in vitro. Além disso, os peptídeos PC7-12, P12-26 e PC7-30 inibiram a citotoxicidade de Stx1 e Stx2 em células Vero. Nós observamos que o peptídeo PC7-30 em forma de loop e o peptídeo P12-26 que é linear produziram as maiores porcentagens de inibição de Stx1 e Stx2 em células Vero, respectivamente. No entanto, o peptídeo P12-26 não inibiu a letalidade em camundongos, enquanto que o peptídeo PC7-30 inibiu a letalidade causada pela toxina Stx1. Nossos resultados indicam que os peptídeos P12-26 e PC7-30 são candidatos promissores para o desenvolvimento de agentes terapêuticos contra as doenças em seres humanos causadas por STEC / Abstract: Shiga toxin-producing Escherichia coli strains are important pathogens for humans. These bacteria are linked with severe diseases such as hemolytic uremic syndrome and produce potent known as Shiga toxins. These toxins, Stx1 and Stx2, share a common cellular receptor called globotriaosylceramide (Gb3) and exhibit the same intracellular biological activity. The development of new neutralizing agents for Stx-induced damage may represent a promising strategy for the treatment of diseases caused by STEC infections. In this study, we developed synthetic peptides that exhibit neutralizing activity against Stxinduced cytotoxicity both in vitro and in vivo and that bind efficiently to the Gb3 receptor. The peptide P12-26 competed efficiently with Stx2 for binding to Gb3 in vitro. Moreover, the peptides PC7-12, P12-26 and PC7-30 inhibited the cytotoxicity of Stx1 and Stx2 in Vero cells. We observed that the loop-constrained peptide PC7-30 and linear peptide P12-26 produced higher percentages of inhibition of Stx1 and Stx2 in Vero cells, respectively. However, the peptide P12-26 did not inhibit lethality in mice, whereas the loopconstrained peptide PC7-30 inhibited the lethality caused by Stx1. Our results indicate that the peptides P12-26 and PC7-30 are promising candidates for the development of therapeutic agents against diseases caused by STEC in humans / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular