Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) is an emerging food- and water-borne pathogen, causing approximately 73,000 annual infections in the United States and an estimated 1.5 million infections globally. E. coli O157:H7, the most frequently associated EHEC strain, is primarily transmitted through consumption of contaminated ground beef and produce and leads to hemorrhagic colitis in humans. In 5% to 15% of infected patients, circulating Shiga toxins (Stx1, Stx2) cause hemolytic uremic syndrome (HUS), characterized by the presence of thrombocytopenia, hemolytic anemia, and thrombotic microangiopathy, contributing to acute kidney injury (AKI). Current treatment is supportive and antibiotic therapy is contraindicative as it increases toxin production. Therapeutics for EHEC-induced HUS need to be identified to minimize kidney injury and uncontrolled coagulopathy. Well-characterized animal models of HUS and EHEC infection are available and provide avenues for potential therapeutic discovery. Baboons (Papio) challenged with endotoxin-free Shiga toxins develop full spectrum HUS, and mice infected with Stx2-producing Citrobacter rodentium (Cr Stx2+), a genetically modified enteric mouse pathogen, develop severe Stx2-mediated kidney injury. Initial studies have shown that soluble thrombomodulin (sTM), an anti-coagulant, is a promising therapeutic in preventing severe kidney injury in pediatric patients. In these studies, we determined whether complement was activated in baboons challenged with Shiga toxins, and evaluated whether intraperitoneal injection of sTM would reduce disease severity from mice infected with Cr Stx2+. D-dimer and cell injury markers (HMGB1, histones) confirmed the presence of coagulopathy and cell injury in Stx challenged baboons. Studies revealed that complement activation is not required for the development of thrombotic microangiopathy and HUS induced by EHEC Shiga toxins in these pre-clinical models. Soluble thrombomodulin treatment in Cr Stx2+ infected mice significantly decreased colonization but did not alter mortality. However, gene expression of kidney injury markers (NGAL, KIM-1) decreased significantly compared to no treatment indicating sTM-associated cytoprotectivity. The C. rodentium mouse model does not develop the coagulopathy seen in HUS patients and sTM treatment may be more effective in the baboon toxemia model. Soluble thrombomodulin is a promising therapeutic for EHEC-induced HUS and should be further evaluated in Stx challenged baboons.

Shiga toxin (Stx) is a bacteriophage (phage)-encoded virulence factor of the Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 implicated in the pathogenesis of renal tissue damage and bloody diarrhoea in human. Cattle are the main asymptomatic reservoir for EHEC O157:H7 with the lymphoid-follicle rich areas of the terminal rectum identified as the primary colonisation site. However, the significance of Stx during bovine intestinal colonisation by EHEC O157:H7 remains unclear with mixed findings described in published studies. The objective of this study was to investigate if Stx and the Stx-encoding phage significantly contribute to EHEC O157:H7 colonisation particularly at the bovine terminal rectum. The expression of Stx receptor, Globotrioasylceramide (Gb3) at the bovine terminal rectum was analysed by fluorescence microscopy, revealing a similar pattern of Gb3 detection in the bovine colon with scattered positive detections limited to sub-epithelial, mesenchymal-associated cells. Purified Stx2 treatment of Gb3+ and Gb3- epithelial cell lines for 6 to 18 hours produced no effect on the cell cycle and proliferation. CD3+/CD8+, CD3+/γδ+ and CD21+ cells were significantly different between calves infected with EHEC O157:H7 Strain 9000 (Stx2a+/Stx2c+) and the uninfected calves, but not in calves with Strain 10671 (Stx2c+). Stx did not interfere with IFN-gamma (IFN-γ)-activation of the JAK/STAT1 pathway in epithelial cells. Bovine EHEC O157:H7 strains isolated from Scottish cattle farms in the IPRAVE study (Phage type 21/28 and 32) were used for a series of bacterial phenotypic characterisation assays. Total Stx production, Verocytotoxicity, growth in a competitive environment, epithelial cell adherence and Galleria mellonella virulence assay were performed to compare the IPRAVE EHEC O157:H7 strains (PT21/28 and PT32) and the isogenic Stx-phage mutants. Stx levels produced by the bovine-originated EHEC O157:H7 strains were significantly lower than that of the human isolated strains. The absence of Gb3 on the bovine terminal rectal epithelium, the non-significant changes in the cell cycle along with the uninterrupted IFN-γ activation of the JAK/STAT1 pathway in intestinal epithelial cells and the minute quantities of Stx generated by EHEC O157:H7 bovine strains suggest that the toxin is not involved in colonisation directly, at least at the intestinal epithelial level. Although future work is required to explain the mechanisms underlying the observed EHEC O157:H7 phenotypic changes particularly in the Stx-phage mutant strains, the work done has proven that the Stx-encoding phage indeed has the ability to exert changes in the bacterial cell leading to changes in bacterial phenotypes, which in turn, might affect the colonisation of the bovine intestine.

The Shiga-like toxins have DNA sequence homology to the toxins accountable for the dysentery brought about by the Shigella species. <i>Escherichia coli</i> which encode and produce shiga-like toxins are referred to as shiga toxin-producing E. coli (STEC). Upon infection with STEC, humans may develop a variety of clinical symptoms ranging in severity from bloody diarrhea to life threatening hemolytic uremic syndrome (HUS). Hemolytic uremic syndrome is the most fatal disease manifestation upon STEC infection for humans and has been documented to occur in up to 20% of patients upon STEC infection [29]. The Shiga toxins (Shiga toxin 1 and 2) are regarded as the principal virulence factor of STEC and are responsible for the clinical manifestations during HUS in humans [49].<p> Cattle are the primary non-human reservoir for STEC and therefore represent an attractive target for pre-slaughter intervention as a means to reduce human infections. To date, vaccination with secreted proteins including Shiga toxin 2 (Stx2), has reduced the numbers of bacteria shed in feces [3]. Even though published data exists supporting vaccination in cattle as a means to reduce STEC, commercially available vaccines are not being used by farms and STEC remain a significant zoonotic pathogen of humans causing disease and death. To further our knowledge about STEC pathogenesis in cattle, we examined the effect of Shiga toxin 2 on bovine immune responses. Bovine lymphocyte function was determined in the presence of Shiga toxin 2 and the magnitude of bovine immunological responses was measure after immunization with Shiga toxin 2. In general, results suggest that Shiga toxin 2 downregulates bovine immune responses suggesting vaccination with effector molecules that exclude Shiga toxin 2 may induce a better immunological response and improve vaccine efficacy.<p> To examine the possibility that Stx2 modulates bovine immune responses, we investigated lymphocyte function in the presence of Stx2. Menge et al [70] have reported that bovine lymphocytes express the Stx receptor and that Shiga toxin 1 inhibits lymphocyte proliferation in vitro. We isolated two populations of lymphocytes, peripheral blood mononuclear cells (PBMCs) and ileal Peyers patch lymphocytes (IPPL) and compared lymphocyte function in the presence and absence of Stx2. We found that Stx2 did not affect IPPL viability in vitro but did inhibit IPPL proliferation after 12 hours of incubation <i>in vitro</i>. In contrast, no altered PBMC function could be observed in the presence of Stx2. These results suggest that receptor-bound Stx2 may inhibit IPPL proliferation and that the two populations of lymphocytes isolated are unique and distinct from each other in their response to Stx2.<p> To determine the effect of Stx2 on bovine immune responses during STEC infection, a bovine ileal ligated loop model was employed. Ligated loops were inoculated with either a Stx2+ STEC strain or an isogenic Stx2- STEC strain. After 24 hours, IPPL populations were isolated from each ligated loop and immunophenotyped. The results indicated a significantly reduced CD4+ T cell population in the presence of Stx2. No differences in the levels of IFNá, TNFá, IL12 or IFNã could be detected between groups. These results suggest that Stx2 modulates bovine immune responses but not as a result of increased production of these cytokines. To extend this finding, we determined the effect of Stx2 on bovine immune responses during active immunization by using ELISA to measure serological responses in the presence and absence of Stx2. Serological responses to secreted proteins, as well as a co-administered antigen (hen egg lysozyme), were significantly reduced in the groups of cattle that were immunized with either purified Stx2 or secreted protein preparations isolated from STEC compared to groups vaccinated with antigens which did not contain the toxin. Bovine proliferative responses were also measured and the results indicated significantly reduced proliferation in the groups vaccinated with the formulations containing Stx2. Therefore, based on these results, we conclude that Stx2 downregulates bovine immune responses and thus may contribute to the colonization and persistence of cattle by STEC.

The Shiga-like toxins have DNA sequence homology to the toxins accountable for the dysentery brought about by the Shigella species. <i>Escherichia coli</i> which encode and produce shiga-like toxins are referred to as shiga toxin-producing E. coli (STEC). Upon infection with STEC, humans may develop a variety of clinical symptoms ranging in severity from bloody diarrhea to life threatening hemolytic uremic syndrome (HUS). Hemolytic uremic syndrome is the most fatal disease manifestation upon STEC infection for humans and has been documented to occur in up to 20% of patients upon STEC infection [29]. The Shiga toxins (Shiga toxin 1 and 2) are regarded as the principal virulence factor of STEC and are responsible for the clinical manifestations during HUS in humans [49].<p> Cattle are the primary non-human reservoir for STEC and therefore represent an attractive target for pre-slaughter intervention as a means to reduce human infections. To date, vaccination with secreted proteins including Shiga toxin 2 (Stx2), has reduced the numbers of bacteria shed in feces [3]. Even though published data exists supporting vaccination in cattle as a means to reduce STEC, commercially available vaccines are not being used by farms and STEC remain a significant zoonotic pathogen of humans causing disease and death. To further our knowledge about STEC pathogenesis in cattle, we examined the effect of Shiga toxin 2 on bovine immune responses. Bovine lymphocyte function was determined in the presence of Shiga toxin 2 and the magnitude of bovine immunological responses was measure after immunization with Shiga toxin 2. In general, results suggest that Shiga toxin 2 downregulates bovine immune responses suggesting vaccination with effector molecules that exclude Shiga toxin 2 may induce a better immunological response and improve vaccine efficacy.<p> To examine the possibility that Stx2 modulates bovine immune responses, we investigated lymphocyte function in the presence of Stx2. Menge et al [70] have reported that bovine lymphocytes express the Stx receptor and that Shiga toxin 1 inhibits lymphocyte proliferation in vitro. We isolated two populations of lymphocytes, peripheral blood mononuclear cells (PBMCs) and ileal Peyers patch lymphocytes (IPPL) and compared lymphocyte function in the presence and absence of Stx2. We found that Stx2 did not affect IPPL viability in vitro but did inhibit IPPL proliferation after 12 hours of incubation <i>in vitro</i>. In contrast, no altered PBMC function could be observed in the presence of Stx2. These results suggest that receptor-bound Stx2 may inhibit IPPL proliferation and that the two populations of lymphocytes isolated are unique and distinct from each other in their response to Stx2.<p> To determine the effect of Stx2 on bovine immune responses during STEC infection, a bovine ileal ligated loop model was employed. Ligated loops were inoculated with either a Stx2+ STEC strain or an isogenic Stx2- STEC strain. After 24 hours, IPPL populations were isolated from each ligated loop and immunophenotyped. The results indicated a significantly reduced CD4+ T cell population in the presence of Stx2. No differences in the levels of IFNá, TNFá, IL12 or IFNã could be detected between groups. These results suggest that Stx2 modulates bovine immune responses but not as a result of increased production of these cytokines. To extend this finding, we determined the effect of Stx2 on bovine immune responses during active immunization by using ELISA to measure serological responses in the presence and absence of Stx2. Serological responses to secreted proteins, as well as a co-administered antigen (hen egg lysozyme), were significantly reduced in the groups of cattle that were immunized with either purified Stx2 or secreted protein preparations isolated from STEC compared to groups vaccinated with antigens which did not contain the toxin. Bovine proliferative responses were also measured and the results indicated significantly reduced proliferation in the groups vaccinated with the formulations containing Stx2. Therefore, based on these results, we conclude that Stx2 downregulates bovine immune responses and thus may contribute to the colonization and persistence of cattle by STEC.

The Shiga-like toxins have DNA sequence homology to the toxins accountable for the dysentery brought about by the Shigella species. Escherichia coli which encode and produce shiga-like toxins are referred to as shiga toxin-producing E. coli (STEC). Upon infection with STEC, humans may develop a variety of clinical symptoms ranging in severity from bloody diarrhea to life threatening hemolytic uremic syndrome (HUS). Hemolytic uremic syndrome is the most fatal disease manifestation upon STEC infection for humans and has been documented to occur in up to 20% of patients upon STEC infection [29]. The Shiga toxins (Shiga toxin 1 and 2) are regarded as the principal virulence factor of STEC and are responsible for the clinical manifestations during HUS in humans [49]. Cattle are the primary non-human reservoir for STEC and therefore represent an attractive target for pre-slaughter intervention as a means to reduce human infections. To date, vaccination with secreted proteins including Shiga toxin 2 (Stx2), has reduced the numbers of bacteria shed in feces [3]. Even though published data exists supporting vaccination in cattle as a means to reduce STEC, commercially available vaccines are not being used by farms and STEC remain a significant zoonotic pathogen of humans causing disease and death. To further our knowledge about STEC pathogenesis in cattle, we examined the effect of Shiga toxin 2 on bovine immune responses. Bovine lymphocyte function was determined in the presence of Shiga toxin 2 and the magnitude of bovine immunological responses was measure after immunization with Shiga toxin 2. In general, results suggest that Shiga toxin 2 downregulates bovine immune responses suggesting vaccination with effector molecules that exclude Shiga toxin 2 may induce a better immunological response and improve vaccine efficacy. To examine the possibility that Stx2 modulates bovine immune responses, we investigated lymphocyte function in the presence of Stx2. Menge et al [70] have reported that bovine lymphocytes express the Stx receptor and that Shiga toxin 1 inhibits lymphocyte proliferation in vitro. We isolated two populations of lymphocytes, peripheral blood mononuclear cells (PBMCs) and ileal Peyer’s patch lymphocytes (IPPL) and compared lymphocyte function in the presence and absence of Stx2. We found that Stx2 did not affect IPPL viability in vitro but did inhibit IPPL proliferation after 12 hours of incubation in vitro. In contrast, no altered PBMC function could be observed in the presence of Stx2. These results suggest that receptor-bound Stx2 may inhibit IPPL proliferation and that the two populations of lymphocytes isolated are unique and distinct from each other in their response to Stx2. To determine the effect of Stx2 on bovine immune responses during STEC infection, a bovine ileal ligated loop model was employed. Ligated loops were inoculated with either a Stx2+ STEC strain or an isogenic Stx2- STEC strain. After 24 hours, IPPL populations were isolated from each ligated loop and immunophenotyped. The results indicated a significantly reduced CD4+ T cell population in the presence of Stx2. No differences in the levels of IFNá, TNFá, IL12 or IFNã could be detected between groups. These results suggest that Stx2 modulates bovine immune responses but not as a result of increased production of these cytokines. To extend this finding, we determined the effect of Stx2 on bovine immune responses during active immunization by using ELISA to measure serological responses in the presence and absence of Stx2. Serological responses to secreted proteins, as well as a co-administered antigen (hen egg lysozyme), were significantly reduced in the groups of cattle that were immunized with either purified Stx2 or secreted protein preparations isolated from STEC compared to groups vaccinated with antigens which did not contain the toxin. Bovine proliferative responses were also measured and the results indicated significantly reduced proliferation in the groups vaccinated with the formulations containing Stx2. Therefore, based on these results, we conclude that Stx2 downregulates bovine immune responses and thus may contribute to the colonization and persistence of cattle by STEC.

abstract: In the United States, Escherichia coli O157:H7 (E. coli O157:H7) is the most frequent cause of hemolytic uremic syndrome (HUS) and it is also the primary cause of acute renal failure in children. The most common route of the infection is ingestion of contaminated meat or dairy product originating from cattle or vegetables contaminated with bovine manure. Since cattle are the main reservoir for human infection with E. coli O157:H7, the reduction of intestinal colonization by these bacteria in cattle is the best approach to prevent human infections. Intimin is an outer membrane protein of E. coli O157:H7 that plays an important role in adhesion of the bacteria to the host cell. Hence, I proposed to express intimin protein in tomato plants to use it as a vaccine candidate to reduce or prevent intestinal colonization of cattle with E. coli O157:H7. I expressed His-tagged intimin protein in tomato plants and tested the purified plant-derived intimin as a vaccine candidate in animal trials. I demonstrated that mice immunized intranasally with purified tomato-derived intimin produced intimin-specific serum IgG1and IgG2a, as well as mucosal IgA. I further demonstrated that mice immunized with intimin significantly reduced time of the E. coli O157:H7 shedding in their feces after the challenge with these bacteria, as compared to unimmunized mice. Shiga toxin is the major virulence factor that contributes to HUS. Since Shiga toxin B subunit has an important role in the attachment of the toxin to its receptor, I fused intimin to Shiga toxin B subunit to create multivalent subunit vaccine and tested the effects upon immunization of mice with the B subunit when combined with intimin. His-tagged intimin, Shiga toxin B subunit, and Shiga toxin-intimin fusion proteins were expressed in E. coli and purified. I demonstrated that this multivalent fusion protein vaccine candidate elicited intimin- and Shiga toxin B-specific IgG1, IgG2a, and IgA antibodies in mice. I also showed a reduction in the duration of the bacterial shedding after the challenge compared to the control sham-immunized groups. / Dissertation/Thesis / Ph.D. Plant Biology 2010

Master of Public Health / Department of Diagnostic Medicine and Pathobiology / Robert Larson / Among animal products consumed by humans, ground beef has been reported as one of the most common vehicles for STEC outbreaks in humans. In the United States, cull dairy cattle contribute as one of the primary sources for ground beef. The objective of this study was to determine the prevalence and concentration of 7 Shiga toxin-producing Escherichia coli serogroups (STEC-7; O26, O103, O111, O121, O45, O145, and O157) and associated virulence genes (Shiga toxin 1 and 2 (stx1, stx2), intimin (eae), and enterohomolysin (ehxA)) in the feces of cull dairy cattle processed in commercial slaughter plants during summer months. Fecal swab samples (n=183) were collected from three processing plants, one in California and two in Pennsylvania. At each plant at least 60 to 65 cattle were selected, and the samples were obtained by swabbing the mucosal surface of the recto-anal junction using a sterile cotton-tipped applicator. To determine prevalence, all samples were subjected to culture-based detection methods that included enrichment, serogroup-specific immunomagnetic separation and plating on selective media, followed by polymerase chain reaction for serogroup confirmation and virulence gene detection. Pre-enriched fecal samples were subjected to spiral plating to determine the concentration of STEC-7. A sample was considered STEC positive if a recovered isolate harbored one of the 7 target O genes, stx1, and/or stx2. Of the 183 fecal swab samples collected, 23 (12.6%) harbored at least one O157, O26, O103, or O111 serogroup, with their associated virulence genes. However, none of the fecal samples from this cattle population carried STEC at high-levels (>10⁴ CFU/g). This study has provided important information on STEC-7 prevalence from dairy cattle that enter the ground beef processing system. However, there is still a need to determine prevalence and concentration of STEC in cull dairy cattle during winter months as well as in other sources of ground beef production (e.g., imported lean beef, cull beef).

Sub-acute/chronic inflammatory diseases are often associated with altered inflammatory response, leading to increased host vulnerability to secondary inflammatory challenges. In the first study, by employing streptozotocin (STZ)-induced diabetes in mice, we further investigate mechanisms leading to enhanced polymorphonuclear leukocytes (PMN) response under hyperglycemia. We show that existence of a proinflammatory state associated with broad increases of macrophages in various organs plays a dominant role in promoting PMN response in diabetic mice. Studies of PMN infiltration during zymosan-induced peritonitis reveal that hyperglycemia enhances PMN recruitment through increasing F4/80+ macrophages in the peritoneal cavity. Insulin reversal of hyperglycemia reduces peritoneal macrophage numbers and ameliorates PMN infiltration. Significantly increased macrophages are also observed in the liver, kidneys, and intestines under hyperglycemia, and are attributable to exacerbated nephropathy and colitis when respective inflammatory conditions are induced. We also find that significant monocytosis of inflammatory F4/80+Gr-1+ monocytes from the spleen and macrophage proliferation in situ synergistically contribute to the increased macrophage population under hyperglycemia. In conclusion, our results demonstrate that STZ-induced hyperglycemic/diabetic mice develop a systemic proinflammatory state mediated by broad infiltration of macrophages. In the second study, we focus on the identification of the carrier that binds to and delivers Shiga toxin 2(Stx2) to the target organ causing hemolytic uremic syndrome (HUS). By employing a murine HUS model through co-injection of LPS-Stx2, we show that, adoptive transfer of CD11b+ leukocytes, but not CD11b- leukocytes, RBC, platelets or plasma, isolated from mice with HUS induces HUS in healthy recipients. Interestingly, we find that LPS priming of mice significantly promotes CD11b+ leukocytes binding to Stx2. Compared to CD11b+ leukocytes from mice without LPS priming, CD11b+ leukocytes isolated from mice after LPS priming demonstrate higher frequencies of toxin binding and augmented potency to induce HUS. In sum, our results demonstrate peripheral CD11b+ myeloid leukocytes act as effective Stx2 carriers that deliver toxin to kidneys causing HUS and that LPS-induced inflammation enhances the carrier capacity and aggravates HUS.

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