Dissertations / Theses: 'Enterohemorrhagic Escherichia coli'

1

Dadgar, Ashraf. "Detection of enterohemorrhagic Escherichia coli (EHEC)." Thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6011.

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Escherichia coli is a natural inhabitant of the intestines of both humans and animals, but there are also several pathogenic types of E. coli which cause disease in humans.

Strains of enterohemorrhagic E. coli (EHEC) have been associated with outbreaks of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome in humans. Most clinical signs of disease arise as a consequence of the production of shigatoxin 1 and 2 or combination of these toxins. Other major virulence factors include EHEC hemolysin and intimin, the product of the eae gene that is involved in attaching and effacing adherence phenotype. EHEC has also been associated with uncomplicated diarrhea.

The capacity to control EHEC disease and to limit the scale of outbreaks is dependent upon prompt diagnosis and identification of the source of infection.

The principal reservoirs of EHEC are cattle and food products, which presumably have come into contact with domestic animal manure and/or are inadequately pasteurised, these are important vehicles of infection.

In the present study, the PCR technique with primers detecting the verocytotoxin genes was shown to be a possible method to screen for and identify EHEC.

In summary stx genes were detected in 16 samples of 228 sampels and the eae gene was detected in 2 samples using PCR.

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2

Yu, Angel Chia-yu. "Structural analysis of an enterohemorrhagic Escherichia coli metalloprotease effector." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/42821.

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Mucins are proteins that contain dense clusters of α-O-GalNAc-linked carbohydrate chains and are the major component of the mucosal barrier that lines the mammalian gastrointestinal tract from mouth to gut. A critical biological function of mucins is to protect the underlying epithelial cells from infection. Enterohemorrhagic Escherichia. coli O157:H7 (EHEC), a bacterial pathogen that causes severe food and water borne disease, is capable of breaching this barrier and adhering to intestinal epithelial cells during infection. StcE (secreted protease of C1-esterase inhibitor) is a ~100 kDa zinc metalloprotease virulence factor secreted by EHEC and plays a pivotal role in remodelling the mucosal lining during EHEC pathogenesis. StcE also dampens the host immune response by targeting the mucinlike region of C1-INH, a key complement regulator of innate immunity. To obtain further mechanistic insight into StcE function, I have determined the crystal structure of the fulllength protease to 2.5Å resolution. This structure shows that StcE adopts a dynamic, multidomain architecture featuring an unusually large substrate binding cleft. Electrostatic surface analysis reveals a prominent polarized charge distribution highly suggestive of an electrostatic role in substrate targeting. The observation of key conserved motifs in the active site allows us to propose the structural basis for the specific recognition of α-O-glycan containing substrates, which have been confirmed by glycan array screening to be Oglycosylation of the mucin-type. Complementary biochemical analysis employing domain variants of StcE further extends our understanding of the substrate binding stoichiometry and distinct substrate specificity of this important virulence-associated metalloprotease.

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3

Treptow, Andrea Lauren. "Investigation of a Thermoregulated Gene in Pathogen Enterohemorrhagic Escherichia coli." Thesis, The University of Arizona, 2014. http://hdl.handle.net/10150/323222.

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4

MacDonald, Leslie Anne. "Antigenic relationship of enterohemorrhagic Escherichia coli hemolysin to other RTX toxins." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ56344.pdf.

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5

Thomassin, Jenny-Lee. "Antimicrobial peptide resistance mechanisms used by Enteropathogenic and Enterohemorrhagic «Escherichia coli»." Thesis, McGill University, 2014. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=121462.

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Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are Gram-negative pathogens that cause diarrheal disease in the developed and developing world. To cause infection, these pathogens must overcome innate host defenses, such as secreted cationic antimicrobial peptides (AMPs). There are two groups of human AMPs: cathelicidins (LL-37) and defensins (α-defensin 5). AMPs are expressed in specific locations of the human body. In the small intestine, the infectious niche for EPEC, human α-defensins 5 and 6 (HD-5 and HD-6) are abundant and there are low levels of LL-37. Conversely in the colon, the infectious niche for EHEC, HD-5 and HD-6 are not expressed and LL-37 is abundant. Pathogens can overcome AMP-killing using several mechanisms, including proteolytic inactivation, producing shielding structures and modifying their lipopolysaccharide (LPS). We hypothesized that EPEC and EHEC use AMP-resistance mechanisms to resist killing by secreted AMPs during infection. Previously, CroP the omptin protease in Citrobacter rodentium, a murine pathogen used to model EPEC and EHEC infections, was shown to degrade murine cathelicidin. Both EPEC and EHEC have a CroP-homologue: OmpT. The contribution of OmpT to LL-37 resistance was analyzed in both pathogens. Peptide cleavage assays showed that EHEC OmpT cleaves and inactivates LL-37 more rapidly than EPEC OmpT. Higher ompT-expression and protein levels in EHEC than EPEC are responsible for the differences observed in LL-37 inactivation rates. Additional studies showed that OmpT was unable to cleave folded α-defensins. These data suggest that EPEC uses other mechanisms to resist killing by the AMPs in its infectious niche. To assess this possibility, surface structures that may shield the bacterial membrane from AMPs were identified. High transcript levels of gfcA, a gene required for group 4 capsule (G4C) secretion, were observed in EPEC but not EHEC. The unencapsulated EPEC ΔgfcA and EHEC wild-type strains were more susceptible to HD-5 killing than EPEC wild-type. Since the G4C is composed of the same sugar repeats as the LPS O-antigen, an O-antigen ligase (waaL) deletion mutant was generated to assess the role of the O-antigen in HD-5 resistance. The EPEC ΔwaaL strain was more susceptible to HD-5 than both the wild-type and ΔgfcA strains. Addition of exogenous polysaccharide increased survival of the ΔgfcAΔwaaL strain in the presence of HD-5, suggesting that HD-5 binds the polysaccharides present on the surface of EPEC. These data show that EPEC relies on both the G4C and O-antigen to resist the bactericidal activity of HD-5. Altogether, these data indicate that EHEC and EPEC differentially regulate AMP-specific resistance mechanisms as an adaptation to their specific infectious niches.
Les Escherichia coli entéropathogènes et entérohémorrhagiques (EPEC et EHEC) sont des bactéries à coloration Gram-négative qui causent des diarrhées dans les pays développés et en développement. Pour causer une infection, ces pathogènes doivent surmonter les défenses de l'immunité innée de l'hôte, tel que les peptides antimicrobiens sécrétés (PAMs). Chez l'humain, les PAMs sont divisés en deux groupes, les cathélicidines (ex. LL-37) et les défensines (ex. α-défensine humaine 5). L'expression des PAMs varie selon les tissus. Dans l'intestin grêle, la niche infectieuse des EPEC, les α-défensines humaines 5 et 6 (HD-5 et HD-6) sont abondantes et le niveau de LL-37 est bas. Inversement, HD-5 et HD-6 ne sont pas exprimées dans le côlon, la niche infectieuse des EHEC, et LL-37 est très abondant. Les pathogènes peuvent résister aux PAMs en utilisant différent mécanismes comme l'inactivation protéolytique, la production de structures recouvrant la cellule bactérienne et la modification du lipopolysaccharide (LPS). Notre hypothèse est que les EPEC et EHEC utilisent des mécanismes de résistance aux PAMs pour établir une infection. Précédemment, il a été démontré que la protéase de type omptin, CroP, de Citrobacter rodentium, un pathogène murin utilisé comme modèle pour les infections des EPEC et EHEC, dégrade la cathélicidine murine. Les EPEC et EHEC possèdent un homologue de CroP, OmpT. La contribution de OmpT à la résistance au LL-37 a été examinée chez ces deux pathogènes. Nos tests de clivage de peptide ont démontré que EHEC OmpT clive et inactive LL-37 plus rapidement que EPEC OmpT. La différence observée a été associée à une plus forte expression et production de OmpT chez les EHEC que chez les EPEC. Des tests supplémentaires ont démontré que OmpT ne peut pas cliver les α-défensines repliées. Ces données suggèrent qu'EPEC utilise d'autres mécanismes de résistance pour surmonter l'activité des PAMs présents dans sa niche infectieuse. Pour tester cette possibilité, les structures recouvrant la cellule ont été identifiées. Un haut niveau de transcription de gfcA, un gène requit pour la sécrétion de la capsule du groupe 4 (G4C), a été observé chez EPEC mais pas chez EHEC. Le mutant EPEC non-encapsulé ΔgfcA et la souche sauvage EHEC sont plus susceptible à l'effet du HD-5 que la souche sauvage EPEC. Étant donné que la G4C est composée des mêmes sucres que l'antigène O, la ligase de l'antigène O, waaL, a été délétée pour déterminer le rôle de l'antigène O dans la résistance au HD-5. La souche EPEC ΔwaaL est plus susceptible au HD-5 que la souche sauvage EPEC et le mutant EPEC ΔgfcA. L'addition de polysaccharide exogène augmente la survie du mutant ΔwaaLΔgfcA en présence de HD-5. Ceci indique que HD-5 se lie aux polysaccharides présents à la surface des EPEC. Ces données démontrent que la résistance à HD-5 chez EPEC repose sur la présence de la G4C et de l'antigène O. Toutes ces données indiquent que EHEC et EPEC utilisent des mécanismes de résistance différents aux PAMs, ce qui démontre une adaptation à leurs niches infectieuses respectives.

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6

Morgan, Jason Kyle. "Genetic basis for the virulence of enterohemorrhagic Escherichia coli strain TW14359." Scholar Commons, 2014. https://scholarcommons.usf.edu/etd/5277.

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Enterohemorrhagic Escherichia coli (EHEC) is a virulent pathotype of E. coli that is associated with major outbreaks of hemorrhagic colitis and the life-threatening kidney disease hemolytic uremic syndrome. For successful host colonization and attachment to the intestinal mucosa, EHEC requires the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system (TTSS) responsible for secreting and translocating effector proteins into host colonocytes. Regulation of the LEE is primarily directed through the first operon, LEE1, encoding the locus encoded regulator (Ler), and occurs through the direct and indirect action of several regulators. The 2006 U.S. spinach outbreak of E. coli O157:H7, characterized by unusually severe disease, has been attributed to a strain (TW14359) with enhanced pathogenic potential including elevated virulence gene expression, robust adherence, and the presence of novel virulence factors. Aim 1 of this dissertation proposes a mechanism for the unique virulence expression and adherence phenotype of this strain, and further expands the role for regulator RcsB in control of the E. coli locus of enterocyte effacement (LEE) pathogenicity island. Proteomic analysis of TW14359 revealed a virulence proteome consistent with previous transcriptome studies that included elevated levels of the LEE regulatory protein Ler and type III secretion system (T3SS) proteins, secreted T3SS effectors, and Shiga toxin 2. Basal levels of the LEE activator and Rcs phosphorelay response regulator, RcsB, were increased in strain TW14359 relative to O157:H7 strain Sakai. Deletion of rcsB eliminated inherent differences between these strains in ler expression, and in T3SS-dependent adherence. A reciprocating regulatory pathway involving RcsB and LEE-encoded activator GrlA was identified and predicted to coordinate LEE activation with repression of the flhDC flagellar regulator and motility. Overexpression of grlA was shown to increase RcsB levels, but did not alter expression from promoters driving rcsB transcription. Expression of rcsDB and RcsB was determined to increase in response to physiologic levels of bicarbonate, and bicarbonate-dependent stimulation of the LEE was shown to be dependent on an intact Rcs system and ler activator grvA. The results of this aim significantly broaden the role for RcsB in EHEC virulence regulation. The bicarbonate ion (HCO3-) has been shown to stimulate LEE gene transcription through the LEE1 promoter, and is predicted to serve as a physiologic signal for EHEC colonization. Results from the previous aim demonstrated that bicarbonate induction of the LEE is mediated through the Rcs phosphorelay, and is dependent upon an intact global regulator of virulence grvA gene. However, the direct mechanism through which RcsB-GrvA regulates ler, and the contribution of GrvA to the virulence of EHEC is unknown. In Aim 2, the RcsB-GrvA regulon of EHEC was determined by RNA sequencing, and the contributions of each to virulence and stress fitness was explored. A significant increase in transcription of the gad genes for extreme acid resistance was observed for both EHEC strains TW14359grvA and TW14359rcsBgrvA compared to TW14359, and corresponded with a significant increase in acid survival for TW14359grvA during exponential growth. Therefore, a model by which RcsB-GrvA coordinate LEE expression with acid resistance through GadE was proposed. Finally, the temporal regulation of both rcsDB and grvAB operons in response to bicarbonate was defined using single copy luxE chromosomal reporter fusions. Taken together, these results demonstrate the role of RcsB and GrvA to EHEC virulence, and reveal a novel role for GrvA in of extreme acid resistance and LEE gene expression and in EHEC. Finally, production of the ECP pilus has been demonstrated in enterohemorrhagic Escherichia coli O157:H7 (EHEC), and has been shown to be required for efficient adherence to epithelial cells during colonization. The first gene of the ecpRABCDE operon encodes a transcriptional regulator (EcpR) that positively regulates its own transcription, and promotes transcription and production of the downstream gene, ecpA, encoding the major ECP subunit EcpA. However, the distance between the ecpR and ecpA genes suggests the presence of regulatory elements that control ecpA directly. Therefore, it was hypothesized that an additional promoter was able to direct transcription of ecpA, independent of the promoter upstream of ecpR. To test this, promoter-lacZ transcriptional reporter fusions were created using the regions upstream of ecpR and ecpA to test for promoter activity, coupled with western blot analysis to detect EcpA in both wild-type and ecpR promoter mutant strains. In Aim 3, we showed that an additional promotable element, downstream of the EHEC O157:H7 strain TW14359 ecpR translational start site, is capable of driving transcription of ecpA, and that its activity is independent of an intact ecpR promoter. In addition, site-directed mutagenesis was used to characterize a TW14359 specific single nucleotide polymorphism within the predicted ecpA promoter region. Overproduction of EcpR was observed to increase cytosolic RcsB and Tir, indicating that ecp production is able to stimulate the LEE, and that the ecpA promoter polymorphism may contribute to intrinsically increased rcsB transcription in TW14359. Taken together, the results, and those obtained in Aims 1 and 2, expand the model for regulation of the ecp operon in EHEC O157:H7 strain TW14359, and broaden the model for EcpR and RcsB in the coordinate regulation of E. coli common pilus and type III secretion.

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7

Tsai, Wan-Ling. "Investigation of Systems For Detection of Enterohemorrhagic Escherichia Coli Contamination In Foods /." The Ohio State University, 1996. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487933245536054.

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8

Eichhorn, Inga [Verfasser]. "Microevolution of epidemiological highly relevant non-O157 enterohemorrhagic Escherichia coli (EHEC) / Inga Eichhorn." Berlin : Freie Universität Berlin, 2016. http://d-nb.info/1115722530/34.

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9

Chong, Yuwen. "Intimate interactions between enteropathogenic and enterohemorrhagic Escherichia coli and intestinal epithelium in vitro." Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1445194/.

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Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are diarrhoeagenic human pathogens that colonize intestinal epithelial cells via an attaching and effacing (A/E) lesion. Intimate adherence is mediated through binding of the intimin adhesin to the bacterial translocated intimin receptor, Tir. EPEC adheres to all regions of the human intestine in the in vitro organ culture (IVOC) system. In contrast, although EHEC is associated with colonic pathology in human infections, a prototypical strain of EHEC has shown a restricted tropism towards follicle-associated epithelium (FAE) of the terminal ileum without evidence of colonic adhesion. This thesis used the human IVOC system to further examine the intestinal interaction of EHEC 0157:H7 and related EPEC serotypes. To address the apparent paradox of non-adherence of EHEC to colonic tissue in the IVOC experimental system, the role of environmental, host and bacterial factors in modulating EHEC colonisation and tissue tropism were studied. No environmental factor (modulation of IVOC system, bicarbonate, serum, and mannose) was found to induce colonic adhesion. The investigation of the hypothesis that prior host-bacterial interactions might enhance subsequent EHEC colonic adhesion found that exposure to FAE promoted subsequent colonic adhesion, but in a non-intimate manner, demonstrating a novel form of interaction with human intestine. Great diversity was found in EPEC and EHEC in relation to the presence and sequence of tir, tccP and espJ fccP-negative strains expressing TireHEc were identified indicating that novel Nck-like molecules may be awaiting discovery. Tir was essential for EPEC and EHEC adhesion in IVOC but its phosphorylation (EPEC) and its interaction with TccP (EHEC) were not necessary for colonisation and A/E lesion formation on IVOC, questioning the role of pedestal formation in enterocyte infection. This is in direct contrast to cell culture findings and demonstrates the importance of IVOC in establishing 0157:H7 human pathogenesis.

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10

Lai, YuShuan (Cindy). "EspFU, an Enterohemorrhagic E. Coli Secreted Effector, Hijacks Mammalian Actin Assembly Proteins by Molecular Mimicry and Repetition: A Dissertation." eScholarship@UMMS, 2014. https://escholarship.umassmed.edu/gsbs_diss/715.

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Enterohemorrhagic E. coli (EHEC) is a major cause of food borne diarrheal illness worldwide. While disease symptoms are usually self-resolving and limited to severe gastroenteritis with bloody diarrhea, EHEC infection can lead to a life threatening complication known as Hemolytic Uremic Syndrome (HUS), which strikes children disproportionately and is the leading cause of kidney failure in children. Upon infection of gut epithelia, EHEC produces characteristic lesions called actin pedestals. These striking formations involve dramatic rearrangement of host cytoskeletal proteins. EHEC hijacks mammalian signaling pathways to cause destruction of microvilli and rebuilds the actin cytoskeleton underneath sites of bacterial attachment. Here, we present a brief study on a host factor, Calpain, involved in microvilli effacement, and an in depth investigation on a bacterial factor, EspFU, required for actin pedestal formation in intestinal cell models. Calpain is activated by both EHEC and the related pathogen, enteropathogenic E. coli (EPEC), during infection and facilitates microvilli disassembly by cleavage of a key membrane-cytoskeleton anchoring substrate, Ezrin. Actin pedestal formation is facilitated by the injection of two bacterial effectors, Tir and EspFU, into host cells, which work in concert to manipulate the host actin nucleators N-WASP and Arp2/3. EspFU hijacks key host signaling proteins N-WASP and IRTKS by mimetic displacement and has evolved to outcompete mammalian host ligands. Multiple repeats of key functional domains of EspFU are essential for actin pedestal activity through proper localization and competition against the an abundant host factor Eps8 for binding to IRTKS.

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11

Lai, YuShuan (Cindy). "EspFU, an Enterohemorrhagic E. Coli Secreted Effector, Hijacks Mammalian Actin Assembly Proteins by Molecular Mimicry and Repetition: A Dissertation." eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/715.

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Enterohemorrhagic E. coli (EHEC) is a major cause of food borne diarrheal illness worldwide. While disease symptoms are usually self-resolving and limited to severe gastroenteritis with bloody diarrhea, EHEC infection can lead to a life threatening complication known as Hemolytic Uremic Syndrome (HUS), which strikes children disproportionately and is the leading cause of kidney failure in children. Upon infection of gut epithelia, EHEC produces characteristic lesions called actin pedestals. These striking formations involve dramatic rearrangement of host cytoskeletal proteins. EHEC hijacks mammalian signaling pathways to cause destruction of microvilli and rebuilds the actin cytoskeleton underneath sites of bacterial attachment. Here, we present a brief study on a host factor, Calpain, involved in microvilli effacement, and an in depth investigation on a bacterial factor, EspFU, required for actin pedestal formation in intestinal cell models. Calpain is activated by both EHEC and the related pathogen, enteropathogenic E. coli (EPEC), during infection and facilitates microvilli disassembly by cleavage of a key membrane-cytoskeleton anchoring substrate, Ezrin. Actin pedestal formation is facilitated by the injection of two bacterial effectors, Tir and EspFU, into host cells, which work in concert to manipulate the host actin nucleators N-WASP and Arp2/3. EspFU hijacks key host signaling proteins N-WASP and IRTKS by mimetic displacement and has evolved to outcompete mammalian host ligands. Multiple repeats of key functional domains of EspFU are essential for actin pedestal activity through proper localization and competition against the an abundant host factor Eps8 for binding to IRTKS.

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12

Willford, John Daniel. "Development of a field-based assay for rapid detection of enterohemorrhagic Escherichia coli (EHEC)." Laramie, Wyo. : University of Wyoming, 2008. http://proquest.umi.com/pqdweb?did=1663059651&sid=1&Fmt=2&clientId=18949&RQT=309&VName=PQD.

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13

Brady, Michael John. "Mechanism and Function of Actin Pedestal Formation by Enterohemorrhagic Escherichia coli O157:H7: A Dissertation." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/342.

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Enterohemorrhagic Escherichia coli O157:H7 (EHEC) and enteropathogenic E. coli O127:H7 (EPEC) induce characteristic F-actin rich pedestals on infected mammalian cells. Each pathogen delivers its own translocated intimin receptor (Tir) to the host cell to act as a receptor for the bacterial outer membrane adhesin, intimin. Interaction of translocated Tir with intimin is essential for mammalian cell binding and host colonization, as well as to induce actin pedestal formation in vitro. In spite of these parallels, EHEC and EPEC Tir appear to generate actin pedestals by distinct mechanisms. Further, while the ability to form actin pedestals is a striking phenotype, the function of pedestals during infection remains unclear. To address these issues, a systematic and quantitative analysis of Tir-mediated actin assembly was conducted. We identified a three-residue Tir sequence involved in actin pedestal formation for both EHEC and EPEC, and developed evidence that the two pathogens trigger a common pathway for actin assembly. Further, the ability of these bacteria to promote actin assembly appears to promote both intimin-mediated bacterial binding in vitro and optimal colonization during experimental animal infection.

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14

Liu, Hui. "Intimin-Tir Interaction in Enterohemorrhagic E. coli: A Dissertation." eScholarship@UMMS, 2000. http://escholarship.umassmed.edu/gsbs_diss/266.

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Enterohemorrhagic E. coli (EHEC) has emerged as an important agent of diarrheal disease in the developed countries. Attachment to host cells, an essential step during intestinal colonization by EHEC, is associated with the formation of a highly organized cytoskeletal structure containing filamentous actin, termed attaching and effacing (A/E) lesion, directly beneath bound bacteria. The outer membrane protein, intimin, is required for the formation of this structure, as is Tir, a bacterial protein that is translocated into the host cell and thought to function as a receptor for intimin. In this thesis, we characterized A/E lesion formation by in vivo and in vitro-grown EHEC, aimed at testing whether bacterial adaptation to the mammalian host included up regulation of A/E lesion formation. Our results showed that actin signaling by EHEC was induced upon bacterial growth in vivo, and this induction was likely due to the up regulation of multiple activities by in vivo-grown EHEC. We also focused on the interaction between intimin and the host cell, an interaction that triggers actin condensation of A/E lesion formation. We evaluated the role of β1 integrins, one of the proposed receptors of intimin, in A/E lesion formation, and demonstrated that β1 integrins are not essential for intimin-mediated cell binding and actin condensation. To better understand intimin function, we mapped the functional domains of intimin, showed that the minimal cell binding domain of intimin correlates with the minimal Tir-binding domain. This minimal Tir-binding domain, when purified and coated on latex beads, was sufficient to trigger actin condensation on preinfected mammalian cells, suggesting that Tir-binding by intimin is critical in the final step of A/E lesion formation. To further demonstrate the significance of the interaction between intimin and Tir in A/E lesion formation, we developed a yeast two-hybrid system to identify intimin mutants diminished in Tir-binding, and then characterized those mutants for the ability to trigger actin condensation, the final step of A/E lesion formation. Finally, as a first step to study the downstream actin signaling pathway after Tir-binding, we mapped the domain of Tir involved in intimin-binding, and showed that the N-terminus and C-terminus of Tir are likely to be localized in the host cell cytoplasm, available to interact with downstream effectors in actin signaling.

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Yang, Yang. "Investigation of enterotoxigenic Escherichia coli (ETEC) vaccine candidates and identification of inhibitor of enterohemorrhagic Escherichia coli (EHEC) Type III secretion system effector NleB." Thesis, Kansas State University, 2017. http://hdl.handle.net/2097/38174.

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Master of Science in Biomedical Sciences
Department of Diagnostic Medicine/Pathobiology
Philip R. Hardwidge
Enterotoxigenic Escherichia coli (ETEC) is the most common cause of diarrhea in travellers and young children in developing countries. We previously characterized three vaccine candidates (MipA, Skp, and ETEC_2479) which effectively protected mice in an intranasal ETEC challenge model after immunization. However, these proteins are conserved not only in multiple ETEC isolates, but also in commensal bacteria. In this study, we examined the potential of these antigens to affect the host intestinal microbiota and subsequently found no significant impact on healthy of host after vaccination. In addition, we also optimized the types of adjuvants and forms of antigens and evaluated the efficacy in a mouse intranasal challenge model. Enterohemorrhagic Escherichia coli (EHEC) is an emerging zoonotic pathogen that cause global public health threads. EHEC possesses the potential to cause gastroenteritis, hemorrhagic colitis and hemolytic uremic syndrome (HUS), which may lead to renal failure. Type III secretion system (T3SS) is a hallmark of EHEC, characterized by the needle-like structure and a variety of effectors injected into host cells. NleB, one of T3SS effectors, is a glycosyltransferase with the ability to catalyze the transfer of N-acetyl-D-glucosamine (N-GlcNAc) to host proteins to suppress the activation of NF-kB signaling pathway. In this study, we employed luminescence-based glycosyltransferase assay and high-throughput screening using a chemical library of various compounds. A total of 128 chemicals was selected with significant inhibition on NleB glycosyltransferase activity for further pharmaceutical study as novel therapy against EHEC infection.

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16

Eißenberger, Kristina [Verfasser], and Herbert [Akademischer Betreuer] Schmidt. "Uptake of enterohemorrhagic Escherichia coli into the roots of lettuce plants / Kristina Eißenberger ; Betreuer: Herbert Schmidt." Hohenheim : Kommunikations-, Informations- und Medienzentrum der Universität Hohenheim, 2020. http://d-nb.info/1214296629/34.

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17

Quintanilla, Lucy Beatriz Zapata. "Anticorpos séricos anti Escherichia coli enterohemorrágica (EHEC) em adultos saudáveis da Grande São Paulo." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-19092006-134239/.

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As gastroenterites ainda são um importante problema de saúde pública nos países em desenvolvimento e as Escherichia colí são as principais causas de diarréias. Adultos brasileiros apresentam anticorpos reativos com E. colí enteropatogênica (EPEC), que apresenta muitas semelhanças com E. colí enterohemorágica (EHEC) e pode ser responsável por complicações como a síndrome hemolítica urêmica e a colite hemorrágica. Ambas as bactérias apresentam um mecanismo patogênico comum: a formação da lesão \"attaching and effacing\" nos microvilus dos enterócitos, mediadas por fatores de virulência, como Intimina, Tir e as Esps. Os lipopolissacarídeos (LPS) são componentes da membrana externa das bactérias gram-negativas, incluindo E. colí. A infecção por EHEC 0157 resulta na produção de anticorpos séricos antiLPS 0157, que geralmente são indicativos de infecção recente. Neste trabalho, nós investigamos a presença de anticorpos séricos IgG e IgM anti-EHEC 0157:H7, EHEC 0111:H- e EPEC 0111:H- em adultos brasileiros, da Grande São Paulo. Amostras de soro foram coletadas de 200 adultos saudáveis, doadores de sangue, e um pool foi formado com 100 amostras. Anticorpos foram determinados em 100 amostras individuais por ELlSA com bactérias íntegras e anti-LPS para a determinação de anticorpos específicos anti-LPS 0111 e 0157 com o \"pool\" de soro como controle, em relação às concentrações de IgG e IgM totais determinadas simultaneamente. Os resultados foram submetidos a análise estatística. O repertório de anticorpos IgG e IgM para as três bactérias foi investigado por \"immunoblotting\" (18). A presença de anticorpos anti-bacterianos e anti-LPS foi confirmada. Existe uma correlação positiva entre os anticorpos reativos com as três bactérias, e entre os anticorpos anti-bactéria e anti-LPS. As concentrações de IgM anti-LPSs foi mais alta que IgG, o que está de acordo com o mecanismo imune esperado para um antígeno timo-independente. As concentrações de anti-LPS 0157 foram altas, apesar da baixa freqüência da bactéria 0157 em nosso meio. Os ensaios de 18 mostraram anticorpos reativos com bandas antigênicas sugestivas de fatores de virulência. A origem destes em nossa população poderia ser o contato com bactérias da microbiota ou do ambiente, de animais ou alimentos. Alternativamente, nossa população pode estar exposta à EHEC mais freqüentemente do que imaginado até agora.
Gastroenteritis is still an important public health problem in developing countries and Escherichia coli are frequent agents of diarrhea. Brazilian adults present antibodies reactive with the principal virulence factors of enteropathogenic E. coli (EPEC), which have many genetic and antigenic similarities with enterohemorrhagic E. coli (EHEC), that may be responsible for complications following diarrhea, as haemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). Both bacteria present a common pathogenic mechanism, with the formation of attaching and effacing lesion in microvilis enterocytes, mediated by virulence factors codified by the patogenicity island LEE, as Intimin, Tir e Esps. Lipopolysaccharides (LPS) are components of outer membrane and important virulence factors of Gram-negative bacteria including E. coli. The infection with EHEC O157 results in the production of serum antibodies to the O157 LPS antigens, and usually they are indicators of recent infection. In this work we investigated IgG and IgM serum antibodies reactive with EHEC O157:H7, EHEC O111:H- and EPEC O111:H- antigens in healthy Brazilian adults, living in São Paulo. Serum samples were collected from 200 healthy adults (blood donors) and a pool was formed with 100 samples. The antibody levels were determined by ELISA for 100 individual serum samples by means of a whole cell ELISA with the three bacteria and an anti-LPS ELISA for the determination of the concentrations of specific antibodies anti-LPS O111 and O157, using the serum pool as control, in relation to the total IgG and IgM concentrations determined simultaneously. The results were submitted to statistical analysis. The repertoire of IgG and IgM antibodies to the three bacteria was investigated by immunoblotting (IB). The presence of anti-bacterial and anti-LPS seric antibodies was confirmed. There is a positive correlation between the titers of antibodies reactive with the three bacteria and between anti-bacteria and anti-LPS antibodies. The concentrations of IgM anti-LPSs were significantly higher than IgG, which is in accordance with the immune mechanism expected to a thymus-independent antigen. Surprisingly the concentrations of anti-LPS O157 were high taking into account the low frequency of O157 bacteria isolation in our country. The IB assays showed the presence of antibodies, mainly IgG, reactive with many antigenic bands suggestive of virulence factors. The origin of anti-EHEC antibodies in our population could be the contact with microbiota or environment bacteria, and animal or human E. coli strains, pathogenic or not. Alternatively, our people may be exposed to EHEC more frequently than previously thought.

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Gardette, Marion. "Virulence des Escherichia coli entérohémmoragiques : rôle central du monoxyde d'azote dans le devenir de l'infection et identification de nouveaux déterminants impliqués dans l'adaptation du pathogène à l'envirronement digestif." Thesis, Université Clermont Auvergne‎ (2017-2020), 2019. http://www.theses.fr/2019CLFAC075.

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Les Escherichia coli entérohémorragiques (EHEC) représentent un enjeu majeur en santé publique. En effet, ces pathogènes sont responsables chaque année de milliers de cas de toxi-infections alimentaires à travers le monde et peuvent engendrer des complications graves, notamment des atteintes rénales chez les jeunes enfants et cérébrales chez les personnes âgées. Actuellement, le principal problème réside dans le fait que les traitements thérapeutiques disponibles sont limités puisque l’antibiothérapie peut favoriser le développement des complications liées à l’infection. Il est donc primordial et d’actualité de mettre en évidence les facteurs bactériens associés à la virulence des EHEC et de comprendre les interactions entre le pathogène et l’hôte afin de développer des stratégies thérapeutiques visant à éliminer le pathogène et limiter l’apparition des symptômes graves. Ainsi, le premier objectif de cette thèse était d’identifier de nouveaux facteurs bactériens potentiellement impliqués dans le processus infectieux. L’utilisation de la technologie RIVET sur la souche de référence O157:H7 EDL933 en modèle murin, a permis de mettre en évidence 31 gènes dont l’expression est spécifiquement induite lors de l’infection. La caractérisation de ces gènes a démontré que certains codent des facteurs de niche qui pourraient accroître le potentiel des souches d’EHEC à s’adapter à l’environnement intestinal et ainsi participer à la virulence du pathogène. Le second volet de cette thèse avait pour but de caractériser in vivo la réponse des EHEC au monoxyde d’azote (NO), un médiateur de la réponse immunitaire de l’hôte, et ainsi d’évaluer le potentiel rôle protecteur du NO lors d’une infection en modèle murin. En utilisant une souche d’EHEC rapportant la présence de NO, nous avons démontré que le NO est produit dès les premiers stades de l’infection et que celui-ci limite l’adhésion du pathogène à la muqueuse colique. En revanche, nous avons également mis en évidence un effet néfaste du NO pour l’hôte puisqu’il favorise la production des Shigatoxines (Stx), le facteur de virulence majeur des EHEC, conduisant au développement d’un dysfonctionnent rénal. Enfin, nous avons montré l’importance de la NO réductase NorVW dans la virulence de certaines souches d’EHEC. En effet, l’inactivation de l’opéron norVW chez la souche O157:H7 620 réduit la capacité du pathogène à coloniser efficacement le tractus digestif et à produire Stx. Cette observation est toutefois souche dépendante et suggère que la réponse des EHEC au stress nitrosant lors d’une infection est complexe et probablement multifactoriel. L’ensemble de ces travaux contribue à une meilleure compréhension du processus infectieux des EHEC, une étape indispensable au développement de futures stratégies anti-infectieuses
Enterohemorrhagic Escherichia coli (EHEC) are a major public health concern. Indeed, these pathogens are responsible for thousands of food-borne illness cases worldwide every year and can lead to serious complications, including kidney damages in young children and brain damages in the elderly. Currently, the main issue is the limited number of available therapeutic treatments since antibiotic therapy can promote the development of infection-related complications. Therefore, it appears essential and topical to identify bacterial factors associated with EHEC virulence and to understand the interactions occurring between the pathogen and the host, in order to develop new anti-infective strategies. The first objective of this thesis was to identify new bacterial factors potentially involved in the infectious process. Application of the RIVET technology to the reference strain O157:H7 EDL933 revealed 31 genes specifically induced during mouse infection. Characterization of these genes showed that some of them encode niche factors potentially involved in the adaptation of EHEC to the intestinal environment, therefore contributing to virulence. The second aim of this thesis was to characterize in vivo the response of EHEC to nitric oxide (NO), a mediator of the host’s immune response, and thus assess the protective role of NO against EHEC infection in a mouse model. By using a NO-sensing reporter EHEC strain, we demonstrated that NO is produced by the host at the early stages of infection and this NO limits adhesion of the pathogen to the colonic mucosa. On the other hand, we also showed that NO is detrimental to the host since it promotes the production of Shigatoxins (Stx), which is the major EHEC virulence factor, and leads to the development of renal dysfunction. Finally, we showed that the NO reductase NorVW is important for the virulence of some, but not all, EHEC strains. Inactivation of the norVW operon in strain O157:H7 620 reduces the ability of the pathogen to efficiently colonize the digestive tract and to produce Stx. However, this observation is strain-specific and this suggests that EHEC response to nitrosative stress during infection is complex and probably multifactorial. This work contributes to a better understanding of the EHEC infectious process, an essential step for the development of future anti-infective strategies

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Radhakrishnan, Padhma. "Role of Intimin and Tir in Actin Signalling by Enterohemorrhagic and Enteropathogenic Escherichia coli: A Dissertation." eScholarship@UMMS, 2003. http://escholarship.umassmed.edu/gsbs_diss/265.

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Enterohemorrhagic Escherichia coli 0157:H7 (EHEC) and Enteropathogenic E. coli (EPEC) are intestinal pathogens that induce characteristic lesions on mammalian cells called actin pedestals. Attachment to host cells by both EPEC and EHEC is an essential step towards colonization and is associated with the formation of highly organized actin cytoskeletal elements termed as attaching and effacing (AE) lesions beneath bound bacteria. The outer membrane protein intimin is required for the formation of these structures and binds its own translocated mammalian cell receptor called Translocated intimin receptor (Tir). These interactions induce a cascade of events that result in actin pedestal formation. In this thesis, we characterized pedestal formation and the requirements of pedestal formation by host adapted and in vitro cultivated EHEC. Our data indicate that growing EHEC in the mammalian host enhances bacterial cell attachment, expression and translocation of virulence effectors and actin signaling, and this enhancement is likely to entail more than one bacterial activity involved in host cell interactions. We also focused on the interaction between the two key bacterial players involved in pedestal formation, intimin and Tir. We randomly mutagenized the Tir-binding domain of intimin and isolated point mutants that disrupted Tir recognition. The ability of intimin mutants to bind to recombinant Tir correlated with their ability to trigger AE lesions on pre-infected mammalian cells. Half of the mutations fell within the previously identified 50 amino acid C-terminal region of intimin, and alanine scanning mutagenesis of this region identified four residues of EHEC intimin that are critical for Tir recognition. In a model of the EHEC intimin-Tir complex that is based on EPEC intimin and Tir, these four amino acids are predicted to be located at the intimin-Tir interface, indicating that these residues play a functional role in intimin recognition by Tir. To identify critical residues involved in intimin recognition and intimin mediated actin signaling, we generated point mutations in the extracellular domain of EHEC Tir. Based on our data, we conclude that Tir-intimin interaction is essential for triggering actin pedestals, and intimin function in the context of Tir signaling can be replaced by proteins that are entirely unrelated to intimin but that bind to Tir. These data are concordant with the model that intimin functions to cluster Tir in the membrane to induce actin assembly. Finally, as a step to study downstream actin signaling processes after Tir translocation, we mapped the domain of Tir involved in host cell signaling. We found that the clustering of a 12 amino acid stretch of C-terminus encompassing the Nck binding sequence of Tir generated actin nucleation indistinguishable from that mediated by the entire C-terminus, and abrogation of Nck binding by mutation of Y474 to Phenylalanine abolished actin assembly. Although these results do not rule out a role for other domains of Tir involved in actin pedestal formation, this suggests that the essential element of Tir consists of the Nck binding domain.

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Skehan, Brian M. "Functional Elements of EspF_u, an Enterohemorrhagic E. coli Effector that Stimulates Actin Assembly: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/443.

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Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an attaching and effacing pathogen that upon attachment to host cells, induce characteristic attaching and effacing lesions and formation of F-actin rich pedestals beneath sites of bacterial attachment. EHEC harbors a Type III secretion system through which it delivers dozens of effectors into the host cell. The two secreted effectors critical for EHEC-mediated actin pedestal formation are the translocated intimin receptor (Tir) and EspFU. EspFU consists of an N-terminal secretion signal and a C-terminus containing six tandem 47-residue proline-rich repeats, each of which can bind and activate the actin nucleation promoting factor N-WASP. Structural and functional analyses described here have identified the mechanism of N-WASP activation by EspFU and the minimal domains and specific residues required for this activity. While EspFU and Tir are the only bacterial effectors required for F-actin pedestal formation, recruitment of EspFU to Tir is mediated by an unidentified putative host factor. To identify the host factor responsible for linking these two effectors, a combination of in vitro and functional assays were used to identify the host factor, IRTKS and the residues required for these interactions were defined. Further, the presence of at least two 47-residue repeats in all characterized clinical isolates of canonical EHEC strains led us to address the minimal requirements for EspFU functional domains to promote recruitment to Tir and N-WASP activation. Here we show that two proline-rich elements of EspFU are required for recruitment of EspFU by IRTKS to sites of bacterial attachment. Furthermore, once artificially clustered at the membrane, a single N-WASP binding element of EspFU can induce actin pedestal formation.

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Pongsri, Tongtawe Wanpen Chaicumpa. "Purification of shiga-like toxins (verocytotoxins) from enterohemorrhagic escherichia coli and the production of specific monoclonal antibodies to the toxins /." Abstract, 1999. http://mulinet3.li.mahidol.ac.th/thesis/2542/42E-PongsriT.pdf.

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Mallick, Emily M. "A New Murine Model For Enterohemorrhagic Escherichia coli Infection Reveals That Actin Pedestal Formation Facilitates Mucosal Colonization and Lethal Disease: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/601.

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Enterohemorrhagic Escherichia coli (EHEC) colonizes the intestine and produces the phage-encoded Shiga toxin (Stx) which is absorbed systemically and can lead to hemolytic uremic syndrome (HUS) characterized by hemolytic anemia, thrombocytopenia, and renal failure. EHEC, and two related pathogens, Enteropathogenic E. coli (EPEC), and the murine pathogen, Citrobacter rodentium, are attaching and effacing (AE) pathogens that intimately adhere to enterocytes and form actin “pedestals” beneath bound bacteria. The actin pedestal, because it is a unique characteristic of AE pathogens, has been the subject of intense study for over 20 years. Investigations into the mechanism of pedestal formation have revealed that to generate AE lesions, EHEC injects the type III effector, Tir, into mammalian cells, which functions as a receptor for the bacterial adhesin intimin. Tir-intimin binding then triggers a signaling cascade leading to pedestal formation. In spite of these mechanistic insights, the role of intimin and pedestal formation in EHEC disease remains unclear, in part because of the paucity of murine models for EHEC infection. We found that the pathogenic significance of EHEC Stx, Tir, and intimin, as well as the actin assembly triggered by the interaction of the latter two factors, could be productively assessed during murine infection by recombinant C. rodentium expressing EHEC virulence factors. Here we show that EHEC intimin was able to promote colonization of C. rodentium in conventional mice. Additionally, previous in vitro data indicates that intimin may have also function in a Tir-independent manner, and we revealed this function using streptomycin pre-treated mice. Lastly, using a toxigenic C. rodentium strain, we assessed the function of pedestal formation mediated by Tir-intimin interaction and found that Tir-mediated actin polymerization promoted mucosal colonization and a systemic Stx-mediated disease that shares several key features with human HUS.

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Stigers, Linnea. "Evaluation of Escherichia coli probiotic candidates for combating EHEC in the food chain using competition analysis in bovine feces." Thesis, Uppsala universitet, Institutionen för kvinnors och barns hälsa, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-353248.

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Enterohemorrhagic E. coli, EHEC, is a verotoxin producing, zoonotic pathogen, which causes diseases in humans such as bloody or watery diarrhea. Microorganisms compete for limited living space, nutrients and other resources and therefore other microorganisms are EHECs biggest competitors. To avoid outbreaks and infections with EHEC, one possible approach is to use harmless but competitive bacteria as probiotics. Therefore, the aim of this study was to evaluate three probiotic E. coli strains and their ability to outcompete EHEC in bovine feces. Ten different cattle fecal samples from three different farms were used to mix with the three probiotic and EHEC strains. The mixture was diluted and cultivated at 0 h as a control and then incubated for 48 h at 20°C and 37°C before dilution and cultivation on CT-SMaC. Colonies was counted and ratios between EHEC and probiotic E. coli before and after incubation were calculated. Kruskal-Wallis test with Dunn’s test as post hoc test were used to see if observed reductions of EHEC were significant or not. In 37°C, strain 10 was the only strain producing a significant reduction of EHEC. In contrast, no significant reduction was observed at 20°C in any of the strains. Future research studying other factors and performed on live cattle models are necessary to confirm the usefulness of the studied probiotic candidates. However, these results indicate probiotics can be a useful tool to avoid infections and big outbreaks of EHEC in the future.

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Jaudou, Sandra. "Metadetect : detection of Shiga toxin-producing Escherichia coli with novel metagenomics approaches and its application on dairy farms in France and Germany." Electronic Thesis or Diss., Maisons-Alfort, École nationale vétérinaire d'Alfort, 2023. http://www.theses.fr/2023ENVA0004.

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Les méthodologies actuelles de caractérisation d'Escherichia coli producteur de toxine Shiga (STEC) nécessitent l'isolement de la souche, ce qui est compliqué par le fait qu'il n'existe pas de milieu d'isolement spécifique qui distingue clairement les STEC des E. coli commensaux non pathogènes. Par conséquent, obtenir des informations sur les souches en utilisant une approche métagénomique éviterait d'isoler les souches pour les caractériser complètement. Dans le cadre du projet, en collaboration avec le BfR en Allemagne, nous évaluerons si de nouvelles approches de métagénomique à lecture longue pourraient déterminer sans ambiguïté si des marqueurs spécifiques d'EHEC typiques (E. coli entérohémorragique) sont co-localisés dans une même souche. Les approches de séquençage hybrides de deuxième et troisième génération seront évaluées. Des pipelines bioinformatiques seront évalués pour analyser les résultats de l'analyse métagénomique. Ces méthodes seront appliquées dans une étude pilote pour étudier le microbiote du lait cru provenant d'exploitations laitières françaises et allemandes et pour identifier un microbiome commun associé aux STEC pathogènes. Nous essaierons de définir un système basé sur l'établissement d'un « score moléculaire » pour qualifier l'état des exploitations
Current methodologies for characterization of Shiga toxin-producing Escherichia coli (STEC) require strain isolation, which is complicated by the fact that there is no specific isolation medium that clearly distinguishes STECs from non-pathogenic commensal E. coli. Therefore, obtaining strain information using a metagenomics approach would avoid isolating a strain to fully characterize it. In the framework of the project, in collaboration with the BfR in Germany, we will evaluate whether new, long-read metagenomics approaches could unambiguously determine whether specific markers of typical EHECs (Enterohemorrhagic E. coli) are co-located in the same strain. Third generation hybrid sequencing approaches will be evaluated. Appropriate bioinformatic pipelines developed in collaboration with the BfR will be evaluated to analyze the metagenomic analysis results. These methods will be applied in a pilot study to study the microbiota of raw milk from French and German dairy farms and to tentatively identify a common STEC-associated microbiome. We aim to define a ‘molecular score' based system to identify the status of the farms, in line with the objective to better precise the notion of ‘STEC molecular risk assessment approach' at the farm level

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Rump, Lydia Vanessa Verfasser], and Markus [Akademischer Betreuer] [Fischer. "Molecular Characterization of Enterohemorrhagic Escherichia coli (EHEC) : O rough strains and the prevalence and importance of IS629 in E. coli O157:H7 / Lydia Vanessa Rump. Betreuer: Markus Fischer." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2011. http://d-nb.info/1020457465/34.

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Chagnot, Caroline. "Colonisation de la viande par Escherichia coli O157∶H7 : caractérisation moléculaire, cellulaire et tissulaire des interactions." Thesis, Clermont-Ferrand 2, 2014. http://www.theses.fr/2014CLF22448/document.

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Escherichia coli O157:H7 est le sérotype le plus souvent incriminé lors de toxi-infection alimentaire par les E. coli entérohémorragiques (EHEC). Il peut être associé, dans les cas les plus graves, à des colites hémorragiques mortelles et au syndrome hémolytique et urémique (SHU), touchant essentiellement les jeunes enfants. Le vecteur alimentaire le plus courant lors de ces contaminations est le boeuf haché. L’étape primaire de la contamination bactérienne se situe lors de l'abattage où les bactéries peuvent être transférées de la peau à la carcasse. Une gaine conjonctive entoure les muscles, sa composition protéique, similaire à la matrice extracellulaire (ECM), pourrait jouer un rôle dans l'adhésion bactérienne. Dans un premier temps, l’étude de l'adhésion et de la colonisation des bactéries aux protéines majeures de l’ECM musculaire, a révélé une forte influence des conditions de croissances sur l’adhésion, l'adhésion étant maximale à 25°C et pH7, en particulier aux collagènes I et III. Chez les EHEC, diverses protéines de surfaces peuvent être potentiellement impliquées dans l’adhésion à l’ECM. Le rôle d'un autotransporteur, l'antigène 43 (Ag43), dans l'autoagrégation, l'adhésion et la formation de biofilm, a été établit chez E. coli O157:H7 EDL933. Par la suite, les interactions entre E. coli O157:H7 et la viande ont été étudiées sur deux muscles modèles de types métabolique et contractile opposés (Soleus oxidatif lent et EDL, glycolytique rapide), caractérisés par microspectroscopie de fluorescence UV couplée au rayonnement synchrotron. Les différents types de fibres musculaires ainsi que l’effet d’une anoxie prolongée simulant la maturation des viandes ont été discriminés par leurs réponses spectrales après une excitation à 275 nm. Un tropisme bactérien plus élevé pour le muscle soleus que pour le muscle EDL a été clairement observé. Bien qu'E. coli O157:H7 adhère de manière similaire aux différents types de fibres musculaires, l'adhésion des bactéries se fait essentiellement au niveau de l'ECM, mettant en évidence le rôle clé de l'ECM et du tissu conjonctif musculaire dans l’adhésion des E. coli O157:H7 à la viande. Ces travaux de recherche sur l’adhésion bactérienne aux muscles squelettiques aux niveaux moléculaires, cellulaires et tissulaires fournissent les premières connaissances sur la physiologie des EHEC lors de la contamination de la viande et constituent un pré-requis indispensable au développement de pratiques et de stratégies innovantes afin de réduire le risque de contamination des viandes
Escherichia coli O157:H7 is the most prevalent serotype involved in foodborne infection by enterohemorrhagic E. coli (EHEC). It is associated with life-threatening hemorrhagic colitis and the hemolyticuremic syndrome (HUS), which essentially affect young children. The major food vector of EHEC contamination is ground beef. The primary bacterial contamination occurs during the slaughter, essentially at dehiding stage where bacteria can be transferred from hides to carcasses. The connective tissue surrounding the muscle, highly similar to extracellular matrix (ECM) could potentially be a support for bacterial adhesion. When investigating the adhesion and colonization to the main muscle fibrous ECM proteins, the great influence of growth conditions on subsequent bacterial attachment was shown. Maximal adhesion to ECM proteins occurred at 25°C and pH 7, especially to collagens I and III. In EHEC, various surface-exposed protein determinants can be expressed and potentially involved in ECM adhesion. Investigating the autoaggregation, bacterial adhesion and biofilm formation, the involvement of Antigen 43 (Ag43), an autotransporter protein, was demonstrated in E. coli O157:H7 EDL933. Then, the attachment of E. coli O157:H7 to the meat was determined on two different model muscles, with different contractile and metabolic characteristic (Soleus oxidative, slow and EDL glycolytic, fast), previously characterized by UV microspectroscopy coupled to synchrotron radiation fluorescence. The different of muscle fiber types and the effect of a prolonged anoxia simulating maturing meat were discriminated by their spectral responses after excitation at 275 nm. It clearly appeared that bacteria displayed differential tropism as function of the muscle types, higher for the Soleus than the EDL muscles. While E. coli O157:H7 adhered similarly to the different types of muscle fibers, bacterial adherence essentially occurred at the ECM, pinpointing the key role of connective tissue for E. coli O157:H7 adhesion to meat. This first comprehensive investigation of bacterial adhesion to skeletal muscles at molecular, cellular and tissue levels provides new insight in the physiology of the colonization of meat by EHEC and constitutes a prerequisite for the development of innovative practices and strategies to minimize the risk of meat contamination

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Cordonnier, Charlotte. "Survie et pathogénicité des EHEC dans l'environnement digestif : Interactions avec le microbiote et l'épithélium intestinal. : Influence de l'administration de levures probiotiques." Thesis, Clermont-Ferrand 1, 2015. http://www.theses.fr/2015CLF1MM21/document.

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Les Escherichia coli entérohémorragiques (EHEC) sont des pathogènes majeurs pour l’homme responsables de toxi-infections alimentaires pouvant évoluer vers des complications potentiellement mortelles. La pathogénicité de ces souches est essentiellement due à la production de Shiga-toxines (Stx), même si d’autres facteurs semblent jouer un rôle important dans la virulence, comme des facteurs d’adhésion. La survie et la régulation des facteurs de virulence des EHEC dans l’environnement digestif humain sont des facteurs clés dans la pathogénicité bactérienne, mais restent à ce jour mal décrits, essentiellement en raison d’un manque de modèles d’étude adaptés. De plus, l’absence de traitement spécifique a conduit à s’intéresser à des moyens préventifs et/ou curatifs alternatifs, comme l’utilisation de probiotiques. L’objectif de ce travail de thèse est (i) de mieux comprendre le comportement de la souche de référence EHEC O157:H7 EDL933 dans l’environnement digestif humain simulé, et en particulier ses interactions avec le microbiote résident et l’épithélium intestinal, et (ii) d’évaluer l’effet antagoniste d’une souche de levure probiotique vis-à-vis de la survie, la virulence et l’interaction du pathogène avec l’épithélium intestinal, à l’aide d’approches in vitro et in vivo complémentaires. En modèles digestifs in vitro, la souche EHEC survit dans l’estomac, voire se multiplie dans les parties distales de l’intestin grêle, alors qu’elle ne se maintient pas dans l’environnement colique. Les gènes de virulence codant les Stx et des adhésines majeurs (intimine et « Long Polar Fimbriae » ou Lpf) sont surexprimés dès les parties hautes du tractus digestifs, et ce, même en absence de cellules épithéliales. Les conditions rencontrées dans le tractus digestif supérieur de l’enfant, comparativement à celui de l’adulte, conduisent à une survie et un niveau d’expression des gènes codant les Stx et les Lpf plus élevés chez l’enfant, ce qui peut contribuer à expliquer la grande sensibilité de cette population aux infections à EHEC. Enfin, les Lpf semblent jouent un rôle clé dans le ciblage spécifique des cellules M et le tropisme des EHEC pour les plaques de Peyer, et ce, à la fois in vitro (cellules M en culture) et in vivo (anses iléales murines). Même si elle ne modifie pas la survie du pathogène dans l’environnement colique, la levure probiotique S. cerevisiae CNCM I-3856 a montré des propriétés antagonistes intéressantes vis-à-vis d’EHEC O157:H7 en (i) modulant favorablement l’activité fermentaire du microbiote intestinal, (ii) diminuant significativement l’expression des gènes codant les Stx et (iii) inhibant la translocation bactérienne au travers des plaques de Peyer et les lésions hémorragiques associées. Par ailleurs, l’effet du pathogène et des probiotiques sur le microbiote colique est individu dépendant, confortant l’hypothèse que des facteurs associés à l’hôte, comme le microbiote, pourraient conditionner l’évolution clinique des infections à EHEC et l’efficacité d’une stratégie probiotique.Ce travail de thèse contribue à une meilleure compréhension du comportement des EHEC dans l’environnement digestif humain et confirme l’intérêt d’une stratégie probiotique dans la lutte contre le pathogène. Une étude plus approfondie du transcriptome du pathogène dans l’environnement digestif et une analyse par des méthodes haut débit du microbiote intestinal permettraient de continuer à mieux décrire la physiopathologie des infections à EHEC et comprendre les mécanismes associés à l’effet antagoniste des probiotiques
The enterohemorrhagic Escherichia coli (EHEC) are major zoonotic pathogens responsible for food-borne infectionwhich leads to life-threatening complications in humans. The main virulence determinant of EHEC is the production of Shigatoxins (Stx), even if other factors seem to play an important role in virulence, such as adhesion factors. Survival and virulenceof EHEC strains in the human digestive environment are a key factor in bacterial pathogenesis but remains unclear owing tolack of relevant model. Moreover, no specific treatment has led to interest in preventative and / or curative alternatives, suchas using probiotics. The objective of this study is to better understand the behavior of the reference strain EHEC O157:H7EDL933 in the entire digestive tract, and in particular its interaction with the resident microbiota and the intestinal epithelium,and to evaluate the antagonistic effect of the probiotic yeast, Saccharomyces cerevisiae CNCM I-3856, using in vitro and in vivo complementary approaches.In vitro, bacterial mortality was noticed in the stomach, whereas bacterial growth resumption was observed in thedistal parts of the small intestine and the pathogen was not able to maintain in the human colonic conditions. Virulence genesencoding Stx and adhesins (intimin and “Long polar fimbriae”) are upregulated in the upper parts of the digestive tract. A ten-time higher amount of cells was found in the ileal effluents of infant compared to adult. stx genes were over-expressed (up to25-fold) in infant conditions compared to the adult ones. This results show that differences in digestive physicochemicalparameters of the upper gastrointestinal tract may partially explain why infants are more susceptible to EHEC infection thanadults. And finally, Lpf seem to play a key role in the interactions of EHEC with murine Peyer’s patches and are needed for anactive translocation of the pathogen across M cells, and both in vitro (M cells culture) and in vivo (murine ileal loops).S. cerevisiae had not effect on EHEC survival in the colonic environment but (i) favorably influenced gut microbiotaactivity through beneficial modulation of short chain fatty acid production, (ii) leading to significantly decrease stx expressionand (iii) significantly reduced EHEC translocation through M cells and inhibited in vivo interactions of the pathogen withPeyer’s patches and the associated hemorrhagic lesions. Probiotic had donor-dependent effect on the gut microbiota strengthenthe hypothesis that host-associated factors such as microbiota could influence the clinical evolution of EHEC infection and theeffectiveness of a probiotic strategy.This work contributes to a better understanding of the behavior of EHEC in the human digestive environment andconfirms the interest of probiotic strategy in controlling EHEC infections. Further transcriptome studies are warranted for thepathogen in the human digestive environment, with or without probiotics for the better understanding of the pathophysiologyof EHEC and so on the mechanisms involved in the antagonistic effect of probiotics

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28

Bansal, Tarun. "Inter-Kingdom Signaling Interactions in Enterohemorrhagic Escherichia coli Infections." Thesis, 2010. http://hdl.handle.net/1969.1/ETD-TAMU-2010-08-8177.

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The overall goal of this research was to understand the role of inter-kingdom signaling in enterohemorrhagic Escherichia coli (EHEC) infections of the human gastro-intestinal (GI) tract from the perspective of both the invading pathogen and the human intestinal epithelial cells, which they colonize. Differential gene expression of EHEC was studied upon exposure to the human neuroendocrine hormones epinephrine and norepinephrine. We determined that these hormones increase EHEC chemotaxis, motility, biofilm formation, colonization of host cells, and virulence gene expression. We also studied the EHEC response to the GI tract commensal bacterial signaling molecules indole and autoinducer-2 (AI-2). We observed that indole decreases all the EHEC phenotypes that are increased by the human hormones and represses EHEC virulence. However, the effect of AI-2 was similar to that observed with hormones and opposite to that observed with indole, i.e. AI-2 increases EHEC virulence phenotypes. We studied changes in host cell transcriptome in the presence of the commensal bacterial signal indole. Indole increases expression of genes involved in tight junction and gap junction formation, and production of mucins and actin cytoskeleton genes. Indole also down-regulates genes encoding for pro-inflammatory cytokines, chemokines, and Toll-like receptors. The gene expression results were confirmed with phenotypic assays where we observed an increase in trans-epithelial resistance, increase in the anti-inflammatory cytokine IL-10, decrease in the pro-inflammatory cytokine IL-8, decrease in the activity of the pro-inflammatory transcription factor NF-κB, and decrease in colonization by EHEC of the indole-pre-treated HCT-8 cells. We established that factors secreted by epithelial cells are important determinants of EHEC virulence. Gene expression studies showed that 34 out of 41 LEE virulence genes were induced when EHEC was cultured in conditioned medium. In addition, the data showed increased expression of the shiga toxin-2 prophage 933W. These changes in gene expression were corroborated by a 5-fold increase in HCT-8 cell colonization and increased intracellular Stx2 phage titers. We determined that the HCT-8-secreted factor(s) was protein-based and that it was greater than 3 kDa in size. In conclusion, we have characterized the pathogen response to various eukaryotic and prokaryotic GI tract signals. We have established, for the first time, that the commensal bacterial signal indole is an inter-kingdom signal for the host epithelial cells. Overall, our studies provide a greater understanding of host-pathogen interactions.

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29

Junkins, Alan Dean. "Studies on adherence of enterohemorrhagic Escherichia coli O157:H7." 1991. http://catalog.hathitrust.org/api/volumes/oclc/24262409.html.

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Thesis (Ph. D.)--University of Wisconsin--Madison, 1991.
Vita. Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 225-272).

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30

Sharp, Faith Christine. "Characterization of QSEA and QSED in the quorum sensing cascade of Enterohemorrhagic Escherichia coli." 2005. http://edissertations.library.swmed.edu/pdf/SharpF081105/SharpFaith.pdf.

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31

Chen, Li-Ming, and 陳立鉻. "Rapid Identification and Molecular Typing of Enterohemorrhagic Escherichia coli O157:H7." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/74064198128034331563.

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碩士
國立臺灣大學
農業化學研究所
88
The DNA sequence that includes entire stx2 gene of E. coli O157:H7 strain Y350-1 isolated from cattle faeces in Taiwan was determined. Both DNA and protein sequences have high identities of 99% with other E. coli O157:H7 stx2 gene in database. DNA sequence has 7 base pair differences whereas protein sequence of subunit A has one amino acid difference with the most similar sequence in database. A multiplex PCR with primers specific to hemolysn, intimin, Stx1, Stx2, O-antigen and flagella antigen gene hlyA, eaeA, stx1, stx2, rfb, and fliC was designed and has a high sensitivity of 103 cells. Specificity was tested with Y350-1, 31 E. coli O157:H7 strains from type culture collection center, in which 9 strains from America outbreak, 9 strains from Japan, and 12 strains from Canada, 35 non-O157 enterovirulent E. coli strains, 2 Shigella sp., and 2 Salmonella sp.. No false positive or negative results were found. The specificity of the multiplex PCR primers is very high. Pulsed-field gel electrophoresis (PFGE) was applied to separate the DNA fragments cut from E. coli genome by restriction enzyme Xba I, and the patterns were analyzed by Phoretix 1D Advanced Version 4.01. The principle of typing is base on fragment differences less than 3 that comes from less than 1 genetic difference, and this is the base that two strains are considered as epidemically related isolates. By this way, all strains are separated into 16 types, and the index of discrimination is 0.944. Compare the fragment differences between E. coli O157:H7 Y350-1 and other strains, we have found that there are no strains epidemical indistinguishable or closely related with Y350-1. In this result, Y350-1 should be a local strain in Taiwan.

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32

Chen, Li-Ming, and 陳立銘. "Rapid Identification and Molecular Typing of Enterohemorrhagic Escherichia coli O157:H7." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/89996932641527413163.

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碩士
國立臺灣大學
農業化學研究所
88
The DNA sequence that includes entire stx2 gene of E. coli O157:H7 strain Y350-1 isolated from cattle faeces in Taiwan was determined. Both DNA and protein sequences have high identities of 99% with other E. coli O157:H7 stx2 gene in database. DNA sequence has 7 base pair differences whereas protein sequence of subunit A has one amino acid difference with the most similar sequence in database. 　　A multiplex PCR with primers specific hemolysn, intimin, Stx1, Stx2，O-antigen and flagella antigen gene hlyA, eaeA, stx1, stx2, rfb, and fliC was designed and has a high sensitivity of 103 cells. Specificity was tested with Y350-1, 31 E.coli O157:H7 strains from type culture collection center, in which 9 strains from America outbreak, 9 strains from Japan, and 12 strains from Canada, 35 non-O157 enterovirulent E. coli strains, 2 Shigella sp, and 2 Salmonella sp.. No false positive or negative results were found. The specificity of the multiplex PCR primers is very high. 　　Pulsed-field gel electrophoresis (PFGE) was applied to separate the DNA fragments cut from E. coli genome by restriction enzyme Xba I, and the patterns were analyzed by Phoretix ID Advanced Version 4.01. The principle of typing is base on fragment differences less than 3 that comes from less than 1 genetic difference, and this is the base that two strains are considered as epidemically related isolates. By this way, all strains are separated into 16 types, and the index of discrimination is 0.944. Compare the fragment differences between E. coli O157:H7 Y350-1 and other strains, we have found that there are no strains epidemical indistinguishable or closely related with Y350-1. In this result, Y350-1 should be a local strain in Taiwan.

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33

Huang, Ling-Hui, and 黃鈴惠. "GrlA Involved in the Pathogenesis of Enterohemorrhagic Escherichia coli O157:H7." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/67207402112893697672.

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碩士
國立陽明大學
微生物及免疫學研究所
93
The locus of enterocyte effacement (LEE) island is one of the virulence factors of enterohemorrhagic Escherichia coli (EHEC) O157:H7 which including proteins of type III secretion system (TTSS). EHEC infects and causes attaching and effacing lesion on host large intestinal cell thorugh the TTSS. Global regulator of LEE-activator (GrlA), which is located within the LEE, is a positive regulator of the LEE operons. However, the regulatory mechanism and the relationships among GrlA and the other two LEE-encoded regulators, GrlR and Ler, still await deciphering. To address this issue, a grlA-deleted mutant (i.e. �孱rlA) was created first, and the deletion resulted in severe damage of the protein synthesis and secretion abilities of the TTSS. To analyze the regulatory mechanism of GrlA and dissect the functions down to a molecular level, series of truncated GrlA molecules were generated. These constructs were transformed to the bacteria, and we analyzed the expression level of LEE proteins by Western blotting. The overexpression results indicated that the 1-138 amino acids of GrlA was sufficient to rescue the synthesis of the type III proteins. By gel-retardation assay and reprotor assay, GrlA was found to activate and bind to the LEE1 promoter region which resulted in upregulated-expression of LEE1 encoded proteins. The GrlA’s binding ability could be inhibited by the presence of GrlR and this observation was consistent with the proved association between GrlA and GrlR. Regions of GrlA critical for this interaction were further defined through the bacterial two hybrid assay. All together, our findings have provided a model that explains how GrlA positively modulates the LEE and counteracts with the inhibitory function of GrlR.

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34

Chang, Shu-Ting, and 張舒婷. "Dissecting the EspA Secretion Mechanism of Enterohemorrhagic Escherichia coli O157: H7." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/47673373893516858561.

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碩士
國立陽明大學
微生物及免疫學研究所
98
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic diarrhea. The major virulent factors reside in the locus of enterocyte effacement (LEE) in the bacterial genome. The LEE island encodes a set of genes for forming a specialized machinery called the type III secretion system (TTSS). TTSS secretes effectors, which collectively cause morphological changes of the infected cell, via a long filamentous structure that connects bacteria to the host cell membrane. This filament is composed of polymerized EspA and forms a hollow channel through which secreted protein may pass. A cytoplasmic protein, CesAB, has been identified as the first chaperone of EspA, a protein that posses an aggregation tendency. However, the translocation and assembly mechanism of EspA from bacterial cytoplasm to extracellular space could be more complicated than previously thought. In previous studies, we documented that L0050 and CesA2, proteins located in the inner-membrane fraction, could interact with EspA, respectively. Here, we further demonstrated that L0050 interacts with CesA2 by co-purification. Co-purification also revealed that EspA, L0050, and CesA2 could form a complex. Furthermore, mapping of L0050 by a bacterial two-hybrid system has shown that the N-terminal region of L0050 is needed for interaction with EspA, and the N-terminal region-deleted L0050 remains active to interact with TTS ATPase (EscN) that is thought to energize the secretion process. We therefore proposed a model that L0050 and CesA2 cooperate to escort EspA to the inner membrane of EHEC. Through the binding of EspA, L0050 interacts with EscN, which provides energy for efficient translocation of EspA.

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35

Yu, Sung-Liang, and 俞松良. "Interaction between Enterohemorrhagic Escherichia coli O157:H7 and Its Specific Phage AR1." Thesis, 1999. http://ndltd.ncl.edu.tw/handle/62010991960089688456.

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博士
國立陽明大學
微生物暨免疫學研究所
87
AR1, a coliphage whose morphology and some essential genes are similar to those of T4 phage, infects Escherichia coli O157:H7 with high specificity. We characterized its two tail fiber proteins gp37 and gp38, and investigated host factors that confer this infection specificity. The host range determinants of T4-like phages and T2-like phages are gp37 and gp38, respectively. It is known that g37 of AR1 does not share similarity with that of any T-even phages except for the 5'' region coding for the first 56 amino acid residues. In the phage particles, gp38 could be detected in the T2-like phages, but not in the T4-like phages. Using specific antibodies, we demonstrated that gp38 was also detected in AR1 particle, a result supporting the previous genetic data showing that g38 is distantly related to that of T2-like phages, particularly in the glycine-rich coding regions. Therefore, we concluded that molecularly AR1 is closer to T2 than T4. We also demonstrated that the outer membrane porin OmpC of E. coli O157:H7 is an important factor that determines the AR1 specificity. OmpC of E. coli O157:H7 was shown to differ from the counterpart of E. coli K-12 that is impermissive to the AR1 infection. A 7% variation of the two OmpC molecules may partly explain the adverse host differences in the AR1 susceptibilities. We deduced this phage-host interaction by (1) transposon mutagenesis to select hosts with potentially critical gene(s) mutated; (2) complementation in the Tn-mutants with plasmids that expresses the putative candidate; (3) generation of knockout mutants by specific gene replacement and further confirming the gene-phenotype relationship using the complementation assay. In addition to OmpC, a gene of waaJ homolog was demonstrated to be important for supporting the infection of AR1. The gene product of waaJ is suggested to be a glucosyltransferase involved in the biosynthesis of R3 type LPS. So far, the LPS type of E. coli O157:H7 has not been characterized. The presence of a waaJ homolog suggests that the LPS organization of E. coli O157:H7 may belong to the R3 type. Furthermore, disturbing the expression of waaJ interfered with the synthesis of OmpC and consequently resulted in the loss of susceptibility to AR1. LPS per se may also contribute to a minor supporting factor of the AR1 infection as mutation affecting the integrity of LPS decreased slightly the plaque forming efficiency of AR1.

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36

Chan, Chen-Hung, and 陳宏展. "Environmental factors on the growth and toxin production of enterohemorrhagic Escherichia coli." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/79777487723155764940.

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37

Wu, Ting-I., and 吳亭儀. "Characterization of l0050 in Pathogenicity Island of Enterohemorrhagic Escherichia coli O157:H7." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/39067890535771273950.

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碩士
國立陽明大學
微生物及免疫學研究所
94
Enterohemorrhagic Escherichia coli (EHEC) is a pathogen that attaches to host intestinal epithelium and efface brush border microvilli, forming A/E lesions. Many key virulence factors reside in the locus of bacterial chromosome named enterocyte effacement (LEE) island. The LEE island contains 41 genes and encodes a type III secretion system (TTSS). TTSS is commonly found in Gram negative pathogenic bacteria. The unique feature is a delivery of bacterial proteins capable of modulating host cellular functions. Recent studies have assigned functions for most genes within this locus. However, the functions of a few LEE genes remain unknown, and l0050 is typically one of them. l0050 is located on LEE1 operon. The intracellular amount of EspA detected was adversely reduced whereas that of EspD was slightly reduced in a l0050 deletion mutant. On the other hand, the secretion of all type III proteins was completely lost. L0050 is hypothesized to be involved in post-transcriptional regulation of EspA. Our results indicated that the stability of EspA in a l0050 deletion mutant was affected. In contrast, there was no apparent difference with the stability of EspD between the wild-type strain and the mutant. Thus, L0050 may be involved in the stabilization of EspA during secretion process, perhaps by acting as a component of TTSS apparatus that bind to and regulate ATPase EscN.

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38

蘇淑薇. "Characterization of gene Loo17 in pathogenicity island of enterohemorrhagic escherichia coli O157:H7." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/93929477561911154515.

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碩士
國立陽明大學
微生物暨免疫學研究所
91
Enterohemorrhagic Escherichia coli O157:H7 causes enterohemorrhagic diarrhea and a serious illness known as hemolytic-uremic syndrome. In the contact with epithelial cells, bacteria tightly associate with cells through a pedestal formation. Underneath the pedestal structure, cytoskeleton rearrangement has been observed. These pathogenic characteristics have been attributed to that this bacterium contains a pathogenic island located on the bacterial chromosome known as the locus for enterocyte effacement (LEE). There is a predicted cluster of 41 open reading frames (ORFs) in LEE, and some of the ORF have been characterized; the encoded proteins are categorized to be components of a type three secretion apparatus, the effector proteins translocated into the contacted host cells, the translocators, the chaperones associated with specific proteins mentioned above, specific transcriptional regulators, and others remained to be characterized. Among the uncharacterized ORFs, L0017 encodes 92 amino acid residues with a predicted pI at 5.46. An L0017-deleted mutant created by homologous recombination gave substantial decrease of EspA and EspD synthesis but not those of EspD and EspB and a complete loss of secreting these proteins. The bacteria also lost the adhesion activity toward host cells and no pedestal formation. By complementation with L0017 expressed from plasmid, the lost phenotypes were recovered. L0017 was not found in the concentrated bacterial media, rather it was found in the bacterial lysate. Fractionation of the bacterial lysate into cytosol, periplasmic proteins, inner membrane and outer membrane fractions indicated that L0017 was mainly located in the inner membrane fraction. Typically, chaperone proteins found in the Gram negative bacteria are acidic protein with small sizes. L0017 appears not belonging to that group. The distribution of L0017 in the inner membrane fraction suggested that this protein act as one of the apparatus components.

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39

Ting-ChenChou and 周廷蓁. "Functional Genomic Analysis of the Virulence of Enterohemorrhagic Escherichia coli in Caenorhabditis elegans." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/36q4v9.

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碩士
國立成功大學
生物化學暨分子生物學研究所
100
Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a major foodborne pathogen causing severe disease in humans worldwide. Three major groups of virulence factors of E. coli O157:H7 have been identified including the Shiga-like toxins, the products of the pathogenicity island called the locus of enterocyte effacement (LEE), and the products of the F-like plasmid pO157. Several strategies for therapy have been studied including the use of antibiotics and vaccination. However, there is no specific treatment for E. coli O157:H7 infection so far and the use of antibiotics may be contraindicated. Therefore, highly effective measures for prevention and control of E. coli O157:H7 infection are essential. Recent reports suggested that the nematode Caenorhabditis elegans can be used as an infection model for diverse groups of bacterial pathogens. Here, we illustrated the C. elegans, which may encounter EHEC in its habitat naturally, as a model host for studying EHEC infection. Our current data suggested that the Shiga-like toxin 1 (Stx1), an important toxin virulence factor for mammalian cells, was required partly for the virulence of E. coli O157:H7 to C. elegans. Moreover, we analyzed the roles of the other well known EHEC virulence factors, which are identified in mammalian cell culture systems, in C. elegans. Finally, we have established a genome-wide transposon mutagenesis library to screen for the bacterial factors that are required for the pathogenesis of EHEC infection in C. elegans. From this genetic screen, we have discovered that the genes involved in lipopolysaccharide (LPS) synthesis may play important roles on the pathogenesis of O157:H7. We also found the component of EHEC LPS is different from that of E.coli OP50. This may suggest that the LPS of EHEC is a key factor on influencing the lifespan of C. elegans. We envision this simple, yet elegant, in vivo system will be useful for identifying previously uncharacterized EHEC virulence factors, and may pave a way for novel treatments beyond traditional therapy.

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40

Wu, Yi-Chih, and 吳宜之. "Analyzing Enterohemorrhagic Escherichia coli O157:H7 Pathogenicity Island at mRNA and Protein Levels." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/32144020522900189279.

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碩士
國立陽明大學
微生物暨免疫學研究所
90
Enterohemorrhagic Escherichia coli (EHEC) has emerged in recent years as the predominant cause of hemorrhagic colitis in humans. During infection, EHEC causes attaching and effacing (A/E) lesions on the intestinal cells. The genes involved in the formation of these A/E lesions are encoded within a chromosomal pathogenecity island named the locus of enterocyte effacement (LEE). The LEE comprises of 41 open reading frames (ORFs) organized in five major operons, LEE1, LEE2, LEE3, tir (LEE5), and LEE4. Among these ORFs, several have been identified, but others whose functions remain unknown. In spite of the new understanding of pathogenesis from recent studies, some questions still remain unclear. What is the correlation between these unknown open reading frames with pathogenesis? How do the LEE genes coordinately express during the process of infection? How do interactions between gene products occur in order to carry out the Type Ⅲ secretion？ To address these questions, we established LEE microarray technology to study the expression profile of genes in LEE island. In the study, we observed a rapid activation of the LEE genes except L0036 under carbon dioxide stimulation. However, secretion of proteins such as EspA, EspB and Tir appeared only after 4-h stimulation of carbon dioxide. We also used Far Western blotting and affinity column to analyze the possible protein-protein interactions between gene products encoded by LEE island as well as bacterial chromosome. The result indicated that L0044 (encoded in LEE island) may interact with some proteins to form complexes. After identifying the major protein with MALDI and Q-TOF, we found a possible target interacting with L0044.

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41

Hsu, Pin-Yuan, and 徐彬源. "Characterization of L0045 in the Pathogenicity Island of Enterohemorrhagic Escherichia coli O157:H7." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/16140202955170438841.

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碩士
國立陽明大學
微生物及免疫學研究所
95
The locus of enterocyte effacement (LEE) island is the vital virulence factors of enterohemorrhagic Escherichia coli (EHEC) O157:H7 which includes proteins of type III secretion system (TTSS). EHEC infects and causes attaching and effacing lesion on host large intestinal epethilial cell thorugh the TTSS. LEE island has three main regulators, including global regulator of LEE-activator (GrlA), Global regulator of LEE-repressor (GrlR) and LEE-encoded regulator (Ler) , GrlA and Ler are the positive regulator of the LEE operons. On the other hand , GrlR is the negative regulator. In order to interpret the roles plays by gene l0045, we do some experiments about the characterization of gene l0045 , in addition, the experiment about the regulatory mechanism and the relationships among these regulators and l0045 need to be done as well. In this issue, it was found that protein L0045 was mainly in the outer membrane fraction.When L0045 overexpress in the EHEC wild type strain, we couldn’t detect L0045 even in the medium which promotes LEE island expression like M9 and DMEM medium. On the contrary, we could detect L0045 in the JM109 wild type strain and EHEC ler deletion strain. Furthermore, we also doubt L0045 has the lysozyme activity, but the experiment result isn’t clear enough to prove it .At last, we propose that GrlR is the negative regulator of the gene l0045.GrlA and Ler may be not the regulator of the gene l0045. GrlR may promote EHEC growth.

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42

莊振樺. "Deducing tir properties that are different between escherichia coli of enterohemorrhagic and enteropathogenic virotypes." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/76322724588390171517.

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43

Yu, Yen-Chi, and 游硯棋. "The Role of l0045 in the Type III Secretion System of Enterohemorrhagic Escherichia coli." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/ctg8ce.

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碩士
國立陽明大學
微生物及免疫學研究所
97
Enterohemorrhagic E. coli (EHEC), which is an etiological agent for food-borne illnesses, is one of the pathogenic E. coli strains. Infection of EHEC often leads to bloody diarrhea and sometimes to serious diseases of hemolytic-uremic syndrome and acute kidney failure. EHEC infects large intestinal cells and forms typical histological lesions called attaching and effacing lesions (A/E lesion). The known mechanism is that the bacteria employ a type III secretion system to deliver several effector proteins into the infected cells. The effector proteins then trigger the formation of pedestal structure that tightens the attachment of the bacteria to the host cells. Most of the genes involved in the formation of A/E lesion reside in the locus of enterocyte effacement (LEE), a pathogen island. The LEE island contains 41 open reading frames. Some of the genes have been well studied as their roles in the type III secretion system are well understood whereas the others are not. l0045 is among the genes that have not been well characterized. To investigate the role of L0045 in the type III secretion system, an l0045-deletion mutant strain was created. The deletion of l0045 results in the attenuation of the type III secretion and decrease of the expression of representative LEE proteins. However, when L0045 expression is driven by a strong promoter, the intracellular levels of Tir and EspA in EHEC were severely suppressed. Furthermore, under this circumstance, the protein level of L0045 in EHEC was hardly detected. And this was in contrast to an observation that a huge amount of L0045 was seen when similarly performed in K-12 of the JM109 strain, a result suggesting that a network must exist in EHEC to tightly regulate the synthesis of L0045. This regulation could link to GrlA, one of the LEE regulator, since deleting grlA from EHEC increased an expression of L0045.

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44

Chiu, Hao-Chiech, and 邱浩傑. "Analysis of EspB Determinants Involved in Type III Secretion System of Enterohemorrhagic Escherichia coli." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/36697333417579270402.

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碩士
國立陽明大學
生物藥學研究所
88
Enterohemorrhagic E. coli (EHEC) can cause different presentations of disease in human. EHEC, like other Gram negative pathogenic bacteria, causes attachment and effacement lesion (A/E lesion) with infected epithelial cells. A type III protein secretion system is necessary for the formation of A/E lesion, and several proteins that are secreted by the system, including EspB (E. coli-secreted proteins B), are involved in inducing the formation of actin pedestals. EspB is secreted outside the bacteria and translocated to the membrane and cytosol of infected epithelial cell. The translocated EspB is also found to affect the signal transduction of infected cell. In this study, we found that the secretion of EspB required at least the first 190 residues and the reporter protein must be fused at the carboxyl terminus of EspB for efficient secretion. We also found that residues 22-117 are important for efficient secretion of EspB. However, the region after residue 117 may also facilitate the secretion. The secretion of the EspB fusion protein was abolished when the central region (119-190) of EspB was changed by frame shifting. Furthermore, without the endogenous EspB, EspB fusion protein can still be secreted by the EHEC. The results above suggest that the secretion of EspB may be controlled by the protein conformation of EspB, particularly an N terminal region. But, the controlling mechanism of secretion remains unexplored.

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45

Hsu, ching-Fang, and 許瀞方. "Detection of enterohemorrhagic Escherichia coli O157:H7 by using multiplex real-time PCR technology." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/17555476995842352456.

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碩士
國立臺灣大學
微生物與生化學研究所
92
Real-time polymerase chain reaction (PCR) assays have been developed for detection and quantification of pathogens in recent years. A multiplex real-time PCR assay was designed and evaluated on the ABI 7700 sequence detection system (TaqMan®) to detect enterohemorrhagic Escherichia coli O157:H7 in pure culture, feces and food samples. Two sets of primers and fluorescent probes were used for amplification and real-time detection of a 192-bp region of the rfbO157 gene encoding E. coli O157:H7-specific O-antigen, and 170-bp segment of stx2 gene encoding Shiga-like toxin 2. Analysis of 217 bacterial strains demonstrated that the multiplex real-time PCR assay successfully distinguished E. coli O157:H7 serotype from non- E. coli O157:H7 serotypes and provided accurate profiling of genes encoding O-antigen and Shiga-like toxin 2. Bacterial strains lacking these genes were not detected by this assay. The quantitative ranges of the real-time PCR assay for the two genes were linear over DNA concentrations corresponding from 103 to 109 CFU/mL of E. coli O157:H7 in pure culture and milk sample. The real-time PCR allowed construction of standard curves that facilitated quantification of E. coli O157:H7 in feces, apple juice, milk and ground beef sample. Detection sensitivity of the real time PCR assay ranged from 104 to 109 CFU/g (or 104 to 109 CFU/mL) of feces or apple juice without enrichment. Detection sensitivity of the real time PCR assay ranged from 105 to 109 CFU/g of ground beef without enrichment.After enrichment of milk and apple juice samples in modified Tryptic Soy Broth, the detection of levels were from 100 to 104 CFU/mL. The real-time PCR assay for rfbO157 and stx2 proved to be a rapid test for detection of E. coli O157:H7 in food matrices and could also be used for quantification of E. coli O157:H7 in foods or fecal samples.

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46

Huang, Hui-Yu, and 黃惠鈺. "The role of EspFu in the pathogenic mechanism of Enterohemorrhagic Escherichia coli O157:H7." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/14691823302222872549.

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碩士
國立陽明大學
微生物及免疫學研究所
94
After enteropathogenic Escherichia coli (EHEC) infects cells, disease presentations such as attaching and effacing (AE) lesions occur. While microbial pathogens are attached to the epithelium of the host, the Tir molecules of bacteria are translocated to the host cell membrane and bind to the bacterial outer membrane adhesion molecule intimin. Translocation of Tir and other effector proteins is mediated by a type III secretion system of EHEC, and the resulting events include a disturbing of cellular signal pathways that regulate actin polymerization at the plasma membrane. Abnormal actin polymerization is necessary to trigger pdedstal formation, which comes together with intercellular cytoskeleton rearrangement and effacement of microvilli. This pathogenic phenotype is the so-called attaching and effacing (AE) lesions. EspFu is encoded by a remote locus away from that codes for Tir, and it is also translocated into host cells in order to produce A/E lesions. To reduce the complexity of other translocated proteins with an aim to define the function of EspFu within the cells, we directly expressed EspFu in the cells by transfection. Immunofluorescence staining using confocal microscopy showed that EspFu was expressed in both cytoplasm and cell nuclei. Morphologically, about 30 percent of transfected cells appear relatively in round shapes. We suggested that EspFu may disturb actin rearrangement in some cells. Furthermore, we removed three proline-rich domain of the C terminus of EspFu and found that this proline-rich carboxyl region is critical for transferring EspFu to nucleus of host cells. According to previous studies, Tir and EspFu of EHEC are critical for the formation of actin pedestals. To examine whether EspFu and Tir are sufficient to induce actin polymerization, Tir and EspFu were co-transfected into host cells and the results were compared to that transfect only with Tir; no differences were observed. Meanwhile, when EspFu over-expressed host cells were infected with wild EHEC, we observed that the degree of pedestal formation and the time of pedestal formation had no substantial changes. When EspFu over-expressing EHEC was used to infect EspFu-transfected cells, the degree of pedestal formation and actin polymerization were less than that observed with untransfected cells. These observations may be explained by pre-exisitng EspFu may excessively compete with those proteins required for actin polymerization. Therefore, EspFu during the EHEC pedestal formation may be preferentially controlled at a sufficient but not excessive level.

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47

Gao, Yi-Shun, and 高義順. "Investigation of Anomalous SDS-PAGE Migration of EscC in Enterohemorrhagic Escherichia coli O157:H7." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/73066695691720237571.

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碩士
國立陽明大學
微生物及免疫學研究所
99
Enterohemorrhagic E. coli (EHEC), a Gram-negative bacterium, which infects human gastrointestinal tract through undercooked beef or contaminated foods, is one of the pathogenic E. coli strains. Infection of EHEC could cause bloody diarrhea and sometimes lead to hemolytic-uremic syndrome and kidney failure. EHEC attacks the epithelial cells of intestinal tracts and results in typical histological lesions called attaching and effacing lesions (A/E lesion). The type three secretion system of EHEC plays an important to this lesion. Bacteria regulate a type three secretion system to deliver effector proteins into the infected epithelial cells. After entering host cells, these effector proteins trigger intracellular signals, cause actin rearrangement and finally bring on the formation of pedestal structure that tightens the attachment of EHEC to infected cells. Most of the genes involved in the formation of A/E lesion reside in the locus of enterocyte effacement island (LEE island), a pathogenic gene locus. The LEE island contains 41 open reading frames, and gene escC is one of these LEE genes. The escC encodes a component of the outer membrane ring of type three secretion system. According to previous study of our laboratory, we expressed plasmid-encoded EscC protein by in different E. coli strain of K-12 and EHEC to compare protein characteristics and found that EscC from EHEC had slower gel mobility than that seen with the K-12 strain in SDS-PAGE electrophoresis. We suggested that this anomalous SDS-PAGE migration could result from EscC protein in EHEC with an additional post-translational modification and tried to find out the possible protein modification for proving this hypothesis. However, we do not find any modification of EscC related with anomalous SDS-PAGE migration. In this study, we ruled out the possibility of protein modification and found that anomalous gel mobility of EscC could arise from the different cell membrane composition between EHEC and K-12 strain. The components in inner membrane of EHEC might change the mobility of EscC in SDS-PAGE electrophoresis. We suggest that the composition of membrane phospholipid in EHEC might different from K-12 strain. The special composition of phospholipid in EHEC membrane, which might lead to anomalous SDS-PAGE migration of EscC, could help EHEC assemble type three secretion system more smoothly.

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48

Hung, Tzu-Chun, and 洪子淳. "Characterization of L0053 Encoded in the Pathogenicity Island of Enterohemorrhagic Escherichia coli O157:H7." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/41396646220190792575.

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碩士
國立陽明大學
微生物及免疫學研究所
100
Enterohemorrhagic Escherichia coli (EHEC) utilizes Type III Secretion System (TTSS) to deliver virulent effectors, a result leading to cytoskeleton rearrangement and pedestal structure formation. This pedestal structure holds EHEC tightly to host cells and causes human epithelial cells becoming loosened and sparse, a pathogenesis named attaching and effacing lesion (A/E lesion). The effector proteins are encoded by genes clustered in the locus of enterocyte effacement (LEE) island in bacterial chromosome. The LEE island contains 41 open reading frames (ORFs) that could be divided into five major operons, LEE1 to LEE5. TTSS is structurally comprised of a membrane basal body and an external needle complex. The needle structure is organized mainly by EspA that possesses a biochemical tendency of self-polymerization. Thus, in the formation of TTSS, EspA needs chaperones or binding proteins for stabilization before the needle structures are assembled. Currently known chaperones are: CesAB (L0052), CesA2 (L0017), and EscL (L0050). Here, we focus on a less characterized protein, L0053, that also causes poor EspA expression when its cognate gene is deleted. L0053 is the fourth ORF in LEE1. By protein fractionation, L0053 is found in the inner membrane and cytosol fractions. Experimentally demonstrated is that L0053 has the ability to interact with one of EspA chaperones, CesA2. Moreover, EHEC strain with l0053 deleted (L53) shows that EspA dramatically diminishes while another secretion protein, EspB, has no apparent effect. However, a consequence is that Tir, EspB, and EspA all disappear from bacteria supernatant. BacterioMatchTM Two-Hybrid-System was used to map how L0053 binds to CesA2 and the N-terminal 19~36 amino acids of CesA2 were concluded to be critical. No binding of L0053 has been observed with any other EspA chaperone. Intriguingly, by the same approach, the same N-terminal region of CesA2 was found critical for the binding of EspA. However, L0053 and CesA2 have no ability to modulate the stability and localization of the expressed proteins to each other. And over-expression CesA2 in EHEC L53 strain couldn’t restore the physiological ability of EspA. Thus, we conclude that EHEC L0053 may serve as a co-chaperone for CesA2 so that CesA2 could execute its interaction with EspA at a right timing after EspA is released from ribosome while moving forwards to the assembly during de novo synthesis.

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49

Buryakina, Tatyana, and 棠雅. "Monitoring of HeLa Cells Revealed through Autofluorescence Lifetime upon Infection with enterohemorrhagic Escherichia coli." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/75206767510233325746.

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博士
國立陽明大學
生醫光電工程研究所
100
Bacterial infections and their influence on cellular physiology are one of the critical interests for researchers in different areas, such as microbiology, medicine, drug design, etc. due to the importance of thorough understanding of the processes related to the development of the efficient therapies. A number of different techniques are being applied for these studies, among which fluorescence spectroscopy has gained wide acceptance because of its high sensitivity and resolution. Being minimum invasive technique, autofluorescence spectroscopy allows studying the processes in live pathogens within their host cells. In particular, fluorescence lifetime imaging microscopy (FLIM) has become an established technique for the monitoring of the functional/conformational states of reduced nicotinamide adenine dinucleotide (NADH) and flavine adenine dinucleotide (FAD), main compounds of cellular metabolism production and oxidative phosphorylation, through the observation of its lifetime dynamics. A gradual shortening of the decaying time constants in both the short and the long lifetime components of NADH’s autofluorescence is detected. The ratio of the short and the long lifetime components’ relative contributions, however, shows a rapid increase, indicating the rise of cellular metabolic activity over the course of infection. Since the majority of the NADH activity is related to mitochondria, observation of the mitochondrial NADH redox state has a potential for detecting the changes in the cellular metabolic state and thus properly characterizing normal and pathological states. The technique is able to reveal changes of the cellular metabolic activity at different time of infection and bacteria concentration. The evolving of NADH’s average autofluorescence lifetime during the 3 hours after infection with enterohemorragic Escherichia coli (EHEC) or STS treatment has been observed. The ratio of the short and the long lifetime components’ relative contributions of NADH increases in time, a fact indicating cellular metabolic activity, such as a decrease of oxydative phosphorylation over the course of infection, while opposite dynamics is observed in FAD. The combination of fluorescence redox ratio and lifetime imaging at high resolution might potentially provide a tool for understanding the morphological and metabolic changes in cell culture.

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50

TSAY, Yu-Wei, and 蔡郁偉. "Comparative genome annotation for pathogenic entericbacteria phage analysis between enterohemorrhagic Escherichia coli and other bacteria." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/33675776228209603945.

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碩士
元智大學
資訊工程學系
92
In decades, the varieties of organisms were sequenced and annotated form laboratory accordingly. The bacterium Escherichia coli is one of the most comprehensive analyzed free-living organisms recently. The sequence、metabolic pathway and regulatory mechanism of these bacterium are well understanding to comparison with other bacterium. However, the mere sequenced of E. coli was not enough to understand the pathogenic mechanism precisely, nothing but it’s physical property or patient’s pathological diagnosis to make a compendium determination. We do not know the exact pathogenic mechanism as well as which protein or genes are related presently. An aspect of compare to other similar entericbacteria, most of the papers are discussing these sequenced bacteria and diversity of E. coli, the comparison between harmless E. coli (MG1655) and the extraintestinal pathogenic E. coli strain. Nowadays, research does not present a remarkable conclusion via computational methods. We offer a comparative query tool based on some assumptions for disease potential E. coli between each entericbacteria. By the way of our computation result and statistics with alternative variety of comparison, we enumerate some highly possible pathogenic proteins and region for the results as our reference material.

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