Shiga toxin-producing E. coli (STEC) comprise a group of pathogenic organisms that elaborate a family of protein exotoxins known as Shiga toxins (Stxs). Intestinal infection with these organisms may lead to hemorrhagic colitis and hemolytic uremic syndrome, a life-threatening condition characterized by thrombocytopenia, non-immune hemolytic anemia, and acute renal failure. Vascular endothelial damage is believed to be a key initiating event in Stx-mediated diseases. At the molecular level, these toxins depurinate human 28S rRNA and inhibit translation. In addition, at concentrations that only minimally affect global protein synthesis, they have been found to alter expression of specific target genes. To better understand the endothelial damage induced by Stx, we investigated the global effects of Stx on endothelial gene expression, and defined a specific group of genes whose expression was altered by the toxin. Of interest, the CXCR4/CXCR7/SDF-1 chemokine pathway, a pathway central to vascular biology, was activated by Stx. In vitro studies demonstrated that Stx enhanced both transcript levels of these molecules, as well as their association with ribosomes. To define the relevance of these findings in vivo, a mouse model was established and key changes were noted in plasma and tissue content of CXCR4/CXCR7/SDF-1 following Stx exposure. Inhibition of CXCR4/SDF-1 interaction decreased indices of endothelial activation and organ injury and improved animal survival. Importantly, in children infected with E. coli O157:H7, plasma SDF-1 levels were significantly elevated in individuals who progressed to hemolytic uremic syndrome. A second pathway critical to endothelial health and function is VEGF signaling. Of interest, our endothelial gene expression analyses revealed changes in this pathway in vitro. VEGF mRNA association with cellular polyribosomes increased following Stx treatment. Further studies in vivo demonstrated decreased cardiac function and blood pressure, and increased vascular permeability in specific tissues. VEGF, an important inducer of vascular permeability, increased in mouse plasma. Additionally, altered mRNA expression was observed in key organs, such as the kidney and heart, following Stx challenge. Inhibition of VEGF significantly improved survival of animals treated with Stx, indicating that VEGF plays a role in Stx-mediated pathogenesis. Moreover, in vitro studies demonstrated that Stx-mediated endothelial permeability was attenuated in the presence of a VEGF inhibitor. Taken together, these data indicate that E. coli-derived Stxs induce pathological changes in two pathways key to vascular biology. These pathways represent novel targets for the development of preventative and therapeutic strategies for complications associated with Shiga toxin-producing E. coli infection.

Shiga toxin-producing E. coli (STEC) comprise a group of pathogenic organisms that elaborate a family of protein exotoxins known as Shiga toxins (Stxs). Intestinal infection with these organisms may lead to hemorrhagic colitis and hemolytic uremic syndrome, a life-threatening condition characterized by thrombocytopenia, non-immune hemolytic anemia, and acute renal failure. Vascular endothelial damage is believed to be a key initiating event in Stx-mediated diseases. At the molecular level, these toxins depurinate human 28S rRNA and inhibit translation. In addition, at concentrations that only minimally affect global protein synthesis, they have been found to alter expression of specific target genes. To better understand the endothelial damage induced by Stx, we investigated the global effects of Stx on endothelial gene expression, and defined a specific group of genes whose expression was altered by the toxin. Of interest, the CXCR4/CXCR7/SDF-1 chemokine pathway, a pathway central to vascular biology, was activated by Stx. In vitro studies demonstrated that Stx enhanced both transcript levels of these molecules, as well as their association with ribosomes. To define the relevance of these findings in vivo, a mouse model was established and key changes were noted in plasma and tissue content of CXCR4/CXCR7/SDF-1 following Stx exposure. Inhibition of CXCR4/SDF-1 interaction decreased indices of endothelial activation and organ injury and improved animal survival. Importantly, in children infected with E. coli O157:H7, plasma SDF-1 levels were significantly elevated in individuals who progressed to hemolytic uremic syndrome. A second pathway critical to endothelial health and function is VEGF signaling. Of interest, our endothelial gene expression analyses revealed changes in this pathway in vitro. VEGF mRNA association with cellular polyribosomes increased following Stx treatment. Further studies in vivo demonstrated decreased cardiac function and blood pressure, and increased vascular permeability in specific tissues. VEGF, an important inducer of vascular permeability, increased in mouse plasma. Additionally, altered mRNA expression was observed in key organs, such as the kidney and heart, following Stx challenge. Inhibition of VEGF significantly improved survival of animals treated with Stx, indicating that VEGF plays a role in Stx-mediated pathogenesis. Moreover, in vitro studies demonstrated that Stx-mediated endothelial permeability was attenuated in the presence of a VEGF inhibitor. Taken together, these data indicate that E. coli-derived Stxs induce pathological changes in two pathways key to vascular biology. These pathways represent novel targets for the development of preventative and therapeutic strategies for complications associated with Shiga toxin-producing E. coli infection.

Cancer cells display aberrant receptors on their surface that can serve as targets for the development of directed drug therapies. As such, our group has utilized two parallel approaches to redirect the cytotoxic properties of a ribosome-inactivating protein (RIP), Shiga-Like Toxin 1 (SLT 1), by altering its receptor specificity to target and kill cancer cells. The first combinatorial protein library was constructed such that a randomized 7 AA long peptide was inserted within the cytotoxic domain (A chain) of SLT-1. A high-throughput protein-based screening campaign identified a novel A chain toxin variant (named SLT 1AIYSNKLM) capable of targeting and killing human melanoma cells. This variant harbours a peptide insert (IYSNKLM) that directs the A chain to kill human melanoma cell lines. Equilibrium binding studies using 125I-radiolabeled SLT-1AIYSNKLM were conducted to determine the equilibrium binding constant and receptor density on 518-A2 human melanoma cells. When injected into SCID mice bearing a human melanoma xenograft, nanoSPECT/CT imaging as well as the biodistribution profile showed marked tumour uptake and retention of the radiolabeled toxin variant. Furthermore, preliminary experiments have shown that the SLT-1AIYSNKLM receptor is a protein, highlighting the potential for this method to be used in the discovery of novel biomarkers. A second approach was employed to demonstrate that our toxin-based combinatorial library system can be adapted to target known cancer biomarkers. Specifically, SLT-1 A chain variants harbouring 12-residue inserts were expressed in a phage display library. The library was screened against cell lines expressing the human colon cancer marker carcinoembryonic antigen (CEA; CD66e; CEACAM-5) to identify candidates that not only targeted, but internalized into cancer cells within a 1 h period. Variant, CSTA-10, was found to kill CEA-expressing BxPC-3 cells. Overall, the directed evolution of an RIP template such as SLT-1 represents a novel and powerful strategy for the identification of tumour-targeted toxin variants.

Subtilase cytotoxin (SubAB) is the prototype of a novel class of AB₅ cytotoxins produced by Shiga-toxigenic Escherichia coli (STEC). The A subunit (SubA) is a serine protease that cleaves the ER chaperone BiP causing cell death by a previouslyundetermined mechanism. The B subunits of AB₅toxins typically recognise host cell glycan receptors and direct the subcellular transport of the A subunit. Although the function of SubA and its intracellular substrate have been elucidated, the B subunit (SubB) is relatively uncharacterised. The subcellular trafficking pathway of SubAB was initially examined. SubAB conjugated to Oregon Green 488 (SubAB-OG) was internalised by Vero cells by 5 min, and co-localised with its ER target BiP within 30 min. When Vero cells were incubated with SubAB-OG and either Alexa Fluor 594-conjugated Cholera toxin B subunit (CtxBAF594) or Texas Red-conjugated Shiga toxin B subunit (StxB-TR), individual cells exhibited differential toxin uptake. This was shown to be cell cycle-dependent, in which, SubAB-OG was preferentially internalised by cells migrating through G1 and early S phases. In contrast, CtxB-AF594 was taken up by cells in S through M phases and by a majority of cells in G1, while StxB-TR endocytosis occurred in cells traversing G1. Fluorescent SubAB co-localised with the clathrin marker transferrin, but not with Caveolin-1 (a marker for cholesterol-associated caveolae) and was subsequently trafficked via a retrograde pathway to the TGN, Golgi and ER. The clathrin inhibitor phenylarsine oxide prevented SubAB entry and BiP cleavage in SubAB-treated Vero, HeLa and N2A cells, while cholesterol depletion did not, demonstrating that, unlike either Stx or Ctx, SubAB internalisation is exclusively clathrin-dependent. Identification of the SubB receptor was initially approached using toxin overlay assays in which Vero cell glycolipid extracts were separated by thin-layer chromatography and overlaid with SubAB. SubAB exhibited a high affinity for particular acidic species in the ganglioside fraction. However, none co-migrated with commercial glycolipid standards. SubAB-OG also exhibited an affinity for the oligosaccharide structures of chimeric LPS from GM₂ and GM₃ bacterial receptor mimic constructs in an LPS toxin overlay assay. Glycan array analysis revealed that SubB possessed a unique affinity for carbohydrate receptors with a terminal Neu5Gcα(2→3)Galβ disaccharide. Monovalent receptor analogues with distal Neu5Gc or Neu5Gcα(2→3)Galβ and highly-sialylated α₁-AGP did not prevent endocytosis of SubAB-OG, BiP cleavage or cytotoxicity in Vero cells. This indicated that SubAB has a greater affinity for the host cell receptors than the receptor analogues and may engage multiple receptors displayed on a lipid bilayer. In addition to mediating toxin binding and subcellular trafficking, CtxB and StxB can also potentiate the immune response to co-administered antigen. Accordingly, the systemic immunomodulatory properties of SubB administered by the i.p. route were assessed in mice. Using SubAA₂₇₂ as a bystander antigen, SubB significantly increased mouse anti-SubAA₂₇₂ titres to levels that were comparable to those obtained using Alum adjuvant. However, when admixed with structurally-unrelated OVA, SubB did not significantly affect anti-OVA titres whereas Alum and CtxB did. This indicated that SubB may function as a systemic carrier protein (rather than an adjuvant) for particular antigens. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1363363 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2009

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

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-10-03Bitstream added on 2014-06-13T19:22:44Z : No. of bitstreams: 1 rodolpho_d_dr_jabo.pdf: 299270 bytes, checksum: 8a1668550cacade414ec08c3350e51f4 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Escherichia coli Shigatoxigênica (STEC) tem sido implicada como agente causador de severas doenças humanas. Amostras de carne moída, moedor de carne e mãos de manipuladores de 23 estabelecimentos comerciais foram testadas para o isolamento de E. coli usando métodos microbiológicos padronizados. Um total de 287 cepas de E. coli isoladas destes diferentes locais foram submetidas ao PCR para detecção de genes stx 1, stx 2 e eae. As cepas positivas para o gene stx foram analisadas verificando se pertenciam ao sorogrupo 0157. Quatro cepas de STEC foram isoladas, sendo 2 de carne moída e 2 de moedor de carne, todas possuíam o gene stx 2, sendo negativas para a presença do gene eae e o sorogrupo 0157. Todas as E. coli isoladas, incluindo as 4 STEC, foram pesquisadas para sua resistência a 12 antibióticos. Altos níveis de resistência frente aos diferentes agentes antimicrobianos foram detectados; as resistências maiores foram observadas para a tetraciclina (76,6%), amoxicilina (64,1 %) e cefalotina (58,8%). Os altos níveis de resistência antimicrobiana salientam a necessidade para a utilização racional destes agentes em bovinos. Foram observadas índices elevados de sensibilidade frente a associação amoxicilina + ácido clavulânico (96,6%), ceftriaxona (92,7%) e gentamicina (90,3%). / Shiga toxigenic Escherichía colí (STEC) has been implicated as the causative agent of several human diseases. Samples from 23 retail meat stores (ground beef, grinding-machine and human hand) were assayed for E. calí isolation using microbiological standard methods. A total of 287 E. colí isolates from these different origins were submitted to polymerase chain reaction for the detection of stx 1, stx 2 and eae genes. The isolates positives for stx gene were serotyped for 0157. Four STEC isolates were recovered, 2 from ground beef and 2 from grinding-machine; ali harbored the stx 2 gene and were negative for the presence of the eae gene and the serogroup 0157. Ali E. colí isolates including the four STEC were screened for antibiotic resistance. High levels of resistance against different antimicrobial agents were detected; those most commonly observed were to tetracycline (76.6%), amoxicillin (64.1 %) and cephalothin (58.8%). Such high levels of antimicrobial agents' resistance highlight the need for a more rational use of these agents in cattle. Susceptibility was high for amoxicillin + clavulanic acid (96,6%), ceftriaxone (92,7%) and gentamicin (90,3%).

Orientador: Cristiano Gallina Moreira / Banca: Waldir Pereira Elias Nunes / Banca: Carla Raquel Fontana Mendonça / Resumo: A sinalização química é o mecanismo através do qual bactérias patogênicas interagem com o hospedeiro e sua microbiota, de modo a promover a regulação dos seus mecanismos de virulência. O estudo da sinalização química, em bactérias entéricas, do sistema de 2-componentes QseBC via autoindutor-3 (AI-3), epinefrina (Epi) e norepinefrina (NE), tem aberto perspectivas para desvendar novos mecanismos. Escherichia coli O104:H4 possui características fenotípicas clássicas de E. coli enteroagregativa (EAEC) e se apresenta positiva para toxina de Shiga, encontrada em cepas de E. coli enterohemorrágica (EHEC). A presença dessa toxina pode levar o hospedeiro ao desenvolvimento de complicações mais graves, como a síndrome hemolítica urêmica (SHU). Dessa forma, essa combinação se torna altamente perigosa e patogênica a humanos, conforme observado no surto epidêmico em 2011 na Europa. O presente estudo teve como objetivo investigar a sinalização química e os mecanismos de patogenicidade em EAEC O104:H4 C227-11, já descritos em EAEC e EHEC, bem como buscar mecanismos ainda não elucidados na literatura. Comparada com cepas protótipos de E. coli diarreiogênicas, os resultados demonstraram que a cepa C227-11 possui um fenótipo de adesão e formação de biofilme acentuados. Em meio ácido, apresentou mais robustez na sobrevivência e maior motilidade em relação à EAEC 042. Também foi possível observar que o nível de expressão gênica para qseC apresentou-se semelhante ao de EHEC e exerce um importante... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Chemical signalling is the mechanism through which pathogenic bacteria interact with the host microbiota, in order to promote regulation of virulence mechanisms. The study of chemical signalling in two-component system QseBC in enteric bacteria, by autoinducer3 (AI-3), epinephrine (Epi) e norepinephrine (NE), has opened perspectives to discover new mechanisms. Escherichia coli O104:H4 has classic phenotypic characteristics of enteroaggregative E. coli (EAEC) and presents itself positive for Shiga toxin, which is found in enterohemorrhagic E. coli strains (EHEC). This toxin may lead the host to the development of more serious diseases, such as the Hemolytic Uremic Syndrome (HUS). Therefore, this combination is highly dangerous and pathogenic to humans, as observed during the epidemic outbreak in Europe in 2011. This study aimed to investigate chemical signalling and mechanisms of pathogenicity in EAEC O104:H4 C227-11, already described in EAEC and EHEC, as well as mechanisms still not elucidated in literature. Compared to prototype strains of diarrheagenic E. coli, the results have shown that C227- 11 has accentuated phenotype of adhesion and biofilm formation. In acid medium, it presented more strength of survival and more motility than EAEC 042. In addition, gene expression level in qseC was found similar to EHEC and it holds an important participation in activation of virulence factors expression, highlighting, thereby, the possibility of its participation in related mechanisms. Through in vivo tests, it was noticed considerable variation of microbiota, compared to C227-11, just as significant differences among other EAEC that mostly did not present great alterations during analysed days, with predominance of Bacteroidetes over Firmicutes Phylum. The conclusion was that its large profile of adhesion and its elevated tolerance... (Complete abstract click electronic access below) / Mestre

Master of Science / Entomology / Ludek Zurek / House flies (Musca domestica L.) and stable flies (Stomoxys calcitrans L.) are insects of medical and veterinary importance. House flies are recognized as mechanical vectors of human foodborne pathogens and stable files are known for their painful bites resulting in reduction of body weight gain and milk production in cattle. The larval development of both fly species takes place in decaying organic materials (primarily animal manure), resulting in large fly populations in confined cattle environments. Shiga toxin-producing Escherichia coli (STEC) are a major foodborne pathogen. Cattle are the asymptomatic reservoir of STEC with bacteria being released to the environment via their feces. STEC O157 is the main serogroup causing human illness. However, infections with non-O157 STEC are increasing: more than 70% of non-O157 infections are caused by six serogroups of non-O157, referred as "Big six" (O26, O45, O103, O111, O121, and O145). In addition, there was a large 2011 outbreak in Europe caused by STEC O104. The objectives of my thesis were: 1) To assess the prevalence of seven serogroups of non-O157 STEC (O26, O45, O103, O104, O111, O121, and O145) (STEC-7) in house flies and stable flies collected from confined cattle environments; 2) To investigate the vector competence of house flies for non-O157 STEC-7. A total of 463 house flies from feedlots and dairies from six states, and 180 stable flies collected from a feedlot in Nebraska were processed for the isolation and identification of STEC-7 using a culture-based approach followed by PCR for the confirmation of serogroups, and virulence genes. A total of 34.3% of house flies and 1.1% of stable flies tested positive for at least one serogroup of E. coli of interest, and 1.5% of house flies harbored STEC with the Shiga-toxin gene (stx1). No STEC were detected in stable flies. Vector competence bioassays for non-O157 STEC revealed that house flies can carry non-O157 STEC for at least six days with the exception STEC O145. Overall, the findings of this research demonstrate that house flies, but not stable flies, likely play an important role in the ecology and transmission of non-O157 STEC in confined cattle environments.

Master of Science / Food Science Institute - Animal Sciences and Industry / Randall K. Phebus / A variety of antimicrobial processes are used to reduce pathogen risks on commercially processed raw beef. Little research has evaluated chlorinated water on beef tissues, especially in a processing water dip scenario. Interest in nanobubble technology has increased due to its proposed surfactant properties, but it is undetermined whether this improves antimicrobial effectiveness of chlorine-based solutions in food applications. Benchtop studies were conducted to evaluate chlorinated nanobubble waters (0 to 11.94 ppm) against Shiga toxin-producing Escherichia coli O26, O45, O103, O111, O121, O145, and O157:H7 (STEC-7), Salmonella spp., and USDA-approved non-pathogenic STEC surrogates 1) in pure culture with the goal of characterizing the lethality contributions of pH (5 or 7), temperature, free available chlorine level (FAC), inclusion of nanobubbles, or a combination thereof; 2) in select chlorinated nanobubble “red water” (water containing 0.1% beef purge) solutions; and 3) on the surface of lean and fat beef tissue. In pure culture solutions, surrogates demonstrated greater resistance (P ≤ 0.05) to chlorinated solutions (3.4-5.5 log CFU/mL reductions) with increased reductions at the higher (11.94 ppm) FAC levels. STEC-7 and Salmonella population reductions were also notably reduced (3.3-7.1 log CFU/mL) by the higher FAC concentrations. No definitive impacts of temperature, nanobubble inclusion, or acidic pH were observed. At an average 5.23 ppm FAC in red water, all microbial populations were reduced by > 6 log CFU/mL after 60 minutes. Reductions of target organisms on inoculated lean and fat tissues were ≤ 1 log CFU/g in red water; likely due to the inability to maintain FAC levels above 0.7 ppm in the presence of organic loading. An in-plant antimicrobial validation study of a proprietary raw beef manufacturing process was conducted to determine the effectiveness of a recirculating acidic nanobubble water system, chlorinated to 5 ppm FAC using EO water generated concentrate, against the USDA-approved STEC surrogates. Preliminarily, inoculated beef trim was introduced into the system targeting 5 ppm FAC; chlorine concentrate reinfusion rates were determined to establish applicable operational parameters and sampling strategies for the system. An optimized in-plant study was conducted. Meat inoculated at ~ 7 log CFU/g was introduced into the recirculating chlorinated nanobubble system every other day over 6 days, achieving an average 1.6 log CFU/g surrogate reduction on inoculated meat throughout the manufacturing process. Approximately 2.7 log CFU/g of residual surrogates were recovered on non-inoculated meat ~35 minutes after inoculated meat entered the system, indicating that harborage of microbial contamination on processing equipment can lead to subsequent contamination carry-over that must be controlled during processing. Surrogate organisms were recovered by enrichment only from non-inoculated meat 24 h after inoculated meat processing on alternate days, likely stemming from inadequately sanitized processing equipment after inoculated batch processing. Control of the residual surrogate population in the system following inoculation was accomplished through daily equipment sanitation and boosting recirculated processing water to 50 ppm during a 4-h sanitation period (no beef entering system). The optimized study will be used as an antimicrobial process validation against STEC and Salmonella spp. in beef manufacturing.

Orientador: José Paes de Almeida Nogueira Pinto / Banca: Márcio Garcia Ribeiro / Banca: Vera Lúcia Mores Rall / Resumo: A carne bovina pode ser um importante veículo de vários patógenos para os humanos, com destaque à Escherichia coli produtora de Shiga-toxina (STEC), associada com diarreia em animais e humanos. Neste estudo, investigou-se em bovinos abatidos no estado de São Paulo, a prevalência dos diferentes patótipos de E. coli diarreiogênica e o perfil de virulência dos isolados de STEC. De um total de 431 animais, STEC foi identificada em 116 (26,9%) amostras de fezes, das quais 111 (25,8%) STEC eae- e 5 (1,2%) STEC eae+. O patótipo EPEC foi detectado em 20 (4,6%) amostras de fezes dos animais testados. Os demais patótipos de E. coli diarreiogênica não foram identificados. Dos 95 isolados de STEC analisados quanto ao perfil de virulência, todos albergavam stx2, enquanto que 28 (29,5%) continham stx1. Os genes iha e saa, que codificam adesinas, foram encontrados em 93,7% (89/95) e 66,3% (63/95), respectivamente. O gene espP, que codifica uma protease que auxilia na colonização intestinal, foi detectado em 61,1% (58/95) e a hemolisina ehxA em 54,7% (52/95). Também foram identificados em menores frequências os genes subAB, nleE e nleB. STEC está amplamente disseminada nos rebanhos bovinos de São Paulo, carreando genes comumente isolados de patógenos humanos, o que reforça a importância da inspeção e fiscalização nos abatedouros / Abstract: Beef can be an important vehicle for various pathogens to humans, especially Shiga toxin-producing Escherichia coli (STEC), associated to human and animal diarrhea. In this study, the prevalence of different pathotypes of diarrheagenic E. coli, and virulence profiles of STEC were investigated among feces of cattle slaughtered in São Paulo state, southeast of Brazil. From a total of 431 animals, STEC was identified from 116 (26,9%) samples, being 111 (25,8%) STEC eae- and 5 (1,1%) STEC eae+. EPEC pathotype was detected among 20 (4,6%) of animals. The other pathotypes of diarrheagenic E. coli were not identified. Of the 95 STEC isolates assessed for virulence profile, all harbored stx2, while 28 (29,5%) contained stx1. Iha and saa, genes encoding adhesins, were found at 93,7% (89/95) and 66,3% (63/95), respectively. EspP, gene which encoding a protease related with intestinal colonization, was detected in 61,1% (58/95) and ehxA hemolysin was present in 54,7% (52/95). SubAB, nleE and nleB genes were also detected in lower rates. STEC is widespread in cattle herds of São Paulo, containing commonly isolated genes from human pathogens, which reinforces the importance of inspection and surveillance in the slaughterhouses / Mestre