Würzburg, Univ., Diss., 2009. / Zsfassung in dt. Sprache.

Le Ca2+ est un messager intracellulaire impliqué dans de nombreux phénomènes physiologiques: sécrétion, fécondation, production d'énergie, ou encore régulation de gène. Suite à une stimulation appropriée de la cellule, on peut mesurer des signaux calciques sous forme d'oscillations. Celles-ci se propagent dans les différents compartiments cellulaires, et y activent pompes et enzymes. A l'inverse, les différents éléments activés par le Ca2+ peuvent à leur tour altérer les propriétés des oscillations. La signalisation du Ca2+ est donc un système complexe, hautement non linéaire, dont il est difficile de dénouer les interactions. C'est pourquoi nous avons adopté une approche de modélisation mathématique pour étudier la dynamique calcique. Dans cette thèse, nous aborderons trois systèmes biologiques différents. Le premier système est la production d'énergie par les cellules. Celle-ci, activée par le Ca2+, a lieu dans les mitochondries. Nous avons construit un modèle reproduisant ce comportement. Le modèle met en évidence l'impact des flux calciques impliquant les mitochondries sur la dynamique des oscillations cytosoliques et pointe le rôle de "pacemaker" joué par les mitochondries. De plus, avec des changements de paramètres et d'équations réalistes, le modèle reproduit la dynamique calcique de set-ups expérimentaux et de types cellulaires différents. Enfin, notre modèle inclut une description du pore de transition de perméabilité mitochondrial (mPTP), basée sur de la bistabilité. Nous suggérons que le mPTP se comporte comme un switch bistable, dont les deux états pourraient correspondre aux deux modes de conductance du mPTP. Le modèle suggère que le cycle d'ouverture/fermeture suit une boucle d'hystérèse dirigée par le potentiel membranaire et le Ca2+, et non pas par les protons. Le deuxième système étudié porte sur les réponses calciques locales et globales induites par la bactérie Shigella dans les cellules hôtes. Nous avons construit un modèle reproduisant ces réponses, et montrons que par rapport au type sauvage, un mutant déficient pour la protéine IpgD induit des réponses calciques plus globales sur des temps d'invasion courts, et plus oscillantes lors de longs temps d'invasion. La protéine atténue donc les réponses calciques. Nos collaborateurs ont réalisé des expériences qui montrent que cela permet à la bactérie de retarder la mort des cellules hôtes. Enfin, nous avons étudié la régulation des hémicanaux de connexines par le Ca2+. Une fois activés, ces canaux s'ouvrent et laissent passer Ca2+ et ATP selon leur gradient. Cet ATP peut à son tour activer des récepteurs purinergiques. Le modèle suggère que l'ouverture des hémicanaux peut altérer la dynamique calcique intracellulaire. De plus, l'ouverture d'un ou deux hemicannaux semble suffisante pour perturber les oscillations de manière significative. / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

Made available in DSpace on 2014-12-17T14:13:51Z (GMT). No. of bitstreams: 1 MariaLSP_DISSERT_1-12.pdf: 307341 bytes, checksum: 501d00488698b866628dca8bc24c0c6e (MD5) Previous issue date: 2010-03-31 / Trata-se de um coorte prospectivo com amostras de leite de 28 m?es da zona rural da Para?ba, durante diferentes dias de amamenta??o exclusiva, com objetivo de avaliar atrav?s do ensaio imunoenzim?tico a presen?a de imunoglobulina A secretora (sIgA) total e espec?fica contra ant?genos de Escherichia coli enteropatog?nica (EPEC) e Shigella flexneri. A reatividade dos anticorpos foi analisada pelo Western blot . Os resultados mostram presen?a da sIgA em todas as amostras, com medianas no colostro de 8,092 g/L(4,546-17,252) e leite de 0,695g/L (0,020-2,830). As medianas nos t?tulos de colostro de IgA anti-EPEC foi 41 (1-659) e anti-Shigella flexneri de 18 (1-4727) enquanto no leite anti-EPEC foi de 8 (1-288) e anti-Shigella flexneri de 6 (1-450). Houve grandes varia??es entre as m?es e entre os dias de amamenta??o. No Western blot os anticorpos sIgA reagiram com prote?nas de EPEC e Shigella flexneri, destacando-se a fra??o antig?nica de 94kDa, correspondente a intimina. Os resultados mostram que a presen?a de sIgA total e de anticorpos IgA contra EPEC e Shigella flexneri no colostro e leite de m?es residentes em zona rural, com prec?rias condi??es s?cioecon?mica e sanit?rias, n?o diferem de estudos realizados com popula??es de ?rea urbana e refor?am a import?ncia do leite materno na defesa contra infec??es ent?ricas. Apesar da aus?ncia na literatura de estudos avaliando o perfil de anticorpos sIgA no leite de m?es residentes em zona rural do Brasil, os resultados demonstraram que a presen?a de sIgA total e de anticorpos IgA contra EPEC e Shigella flexneri no colostro e leite de m?es residentes em zona rural, com prec?rias condi??es s?cio-econ?mica e sanit?rias, n?o diferem de estudos realizados com popula??es de ?rea urbana e refor?am a import?ncia do leite materno na defesa contra infec??es ent?ricas

Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-10-18T15:48:16Z No. of bitstreams: 3 Dissertação Parcial - Mayana Cristina S. Pardo.pdf: 2758393 bytes, checksum: 9b78b369931f18a430b77faca7be9c89 (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-10-18T15:48:43Z (GMT) No. of bitstreams: 3 Dissertação Parcial - Mayana Cristina S. Pardo.pdf: 2758393 bytes, checksum: 9b78b369931f18a430b77faca7be9c89 (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-10-18T15:48:43Z (GMT). No. of bitstreams: 3 Dissertação Parcial - Mayana Cristina S. Pardo.pdf: 2758393 bytes, checksum: 9b78b369931f18a430b77faca7be9c89 (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-11-18 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / Shigella sp. is responsible for one of the leading causes of child morbidity and mortality, especially in developing countries. Infection by this bacterium is known as shigellosis or bacillary dysentery, a highly contagious inflammatory diarrhea. Antibiotic therapy against shigellosis has become a challenge due to the increasing resistance to antibiotics presented by clinical isolates. Thus, immunoprophylaxis directed towards the development of vaccines has become a priority for the World Health Organization in the fight against shigellosis. Considering the need to develop new vaccination strategies for the control of shigellosis and the absence of a licensed and safe vaccine, the present work proposed to identify B cell epitopes of the OmpA and FimH proteins of Shigella flexneri with immunogenic potential, using in silico analyzes for the prediction, besides evaluating the humoral response of mice immunized with synthetic peptides that mimic these epitopes. Through the IEDB program, 11 epitopes (5 for OmpA and 6 for FimH) were predicted and the corresponding peptides were synthesized. The 3D structure of these antigens was also constructed to facilitate the prediction of epitopes. Peptides that reacted to the anti-Shigella antiserum were selected for immunization of immunocompetent mice. The anti-peptide antisera P1 (positive control), P2, P3 and P4 were produced and tested against 13 wild isolates of Shigella flexneri, and showed 92.3% (12/13) of recognition by the corresponding epitope in the native protein present in the strains Tested. The most immunodominant epitope was P2 with a statistically significant result (P <0.0422), when the level of recognition of P2 was compared with P1, significant differences were also observed (P <0.0155). The P4 epitope was the second immunodominant epitope and is located in one of the loops on the surface of the OmpA, whereas the P2 epitope which was the most immunodominant is located in the globular domain in the periplasmic space. However, the antibodies produced against the P2 and P4 peptides were able to recognize wild isolates of S. flexneri, this result makes it possible to infer that these peptides have potential for a vaccine candidate. / Shigella sp. é responsável por uma das principais causas de morbidade e mortalidade infantil, especialmente em países em desenvolvimento. A infecção por esta bactéria é conhecida como shigelose ou disenteria bacilar, uma diarreia inflamatória altamente contagiosa. A antibioticoterapia contra shigelose tem se tornado um desafio devido à crescente resistência aos antibióticos apresentada pelos isolados clínicos. Dessa maneira, a imunoprofilaxia direcionada para o desenvolvimento de vacinas tem se tornado prioridade pela Organização Mundial de Saúde no combate a shigelose. Considerando a necessidade de desenvolver novas estratégias de vacinação para o controle da shigelose, uma vez que ainda não existe uma vacina licenciada segura e eficaz, o presente trabalho objetivou identificar epitopos de célula B das proteínas OmpA e FimH de Shigella flexneri com potencial imunogênico, utilizando de análises in silico para a predição, além de avaliar a resposta humoral de camundongos imunizados com peptídeos sintéticos que imitam estes epitopos. Através do programa IEDB foram preditas 11 epitopos (5 para OmpA e 6 para FimH) e os peptídeos correspondentes foram sintetizados. A estrutura 3D desses antígenos também foi construída para facilitar a predição dos epitopos. Os peptídeos que reagiram ao antissoro anti-Shigella, foram selecionados para imunização de camundongos imunocompetentes. Os antissoros antipeptídeos P1 (controle positivo), P2, P3 e P4 foram produzidos e testados contra 13 isolados selvagens de Shigella flexneri, e apresentaram 92,3% (12/13) de reconhecimento pelo epitopo correspondente na proteína nativa presente nas cepas testadas. O epitopo mais imunodominante foi o P2 com resultado estatisticamente significativo (P<0,0422), quando o nível de reconhecimento de P2 foi comparado com P1, diferenças significativas também foram observadas (P<0,0155). O epitopo P4 foi o segundo epitopo imunodominante e está localizado em uma das alças na superfície da OmpA, enquanto o epitopo P2 que foi o mais imunodominante está localizado no domínio globular no espaço periplasmático. Entretanto, os anticorpos produzidos contra os peptídeos P2 e P4 foram capazes de reconhecer os isolados selvagens de S. flexneri, este resultado possibilita inferir que esses peptídeos possuem potencial para um candidato a vacina.

The Shigella species cause millions of cases of watery or bloody diarrhea each year in developing countries, with children under the age of five years most vulnerable to infection. Emerging strains of multidrug resistant Shigella emphasize the need for a comprehensive and cost-efficient vaccine; however, an effective vaccine has yet to be produced despite years of research. Several studies have demonstrated that Shigella utilizes host physiology, specifically bile salts as signals for invasion and virulence gene expression. This study aimed to build upon previous research analyzing the bile salts transcriptional profile of Shigella flexneri 2457T, in which an induction of the uncharacterized gene was demonstrated during bile salts exposure. Here, a mutant and wild-type 2457T strains were used in infection of HT-29 colonic epithelial cells to compare invasion ability and intracellular replication. The Congo red (CR) secretion assay was also used as a measure of virulence protein secretion from the type-III secretion system (T3SS), while interleukin-8 (IL-8) secretion from infected HT-29 cells was measured as a marker for the epithelial cell response to infection. Infection analyses included subculturing the strains in media with 0.4% bile salts to mimic small intestine physiology and gastrointestinal transit of S. flexneri prior to infection. The mutant strain displayed both increased invasion of and intracellular replication in HT-29 cells compared to 2457T. The presence of bile salts enhanced both invasion and intracellular replication in both strains when compared to wild-type without bile salts exposure during subculture. The CR assays revealed increased protein secretion from the mutant compared to 2457T, and that bile salts increased T3 secretion in both strains. Increased IL-8 secretion from infected HT-29 cells was detected when both strains were subcultured in bile salts; however, a decrease in IL-8 secretion was observed following infection with the mutant subcultured without bile salts. Overall, the data suggest that this bile salt-induced gene encodes a negative regulator of virulence, and that the gene product likely prevents a hypervirulence phenotype that would compromise the ability of S. flexneri to control infection and regulate the host immune response. This work has provided insights into the function of this uncharacterized gene, which could serve as a novel target for future therapeutic development. / 2027-06-30T00:00:00Z

Several bacterial pathogens including Shigella (shigellosis), Escherichia coli (urinary tract infections), Pseudomonas (lung infections), Salmonella (food poisoning), and Yersinia (plague) critically rely on a complex type three secretion system (T3SS) for infection. With the rise in multi-antibiotic resistant strains of several of these pathogens, we turn to the T3SS as a promising target for the development of novel therapeutics. The Dickenson lab at Utah State University has been the first to identify and characterize the ATPase Spa47, the energy source of the Shigella infection system. We show that Spa47 is necessary for proper T3SS formation and function, being ultimately responsible for overall Shigella virulence. We find that proper ATPase function and in turn T3SS apparatus formation can be affected by something as simple as a single mutation to the removal of a non-catalytic domain. The insights gained from this work expands our understanding of the powerhouse that fuels these infection systems and brings us a step closer to developing novel therapeutics to combat infection.

The Shigella species are Gram-negative enteropathogens that produce severe diarrhea, cramping, and dehydration in millions of people annually. The pathogens most commonly infect children under the age of 5 years in developing nations, where the rise of multidrug-resistant species is increasingly problematic. Despite several attempts to develop a vaccine against these pathogens, no successful vaccine has been produced. In order to achieve this goal, several characteristics of Shigella must be further elucidated. Namely, we must better understand the mechanisms Shigella employs in order to circumvent the immune response. A key way in which Shigella circumvents the innate defenses of the host is through resistance to bile salts, the principal component of bile, a substance found in the small intestine that is required for digestion. One such bile salt resistance mechanism of Shigella involves lipopolysaccharide (LPS), an extracellular structure composed of three regions: a transmembrane lipid, a polysaccharide core, and an O-antigen. LPS and LPS modifications have been implicated in bile salts resistance in other enteropathogens. Thus, the goal of this study was to build from preliminary findings to understand the role of LPS in conferring bile salts resistance in Shigella. Two Shigella flexneri mutants were studied to understand the roles of the polysaccharide core and O-antigen on bacterial growth and LPS modifications during exposure to bile salts. Growth comparisons of the mutants relative to wild type bacteria in the presence of bile salts were performed, including analysis of growth with exposure to bile salts and with varying levels of environmental glucose. Additionally, LPS was extracted from wild type and mutant bacteria grown in these conditions for analysis by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The growth curves demonstrated that both the O-antigen and polysaccharide core mutants exhibited slow growth with exposure to bile salts, while the SDS-PAGE analyses revealed changes in the LPS profile of wild type and both LPS mutants when grown in bile salts. These data indicate that the O-antigen likely has an important role in conferring bile salts resistance and that the polysaccharide core may also facilitate resistance. This study allows us to better understand how LPS contributes to bile salts resistance in S. flexneri, which may enhance efforts to develop an effective vaccine against this pathogen. / 2023-12-10T00:00:00Z

The human-bacterial pathogen interaction is a complex process that results from a prolonged evolutionary arms race in the struggle for survival. The pathogen employs virulence strategies to achieve host colonization, and the latter counteracts using defense programs. The encounter of both organisms results in drastic physiological changes leading to stress, which is an ancient response accompanying infection. Recent evidence suggests that the stress response in the host converges with the innate immune pathways and influences the outcome of infection. However, the contribution of stress and the exact mechanism(s) of its involvement in host defense remain to be elucidated. Using the model bacterial pathogen Shigella flexneri, and comparing it with the closely related pathogen Salmonella Typhimurium, this study investigated the role of host stress in the outcome of infection. Shigella infection is characterized by a pronounced pro-inflammatory response that causes intense stress in host tissues, particularly the intestinal epithelium, which constitutes the first barrier against Shigella colonization. In this study, inflammatory stress was simulated in epithelial cells by inducing oxidative stress, hypoxia, and cytokine stimulation. Shigella infection of epithelial cells exposed to such stresses was strongly inhibited at the adhesion/binding stage. This resulted from the depletion of sphingolipidrafts in the plasma membrane by the stress-activated sphingomyelinases. Interestingly, Salmonella adhesion was not affected, by virtue of its flagellar motility, which allowed the gathering of bacteria at remaining membrane rafts. Moreover, the intracellular replication of Shigella lead to a similar sphingolipid-raft depletion in the membrane across adjacent cells inhibiting extracellular bacterial invasion. Additionally, this study shows that Shigella infection interferes with the host stress granule-formation in response to stress. Interestingly, infected cells exhibited a nuclear depletion of the global RNA-binding stress-granule associated proteins TIAR and TIA-1 and their accumulation in the cytoplasm. Overall, this work investigated different aspects of the host stress-response in the defense against bacterial infection. The findings shed light on the importance of the host stress-pathways during infection, and improve the understanding of different strategies in host-pathogen interaction. / Die Interaktion von Mensch und bakteriellem Krankheitserreger ist ein komplexer Prozess, der aus dem anhaltenden evolutionären Wettrüsten im Kampf ums Überleben resultiert. Der Erreger setzt Virulenzstrategien zur Kolonisierung des Wirtes ein und dieser nutzt Verteidigungsprogramme um dem entgegenzuwirken. Die Begegnung der beiden Organismen resultiert in drastischen physiologischen Veränderungen, welche zu Stress führen, der eine klassische infektionsbegleitende Reaktion ist. Neuere Untersuchungen deuten darauf hin, dass die Stressantwort des Wirtes mit den Signalwegen der angeborenen Immunantwort konvergiert und im Ergebnis die Infektion beeinflusst. Jedoch bleiben die Bedeutung des Stresses und der exakte Mechanismus wie Stress an der Verteidigung des Wirtes beteiligt ist, noch zu klären. In dieser Studie dienten der bakterielle Krankheitserreger Shigella flexneri und vergleichend dazu der nah verwandte Erreger Salmonella Typhimurium als Modellorganismen, um die Rolle von Wirtszellstress für den Ausgang der Infektion zu untersuchen. Die Infektion mit Shigellen ist durch eine ausgeprägte pro-inflammatorische Reaktion gekennzeichnet. Diese versursacht in den Wirtsgeweben, insbesondere im Darmepithel, einen starken Stress, der die erste Barriere gegen die Besiedelung mit Shigellen darstellt. In der vorliegenden Arbeit wurde entzündlicher Stress in Epithelzellen durch die Induktion von oxidativem Stress, Hypoxie und Zytokinstimulation simuliert. Die Shigelleninfektion von Epithelzellen, die solchen Belastungen ausgesetzt waren, war stark im Adhäsions-/ Bindungsstadium gehemmt. Dies resultierte aus der Verarmung von Sphingolipidflößen in der Plasmamembran durch stressaktivierte Sphingomyelinasen. Interessanterweise wurde die Adhäsion von Salmonellen, aufgrund ihrer Flaggellenvermittelten Beweglichkeit, nicht beeinträchtigt und ermöglichte so die Ansammlung von Bakterien an den verbleibenden Membranflößen. Darüber hinaus führte die intrazelluläre Replikation von Shigellen zu einer ähnlichen Verminderung von Sphingolipidflößen in der Membran benachbarter Zellen, wodurch die extrazelluläre bakterielle Invasion gehemmt wurde. Zusätzlich zeigt diese Studie, dass eine Infektion mit Shigellen mit der Bildung von Stressgranula in der Wirtszelle interferiert. Interessanterweise zeigten infizierte Zellen eine nukleäre Depletion der globalen RNA-bindenden und Stressgranula assoziierten Proteine TIAR und TIA-1 sowie deren Akkumulation im Zytoplasma. Insgesamt untersuchte diese Arbeit verschiedene Aspekte der Stressreaktion der Wirtszelle bei der Verteidigung gegen bakterielle Infektionen. Die Ergebnisse beleuchten die Bedeutung der Stresssignalwege im Wirt während der Infektion und verbessern das Verständnis der verschiedenen Strategien in der Interaktion von Wirt und Krankheitserreger.

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

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