Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Orientadores: Wanderley Dias da Silveira, Eliana Guedes Stehling / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T01:02:36Z (GMT). No. of bitstreams: 1 Mobilon_Cristiane_D.pdf: 11609484 bytes, checksum: 06504829a16dd9fe15e1e7aff6ed5392 (MD5) Previous issue date: 2012 / Resumo: A shigelose, uma infecção intestinal grave, causada por bactérias Gram-negativas pertencentes ao gênero Shigella, é uma patologia com incidência significativa, principalmente em países em desenvolvimento, onde ocorrem 99% dos casos relatados, sendo que, 69% deles acometem crianças menores de 5 anos de idade. A alta incidência de Shigella em países em desenvolvimento é, geralmente, atribuída à falta de saneamento básico, condições de higiene inadequadas, subnutrição e alto custo dos antimicrobianos. O modo predominante de transmissão ocorre por via fecal-oral. Os mecanismos de patogenicidade descritos para as diferentes espécies de Shigella incluem numerosos genes de virulência, sendo a maioria encontrada em um plasmídio (pINV) de 220 kb. O plasmídio pINV de S. flexneri codifica 2 operons cruciais para seu fenótipo invasivo: o operon ipa e o operon mxi-spa. O operon ipa codifica os antígenos plasmidiais de invasão denominados IpaA, IpaB, IpaC e IpaD, os quais são efetores para o processo de invasão bacteriana. O operon mxi-spa juntamente com as proteínas IpaB, IpaC e IpaD são essenciais para a invasão in vitro de células epiteliais. Sabe-se, também, que essas proteínas, ou antígenos plasmidiais de invasão, podem ser altamente imunogênicos. Neste trabalho, clonou-se o gene ipaC no vetor pET28a para expressão em Escherichia coli e em vetores constitutivos para bactérias lácticas com a finalidade de se verificar a capacidade imunogênica da proteína expressa por este gene. Além disso, verificou-se que os anticorpos produzidos pela resposta à proteína IpaC foram capazes de inibir a adesão e invasão de Shigella flexneri em culturas celulares cultivadas in vitro confirmando, assim, a importância dessa proteína no processo invasivo da bactéria e seu possível uso no desenvolvimento de futuras vacinas a serem usadas contra a shigelose / Abstract: Shigellosis is a severe intestinal infection caused by Gram-negative bacteria which belongs to the genus Shigella. It is a pathology with high incidence and it is known that 99% of cases occur in underdevelopment countries. Among these cases, 69% of them affect children under 5 years old. The high incidence of Shigella in underdevelopment countries, is mainly atribbuted to substandard hygiene, unsafe water supplies, subnutrition and antibiotic high cost. The predominant mode of transmission is by fecal-oral contact. The pathogenicity mechanisms described for Shigella species include several virulence genes, most of them been encoded in a plasmid (pINV) with 220 kb. This plasmid encodes 2 important operons which are essential for the invasive phenotype: the operon ipa and the operon mxi-spa. The ipa operon encodes the invasion plasmid antigens called IpaA, IpaB, IpaC and IpaD, which are effector proteins required for the bacteria invasion process. The mxi-spa operon together with IpaB, IpaC and IpaD are essential to epithelial in vitro cell invasion. It is also known that these proteins or invasion plasmid antigens may be highly immunogenic. During the development of this work, ipaC gene was cloned in the plasmidial vector pET28a for expression in Escherichia coli and in constitutive vectors for lactic acid bacteria to prove its immunogenic importance. Furthermore it was verified that the antibodies produced in response to IpaC protein were able to inhibit Shigella flexneri adhesion and invasion in in vitro cultured cells, confirming its important hole in bacteria invasion process and its possible usage in vaccine development against shigellosis / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

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The Shigella species is responsible for many diarrheal infections and deaths across the world each year, with the largest impact on less industrialized countries, especially in children under 5 years of age. The battle between the lack of a targeted treatment or vaccine and the significant rise of antibiotic resistance in Shigella underscores the importance of fully understanding mechanisms of Shigella virulence. Past research clearly demonstrates that Shigella flexneri strain 2457T utilizes host physiology to regulate pathogenesis, including increasing virulence during exposure to bile salts at concentrations found in the small intestine. This study aimed to further characterize the effects of bile salts exposure in Shigella by focusing on a particular gene induced in the presence of bile salts. Growth curve analyses were performed with S. flexneri wild-type and mutant strains to examine the role of the unknown protein in the growth of Shigella during bile salts exposure. To examine the effects of the mutation on virulence, a Congo red secretion assay was also used as a measure of type-III secretion system function as well as invasion assays, both of which used bile salts in the subculture conditions to mimic small intestinal transit of wild-type and the mutant strain prior to infection in the colon. The mutant displayed no change in growth patterns in comparison to WT in the presence or absence of bile salts. However, the mutant displayed increased protein secretion and invasion rates relative to wild-type. Overall, the data suggest that this bile salts-induced gene encodes a protein that negatively regulates S. flexneri virulence, likely providing protection against a hypervirulent phenotype of Shigella. This work has succeeded in further characterizing an unknown protein that is induced by bile salts, and could provide insight for future therapeutic and vaccine development. / 2025-01-19T00:00:00Z

Made available in DSpace on 2015-04-11T13:38:31Z (GMT). No. of bitstreams: 1 Maria Scheffer.pdf: 2086782 bytes, checksum: 928db75671ab568b474a01a176b2665e (MD5) Previous issue date: 2012-12-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Shigellosis is a disease manifested from mild diarrhea to severe ysentery. The ability to invade cells, cause injury and imbalance in the homeostasis to the intestinal mucosa and modulate the inflammatory response has been attributed to several factors of pathogenicity of Shigella spp. In this study, thirty-six isolates of Shigella were identified from an etiologic study of childhood diarrhea in 1,500 children aged 0-10 years who were admitted to hospitals in Manaus (East-South-and-West Zones) between August 2007 and July 2009. Shigella flexneri was the most common (65%), followed by Shigella sonnei (20%), Shigella boydii (12.5%) and Shigella dysenteriae (2.5%). Among the isolates, one isolate (intermediate) resistant to ciprofloxacin and 1 (intermediate) to ceftriaxone, which are antibiotics recommended by WHO. Molecular typing of the pathogenicity factors showed evidence of the pathogenic potential of shet- 1B subunit of Shigella and can lead to complications related to dehydration. Frequencies of the IpaBCD (the most prevalent factors among isolates) followed by IpaH suspect associations to fever and bowel injury. The IpaH was clearly associated to bloody diarrhea. And finally, differences in the frequencies of Shet-2 positives between febrile-based groups reinforce its role in the inflammatory response. This study has contributed to the few data in the literature on the pathogenicity of wild Shigella isolates associated to shigellosis and consolidate the importance of finding ways to block the toxicity of these major pathogenicity factors / A shigelose é uma doença manifestada desde uma diarreia leve até uma severa disenteria. A habilidade de invadir células e provocar lesão na mucosa intestinal, bem como levar ao desequilíbrio na homeostase e modular a resposta inflamatória em nível intestinal tem sido atribuídos a diversos fatores de patogenicidade das Shigella spp. Neste trabalho, trinta e seis isolados de Shigella foram identificados a partir de um estudo etiológico de diarreia infantil em 1.500 crianças com idade entre zero a dez anos que deram entrada nos hospitais de Manaus (Zona Leste, Zona Sul e Zona Oeste) com quadro diarreico, no período entre agosto de 2007 a julho de 2009. Shigella flexneri foi à espécie mais frequente (65%), seguida por S. sonnei (20%), S. boydii (12,5%) e S. dysenteriae (2,5%). Entre os isolados, merece destaque o aparecimento de um isolado resistente (intermediária) à ciprofloxacina e 1 resistente (intermediária) à ceftriaxona, que são os antibióticos recomendados pela OMS. A tipagem molecular dos fatores de patogenicidade mostrou evidências sobre o potencial patogênico da subunidade shet-1B das Shigellas, podendo levar a complicações relacionadas à desidratação, de IpaH na febre e na lesão do intestino evidenciado pelo diarreia sanguinolenta. IpaBCD foram os fatores de virulência mais predominantes seguido de IpaH. E uma frequência maior de Shet-2+ em crianças febris em relação a não febris foi verificada. Este estudo vem contribuir com a pouca literatura que existe em relação aos fatores de patogenicidade de isolados de Shigella selvagens com quadro diarreico com ou sem shigelose e consolidar a importância de encontrar caminhos para bloquear a toxicidade destes principais fatores de patogenicidade

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Source: Masters Abstracts International, Volume: 47-01, page: 0319. Advisers: Irina Cech; Michael Smolensky. Includes bibliographical references (leaves xx-xx).

No description available.

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Shigella est l'agent causal de la shigellose, une maladie à transmission oro-fécale, et responsable d'une grande partie des cas diarrhéiques dans les pays en voie de développement. L'infection par Shigella résulte en la colonisation et l'inflammation de la muqueuse colique. Sa virulence est liée à son Système de Sécrétion de Type 3 (SST3) qui agit comme une "seringue" moléculaire pour l'injection des protéines directement dans le cytoplasme de la cellule hôte, via un pore de translocation, provoquant la subversion de sa physiologie et l'internalisation de la bactérie. Le SST3 est composé d'un bulbe cytoplasmique, d'un corps basal et d'une aiguille extracellulaire assemblée suite à la polymérisation hélicoïdale des sous-unités MxiH, formant ainsi un canal qui permet le transit des substrats du SST3. L'extrémité de l'aiguille comprend les translocateurs IpaBCD qui constituent le complexe d’extrémité. IpaD module l'insertion membranaire des protéines hydrophobes IpaB et IpaC, qui forment le pore de translocation, et prévient, avec IpaB, la sécrétion prématurée des effecteurs avant le contact cellulaire. La sécrétion est également contrôlée à la base du SST3 par la protéine cytoplasmique MxiC qui perçoit le contact cellulaire par un signal transmis à travers l'aiguille. Ces deux dispositifs de contrôle participent à l'établissement d'une hiérarchie de sécrétion entre translocateurs, effecteurs précoces et tardifs. <p>Lors de cette étude, nous avons essayé de mieux comprendre le fonctionnement du SST3 en ciblant la protéine IpaD. Des variants obtenus par délétions de 10 acides aminés (Schiavolin, 2013) et par mutations ponctuelles (Meghraoui, 2014) d’IpaD ont été générés pour caractériser leurs effets sur l'exposition à la surface des translocateurs, le contrôle de sécrétion, la formation du pore de translocation, et enfin l'invasion cellulaire. Nos résultats ont permis d’identifier trois phénotypes correspondant à i) une sécrétion contrôlée, similaire à la souche sauvage, ii) une sécrétion constitutive de tous les substrats, et iii) un phénotype de sécrétion intermédiaire. Les variants par délétions nous ont permis de comprendre l'importance de la localisation d'IpaD et IpaB à la surface pour le fonctionnement du SST3. Les variants par mutations ont révélé l'indépendance entre le contrôle de sécrétion et l’invasion cellulaire, ainsi qu’une corrélation entre la sécrétion prématurée des translocateurs/effecteurs précoces et l'augmentation de la virulence in vitro. Nous avons aussi étudié les partenaires d'interaction d'IpaD (Meghraoui, in prep) et MxiC (Cherradi, 2013) respectivement à l'extrémité de l'aiguille et à la base du SST3. L'interaction d'IpaD avec elle-même et avec MxiH a pu être montrée uniquement après délétion du domaine auto-chaperon d’IpaD. Ce domaine semble essentiel au maintien d'IpaB à l'extrémité de l'aiguille, au contrôle de sécrétion, à l'insertion du translocon, mais pas à l'invasion cellulaire. D'autre part, nous avons démontré que l'interaction entre MxiC et le composant de la tige interne MxiI participe au contrôle interne de la sécrétion. En conclusion, nos travaux contribuent à une meilleure compréhension du lien entre différents composants et fonctions du SST3 et de l'implication d'IpaD dans la régulation allostérique de la sécrétion. Ces résultats dépassent le cadre de Shigella puisque les composants étudiés dans le cadre de cette thèse sont conservés chez d’autres bactéries utilisant le SST3 comme dispositif principal de virulence./<p><p>Shigella is the causative agent of shigellosis, an oro-fecally transmitted disease, among major causes of diarrhoea in developing countries. Infection by Shigella results in the colonisation and inflammation of colonic mucosa. The virulence of this bacterium is related to a Type 3 Secretion System (T3SS) that acts as a molecular syringe to inject proteins directly into host cell cytoplasm, through a translocation pore, leading to subversion of cell physiology and bacterial internalisation. The T3S apparatus (T3SA) is composed of a cytoplasmic bulb, a basal body and an extracellular needle. The needle is assembled through the helical polymerisation of MxiH subunits that form a channel allowing the passage of T3S substrates and topped by the translocators IpaBCD (needle tip complex). IpaD is a hydrophilic protein that modulates the membrane insertion of the hydrophobic IpaB and IpaC (translocation pore) and prevents, along with IpaB, the leakage of proteins before cell contact. Secretion is also controlled at the base of the T3SA by cytoplasmic MxiC that senses cell contact through the transmission of a signal along the needle. These two control devices are involved in the establishment of a secretion hierarchy between translocators, early and late effectors.<p>In this study, we aimed to better understand the function of the T3SS by targeting the tip protein IpaD. Ten amino-acid deletion (Schiavolin, 2013) and point mutation (Meghraoui, 2014) variants of IpaD were generated to characterise their effects on translocators exposure, secretion control, pore formation and cell invasion. Three secretion phenotypes were identified and correspond to wild-type like secretion control, constitutive secretion and an intermediate secretion phenotype. Deletion variants allowed us to understand the requirement of IpaB and IpaD surface exposure for the T3SS functions. Mutation variants highlighted the uncoupling between secretion control and cell entry and the correlation between the premature secretion of translocators/early effectors and the enhanced in vitro virulence. We also studied interaction partners of IpaD (Meghraoui, in prep) and MxiC (Cherradi, 2013) at the needle tip and T3SA base, respectively. The bindings of IpaD to itself and to the needle subunit MxiH were only possible after deletion of the self-chaperoning domain. This domain was essential for the correct maintenance of IpaB at the needle tip, the secretion control, the insertion of the translocon, but not for cell entry. Besides, we showed that the interaction of MxiC with inner rod component MxiI participates in the internal control of secretion. In conclusion, these observations facilitated our understanding of the links between the different components and functions of the T3SS and the involvement of IpaD in the allosteric regulation of secretion. Our work is relevant beyond the Shigella field as genes studied here are conserved among several pathogenic bacteria using T3SS as a virulence weapon. <p><p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished