Orientador: Marcelo Brocchi / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T03:47:25Z (GMT). No. of bitstreams: 1 Gregoracci_GustavoBueno_D.pdf: 6197096 bytes, checksum: 05eb5e18d8084e5c5f742c6ec5afd63d (MD5) Previous issue date: 2010 / Resumo: Bacteriófagos são vírus que infectam bactérias e arqueias, representando as entidades biológicas mais abundantes do mundo e influenciando de maneira marcante populações naturais de seus hospedeiros. A terapia com bacteriófagos, que representa uma das primeiras formas modernas de combate a infecções bacterianas, foi recentemente redescoberta e vem sendo reavaliada seguindo metodologias atuais quanto à sua viabilidade terapêutica. Para completar a caracterização dos fagos de nossa coleção, sequenciamos completamente o genoma da maior parte destes, através da metodologia multiplex pair-ended utilizando a plataforma Illumina. Visando contribuir para verificação da viabilidade terapêutica de bacteriófagos testamos os efeitos protetor e terapêutico dos fagos Shfl1, Saen1v2 e Saen1v4, pertencentes à coleção de nosso laboratório, em modelos biológicos relevantes. O fago Shfl1, lítico contra Shigella flexneri, foi testado em ensaio de invasão em células HeLa. A redução de bactérias intracelulares foi mensurada independentemente através de plaqueamento e citometria de fluxo, além da observação direta por microscopia de fluorescência. O fago Saen1v2, lítico contra Salmonella Typhimurium, foi estudado quanto à biodistribuição e meia-vida em modelo murino, e uma variante viral com maior persistência in vivo foi selecionada. Essa variante, denominada Saen1v2p5, e o fago Saen1v4, lítico contra Salmonella Typhi, foram testados em modelo murino de infecção tifoide, contra seus respectivos hospedeiros. Encontramos similaridade genômica a fagos conhecidos, como T4, T7, T1 entre outros, em maior ou menor grau. Obtivemos um efeito protetor e terapêutico contra Shigella flexneri utilizando o fago Shfl1 em ensaio de invasão em cultura de células HeLa, verificado por todas as metodologias empregadas. Não verificamos efeito antimicrobiano in vivo do fago Saen1v2p5 em modelo murino de infecção por Salmonella Typhimurium. Por outro lado, observamos efeito terapêutico e protetor dose dependente utilizando o fago Saen1v4 em modelo murino de infecção por Salmonella Typhi. O sucesso obtido com baixas multiplicidades de infecção sugere um possível efeito indireto ou estimulação imune inespecífica / Abstract: Bacteriophages are viruses that infect Bacteria and Achaea, representing the most abundant biological entities in the world and markedly influencing natural host populations. Phage therapy, which represents one of the first modern ways to fight bacterial infections, was recently rediscovered and is being re-evaluated according to current methodologies regarding its therapeutic viability. In order to complete phage characterization in our collection, we sequenced completely the genomes of most of these, through the multiplex pair-ended methodology using the Illumina platform. Aiming to contribute to the therapeutic viability verification of bacteriophages we tested phage protective and therapeutic effects of Shfl1, Saen1v2 and Saen1v4, which belong to our collection, in biologically relevant models. Phage Shfl1, lytic against Shigella flexneri, was tested in a HeLa invasion assay. Intracellular bacteria reduction was measured independently through plating and flow cytometry, besides direct observation through fluorescent microscopy. Phage Saen1v2, lytic against Salmonella Typhimurium, was studied about its bio-distribution and half-life in murine model, and a viral variant with longer in vivo persistence was selected. This variant, denominated Saen1v2p5, and phage Saen1v4, lytic against Salmonella Typhi, were tested in murine typhoid model, against their respective hosts. Genomic similarity to known phages such as T4, T7, T1 among others, was found, in various degrees. We obtained both protective and therapeutic effect against Shigella flexneri using phage Shfl1 in the HeLa invasion assay, through all methodologies utilized. We could not verify in vivo antimicrobial effect of phage Saen1v2p5 in the murine model of Salmonella Typhimurium infection. On the other hand, we observed both therapeutic and protective dose dependent effect using phage Saen1v4 in Salmonella Typhi murine infection model. The success obtained with low multiplicities of infection may suggest a possible indirect effect or unspecific immune stimulation / Doutorado / Microbiologia / Doutor em Genetica e Biologia Molecular

Neste trabalho, esclarecemos tópicos da patogenicidade de EIEC que sustentam a sua menor virulência quando comparada à S. flexneri, e mostramos a importância das células dendríticas (CD) nesse processo. Estudou-se o comportamento de EIEC e S. flexneri quando em contato com células Caco-2, avaliando-se uma cinética de expressão dos genes envolvidos na invasão e disseminação bacteriana. Em geral, todos os genes foram menos expressos em EIEC, fato corroborado pelo fenótipo de disseminação bacteriana, onde EIEC foi menos eficiente do que Shigella. Também foi avaliada a modulação da resposta inflamatória de células dendríticas intestinais murinas pela produção de citocinas, expressão de moléculas co-estimulatórias e apresentação de antígenos, após desafio das células com as bactérias. Os resultados sugerem que EIEC induz a uma resposta protetora ao hospedeiro, enquanto que Shigella estaria \"driblando\" o sistema imune, além de provavelmente super-estimular o sistema imune adaptativo, fato que poderia levar a um agravamento da doença. As ações integradas das células Caco-2, células dendríticas e estímulos bacterianos foram estudadas em co-cultura celular. Observou-se que EIEC e suas proteínas secretadas induzem a migração das CDs ao compartimento apical da co-cultura; nada foi observado quando o desafio se deu com Shigella. Também foram avaliadas as concentrações de citocinas inflamatórias no microambiente infeccioso formado. A citocina TNF-α, bem como CCL20 e MCP-1 foram mais proeminentes após estímulo com EIEC, fato que poderia explicar parcialmente a migração das CDs ao lado apical da co-cultura após estímulo com EIEC e suas proteínas secretadas. Nossas evidências experimentais indicam que a doença desencadeada por EIEC é mais restrita a um determinado local da infecção, ou seja, não é capaz de se disseminar a ponto de estender a lesão tecidual de forma mais drástica, como Shigella. Esse fenômeno pode estar associado à menor expressão de seus dos fatores de virulência e à resposta imune inata induzida no sítio de infecção, o que levaria, fatalmente, à resolução da doença. / In this study, we clarify topics of pathogenicity from EIEC that support its lower level of virulence when it is compared to S. flexneri, and we have shown the importance of dendritic cells (DC) in this process. We studied the conduct of EIEC and S. flexneri when they were in contact with Caco-2 cells and we analyzed the kinetics of the genes expression that was involved in the spread and invasion of the bacteria. In general, all genes were expressed less in EIEC, as demonstrated by the phenotype of the bacterial spread, where EIEC was less efficient than Shigella. We also analyzed the modulation of the inflammatory response by the murine intestinal dendritic cells by the production of cytokine, expression of co-stimulators molecules and antigens presentation, after the interaction of the cells with the bacteria. The results showed that EIEC induces a response that protects the host while Shigella manipulate the host intestinal innate and adaptive immune system and it probably over-stimulates the adaptive immune system which could let the disease worse. The integrated actions of Caco-2 cells, dendritic cells and bacterial stimulus, were studied in a co-culture cell. We observed that EIEC and its secreted proteins induce the migration of the DCs to the apical compartment of the co-culture; nothing was observed related to Shigella. We also evaluated the concentrations of the inflammatory cytokines at the infective micro environment that was formed. The cytokine TNF-α, as CCL20 and MCP-1 were more prominent after been stimulated with EIEC, a fact that could partially explain the migration of DCs to the apical side of the co-culture after the stimulus with EIEC and its secreted proteins. Our experimental evidence shows that the disease triggered by the EIEC is more restricted at a definite infection place, which means that it is not capable of disseminating beyond a certain point to extend the tissue\'s injury and let it worsen, as Shigella do. This phenomenon can be associated with the lower level of expression of its virulence factors and to the immune response induced in the infection site, what could finally lead to the eradication of the disease.

Das Typ III Sekretionssystem (T3SS) ist ein Proteinkomplex den Gramnegative Bakterien nutzen um in einem Schritt Effektorproteine (Effektoren) aus dem Zytosol über die Doppelmembran zu sekretieren. Für viele Bakterien ist das T3SS ein essenzieller Virulenzfaktor, der es ihnen erlaubt mit ihrem Wirt zu interagieren und diesen zu manipulieren. Charakteristisch für das T3SS ist die strukturelle Komponente, der Nadelkomplex. Dieser ähnelt strukturell einer Spritze, deren Basalkörper die bakteriellen Membranen und das Periplasma durchspannt und einer Nadel, die vom Basalkörper aus dem Bakterium ragt. Basierend auf dem Modell einer Spritze wird angenommen, dass Effektoren entfaltet und anschließend durch Basalkörper und Nadelkanal sekretiert werden. Trotz der kontinuierlichen Forschung an T3SS entbehrt dieses Modell einer experimentellen Grundlage und der Mechanismus ist nicht vollständig erklärt. Ziel der Arbeit war es, eine experimentelle Basis für den Sekretionsmechanismus des T3SS zu schaffen. Um zu verstehen, wie das T3SS Effektoren sekretiert, wurden zunächst Fusionsproteine konstruiert, welche aus einem Effektor und einem stabil gefalteten Knotenprotein bestehen. Aufgrund des Knotens in der Fusion ist davon auszugehen, dass dieser während der Sekretion nicht entfalten kann. Die Effektordomäne wird sekretiert während der Knoten im Kanal verbleibt und diesen verstopft. Nach unseremWissen ist diese Arbeit die erste Visualisierung von Effektorfusionen an isolierten Nadelkomplexen. Die Effektorfusion wird N-terminal voran durch den Kanal sekretiert, wobei der Kanal das Substrat umschließt und gegen Proteasen und chemische Modifikationen abschirmt. Die Ergebnisse dieser Arbeit untermauern eine Grundidee der Funktionsweise des T3SS und liefern eine vielversprechende Strategie für in situ-Strukturanalysen. Dieser Ansatz lässt sich auch auf andere Proteinsekretionssysteme übertragen, bei welchen Substrate vor dem Transport entfaltet werden müssen. / The Type III Secretion System (T3SS) is a complex used by Gram-negative bacteria to secrete effector proteins from the cytoplasm across the bacterial envelope in a single step. For many pathogens, the T3SS is an essential virulence factor that enables the bacteria to interact with and manipulate their respective host. A characteristic structural feature of the T3SS is the needle complex (NC). The NC resembles a syringe with a basal body spanning both bacterial membranes and a long needle-like structure that protrudes from the bacterium. Based on the paradigm of a syringe-like mechanism, it is generally assumed that effectors are unfolded and secreted from the bacterial cytoplasm through the basal body and needle channel. Despite extensive research on T3SS, this hypothesis lacks experimental evidence and the mechanism of secretion is not fully understood. This work aimed to provide an experimental basis for the model of the T3SS mechanism. In order to elucidate details of the effector secretion mechanism, fusion proteins consisting of an effector and a bulky protein containing a knotted motif were generated. It is assumed that the knot cannot be unfolded during secretion of the chimera. Consequently, these fusions are accepted as T3SS substrates but remain inside the NC channel and obstruct the T3SS. This is, to our best knowledge, the first time effector fusions have been visualized together with isolated NCs and it demonstrates that effector proteins are secreted directly through the channel with their N-terminus first. The channel encloses the substrate and shields it from a protease and chemical modifications. These results corroborate an elementary understanding of how the T3SS works and provide a powerful tool for in situ-structural investigations. This approach might also be applicable to other protein secretion systems that require unfolding of their substrates prior to secretion.

O presente estudo avaliou a capacidade de invasão de hepatócitos de rato por Shigella flexneri (S. flexneri) nas condições de normóxia e hipóxia. O estudo do microambiente de hipóxia tem grande importância, por estar presente em muitas doenças hepáticas, além de aumentar a translocação quando presente no lúmen intestinal. Bactérias invasivas como S. flexneri podem romper a barreira intestinal e chegar ao fígado através da circulação portal. O efeito da invasão bacteriana das células hepáticas é pouco conhecido. Neste trabalho buscamos pesquisar as alterações morfológicas e funcionais de hepatócitos de rato após infecção por S. flexneri na presença e na ausência de hipóxia. Para esta finalidade foram utilizados hepatócitos de rato cultivados pela técnica de cultura primária. Vários parâmetros foram analisados, tais como: taxa de invasão celular pela bactéria, quantificação da produção e liberação de DHL, produção de TNF-, taxa de morte celular por apoptose e a expressão do fator de transcrição HIF-1a. Os resultados mostraram que a metodologia empregada para a obtenção do microambiente hipóxico foi satisfatória, com redução de 70% da pO2 inicial (atingindo 43.2 mmHg in vitro ou 6.5% O2). A invasão de hepatócitos de rato por S. flexneri foi menor nas células previamente expostas à hipóxia quando comparada com a invasão das células cultivadas em normóxia. A viabilidade dos hepatócitos não apresentou diferenças significativas entre os grupos experimentais, variando entre 74 e 86%. A liberação de TNF- nas situações de normóxia e hipóxia foi similar, embora as células infectadas em normóxia tenham aumentado a liberação desta citocina. Na condição de hipóxia + infecção a liberação de TNF- foi menor do que na condição de normóxia + infecção, porém ambos os grupos produziram aumento significativo da citocina em relação aos controles normóxicos e hipóxicos. Este resultado sugere que a presença da bactéria no interior das células aumenta significativamente a liberação de TNF-pelos hepatócitos. A produção de DHL também foi maior de forma significativa no grupo hipóxico em relação ao grupo normóxico, porém não apresentou alteração nos grupos infectados por S. flexneri após uma hora. As taxas de apoptose aumentaram nos grupos hipóxia e nos grupos infectados com S. flexneri de maneira similar, variando entre 24 e 31%, quando comparados aos grupos controle em normóxia. A expressão do fator de transcrição HIF ocorreu nos grupos: hipóxia, normóxia + infecção e hipóxia + infecção, evidenciando que a infecção por S. flexneri induz a expressão deste fator. Em seu conjunto, nossos resultados buscam contribuir para o maior conhecimento da interação entre S. flexneri e hepatócitos em condição de hipóxia e normóxia. Tal conhecimento poderá ser útil na construção de futuras estratégias para auxiliar no combate a esta importante bactéria invasiva, principalmente nos casos de septicemia / This study evaluated the invasiveness of rat hepatocytes by Shigella flexneri (S. flexneri) in normoxia and hypoxia conditions. The study of hypoxia microenvironment is of great importance, since hypoxia is present in many liver diseases and increases bacterial translocation when present in intestinal lumen. Invasive bacteria such as S. flexneri can disrupt the intestinal barrier and reach the liver through portal circulation. The effect of bacterial invasion in liver cells is poorly understood. In this study we investigated the morphological and functional changes of rat hepatocytes after infection with S. flexneri in the presence and absence of hypoxia. For this purpose we used primary cultures of rat hepatocytes. Several parameters were analyzed, such as: bacterial invasion cell rate, quantification of LDH production and release, TNF-a production, cell death rate by apoptosis and expression of the transcription factor HIF-1a. The results showed that the methodology used to obtain the hypoxic microenvironment was satisfactory, with 70% reduction of initial pO2 (to 43.2 mmHg in vitro or 6.5% O2). The invasion of rat hepatocytes by S. flexneri was lower in cells previously exposed to hypoxia compared with the invasion of cells grown in normoxia. The viability of hepatocytes showed no significant differences between experimental groups, ranging between 74% and 86%. The release of TNF-a in situations of normoxia and hypoxia was similar, although the infected cells in normoxia have increased the release levels of this cytokine. In hypoxia + infection condition the release of TNF-a was lower than normoxia + infection condition, but both groups produced a significant increase in cytokine release when compared to normoxic and hypoxic controls. This result suggests that the presence of bacteria inside the cells significantly increases the release of TNF-a by hepatocytes. DHL production was also significantly greater in the hypoxic group compared to the normoxic group, but had no change in the groups infected with S. flexneri after an hour. The apoptosis rates increased in hypoxia and infected groups in a similar way, varying between 24% and 31% when compared with control group in normoxia. The expression of HIF- 1a transcription factor occurred in hypoxia, normoxia + infection and hypoxia + infection groups, indicating that infection with S. flexneri induces the expression of this factor. Overall, our results sought to contribute to a greater understanding of the interaction between S. flexneri and hepatocytes under hypoxia and normoxia conditions. Such knowledge may be useful in building future strategies to assist in combating these major invasive bacteria

O presente estudo avaliou a capacidade de invasão de hepatócitos de rato por Shigella flexneri (S. flexneri) nas condições de normóxia e hipóxia. O estudo do microambiente de hipóxia tem grande importância, por estar presente em muitas doenças hepáticas, além de aumentar a translocação quando presente no lúmen intestinal. Bactérias invasivas como S. flexneri podem romper a barreira intestinal e chegar ao fígado através da circulação portal. O efeito da invasão bacteriana das células hepáticas é pouco conhecido. Neste trabalho buscamos pesquisar as alterações morfológicas e funcionais de hepatócitos de rato após infecção por S. flexneri na presença e na ausência de hipóxia. Para esta finalidade foram utilizados hepatócitos de rato cultivados pela técnica de cultura primária. Vários parâmetros foram analisados, tais como: taxa de invasão celular pela bactéria, quantificação da produção e liberação de DHL, produção de TNF-, taxa de morte celular por apoptose e a expressão do fator de transcrição HIF-1a. Os resultados mostraram que a metodologia empregada para a obtenção do microambiente hipóxico foi satisfatória, com redução de 70% da pO2 inicial (atingindo 43.2 mmHg in vitro ou 6.5% O2). A invasão de hepatócitos de rato por S. flexneri foi menor nas células previamente expostas à hipóxia quando comparada com a invasão das células cultivadas em normóxia. A viabilidade dos hepatócitos não apresentou diferenças significativas entre os grupos experimentais, variando entre 74 e 86%. A liberação de TNF- nas situações de normóxia e hipóxia foi similar, embora as células infectadas em normóxia tenham aumentado a liberação desta citocina. Na condição de hipóxia + infecção a liberação de TNF- foi menor do que na condição de normóxia + infecção, porém ambos os grupos produziram aumento significativo da citocina em relação aos controles normóxicos e hipóxicos. Este resultado sugere que a presença da bactéria no interior das células aumenta significativamente a liberação de TNF-pelos hepatócitos. A produção de DHL também foi maior de forma significativa no grupo hipóxico em relação ao grupo normóxico, porém não apresentou alteração nos grupos infectados por S. flexneri após uma hora. As taxas de apoptose aumentaram nos grupos hipóxia e nos grupos infectados com S. flexneri de maneira similar, variando entre 24 e 31%, quando comparados aos grupos controle em normóxia. A expressão do fator de transcrição HIF ocorreu nos grupos: hipóxia, normóxia + infecção e hipóxia + infecção, evidenciando que a infecção por S. flexneri induz a expressão deste fator. Em seu conjunto, nossos resultados buscam contribuir para o maior conhecimento da interação entre S. flexneri e hepatócitos em condição de hipóxia e normóxia. Tal conhecimento poderá ser útil na construção de futuras estratégias para auxiliar no combate a esta importante bactéria invasiva, principalmente nos casos de septicemia / This study evaluated the invasiveness of rat hepatocytes by Shigella flexneri (S. flexneri) in normoxia and hypoxia conditions. The study of hypoxia microenvironment is of great importance, since hypoxia is present in many liver diseases and increases bacterial translocation when present in intestinal lumen. Invasive bacteria such as S. flexneri can disrupt the intestinal barrier and reach the liver through portal circulation. The effect of bacterial invasion in liver cells is poorly understood. In this study we investigated the morphological and functional changes of rat hepatocytes after infection with S. flexneri in the presence and absence of hypoxia. For this purpose we used primary cultures of rat hepatocytes. Several parameters were analyzed, such as: bacterial invasion cell rate, quantification of LDH production and release, TNF-a production, cell death rate by apoptosis and expression of the transcription factor HIF-1a. The results showed that the methodology used to obtain the hypoxic microenvironment was satisfactory, with 70% reduction of initial pO2 (to 43.2 mmHg in vitro or 6.5% O2). The invasion of rat hepatocytes by S. flexneri was lower in cells previously exposed to hypoxia compared with the invasion of cells grown in normoxia. The viability of hepatocytes showed no significant differences between experimental groups, ranging between 74% and 86%. The release of TNF-a in situations of normoxia and hypoxia was similar, although the infected cells in normoxia have increased the release levels of this cytokine. In hypoxia + infection condition the release of TNF-a was lower than normoxia + infection condition, but both groups produced a significant increase in cytokine release when compared to normoxic and hypoxic controls. This result suggests that the presence of bacteria inside the cells significantly increases the release of TNF-a by hepatocytes. DHL production was also significantly greater in the hypoxic group compared to the normoxic group, but had no change in the groups infected with S. flexneri after an hour. The apoptosis rates increased in hypoxia and infected groups in a similar way, varying between 24% and 31% when compared with control group in normoxia. The expression of HIF- 1a transcription factor occurred in hypoxia, normoxia + infection and hypoxia + infection groups, indicating that infection with S. flexneri induces the expression of this factor. Overall, our results sought to contribute to a greater understanding of the interaction between S. flexneri and hepatocytes under hypoxia and normoxia conditions. Such knowledge may be useful in building future strategies to assist in combating these major invasive bacteria

Neste trabalho, esclarecemos tópicos da patogenicidade de EIEC que sustentam a sua menor virulência quando comparada à S. flexneri, e mostramos a importância das células dendríticas (CD) nesse processo. Estudou-se o comportamento de EIEC e S. flexneri quando em contato com células Caco-2, avaliando-se uma cinética de expressão dos genes envolvidos na invasão e disseminação bacteriana. Em geral, todos os genes foram menos expressos em EIEC, fato corroborado pelo fenótipo de disseminação bacteriana, onde EIEC foi menos eficiente do que Shigella. Também foi avaliada a modulação da resposta inflamatória de células dendríticas intestinais murinas pela produção de citocinas, expressão de moléculas co-estimulatórias e apresentação de antígenos, após desafio das células com as bactérias. Os resultados sugerem que EIEC induz a uma resposta protetora ao hospedeiro, enquanto que Shigella estaria \"driblando\" o sistema imune, além de provavelmente super-estimular o sistema imune adaptativo, fato que poderia levar a um agravamento da doença. As ações integradas das células Caco-2, células dendríticas e estímulos bacterianos foram estudadas em co-cultura celular. Observou-se que EIEC e suas proteínas secretadas induzem a migração das CDs ao compartimento apical da co-cultura; nada foi observado quando o desafio se deu com Shigella. Também foram avaliadas as concentrações de citocinas inflamatórias no microambiente infeccioso formado. A citocina TNF-α, bem como CCL20 e MCP-1 foram mais proeminentes após estímulo com EIEC, fato que poderia explicar parcialmente a migração das CDs ao lado apical da co-cultura após estímulo com EIEC e suas proteínas secretadas. Nossas evidências experimentais indicam que a doença desencadeada por EIEC é mais restrita a um determinado local da infecção, ou seja, não é capaz de se disseminar a ponto de estender a lesão tecidual de forma mais drástica, como Shigella. Esse fenômeno pode estar associado à menor expressão de seus dos fatores de virulência e à resposta imune inata induzida no sítio de infecção, o que levaria, fatalmente, à resolução da doença. / In this study, we clarify topics of pathogenicity from EIEC that support its lower level of virulence when it is compared to S. flexneri, and we have shown the importance of dendritic cells (DC) in this process. We studied the conduct of EIEC and S. flexneri when they were in contact with Caco-2 cells and we analyzed the kinetics of the genes expression that was involved in the spread and invasion of the bacteria. In general, all genes were expressed less in EIEC, as demonstrated by the phenotype of the bacterial spread, where EIEC was less efficient than Shigella. We also analyzed the modulation of the inflammatory response by the murine intestinal dendritic cells by the production of cytokine, expression of co-stimulators molecules and antigens presentation, after the interaction of the cells with the bacteria. The results showed that EIEC induces a response that protects the host while Shigella manipulate the host intestinal innate and adaptive immune system and it probably over-stimulates the adaptive immune system which could let the disease worse. The integrated actions of Caco-2 cells, dendritic cells and bacterial stimulus, were studied in a co-culture cell. We observed that EIEC and its secreted proteins induce the migration of the DCs to the apical compartment of the co-culture; nothing was observed related to Shigella. We also evaluated the concentrations of the inflammatory cytokines at the infective micro environment that was formed. The cytokine TNF-α, as CCL20 and MCP-1 were more prominent after been stimulated with EIEC, a fact that could partially explain the migration of DCs to the apical side of the co-culture after the stimulus with EIEC and its secreted proteins. Our experimental evidence shows that the disease triggered by the EIEC is more restricted at a definite infection place, which means that it is not capable of disseminating beyond a certain point to extend the tissue\'s injury and let it worsen, as Shigella do. This phenomenon can be associated with the lower level of expression of its virulence factors and to the immune response induced in the infection site, what could finally lead to the eradication of the disease.

Les bactéries du genre Shigella sont responsables de la shigellose, une maladie diarrhéique invasive du colon. L’entrée et la dissémination de Shigella à travers l’épithélium colique sont médiées par un système de sécrétion de type III (SST3) codé par un plasmide de virulence. Au sein de ce plasmide se trouve une région de 30-kb comportant les gènes impliqués dans l’entrée de la bactérie dans les cellules hôtes. Ces gènes sont regroupés en deux loci :le locus ipa-ipg qui code pour les protéines sécrétées et leurs chaperons ainsi que le locus mxi-spa codant pour les composants de l’appareil de sécrétion de type III (AST3), constitué d’un bulbe cytoplasmique, d’un corps basal transmembranaire et d’une aiguille se projetant au niveau extracellulaire. Ce système permet la sécrétion ordonnée et hiérarchique de différentes classes de protéines et la translocation de certaines d’entre elles (appelées effecteurs) dans le cytoplasme de la cellule hôte où elles interfèrent avec les voies de signalisation cellulaires. Avant le contact avec la cellule hôte, l’AST3 est inactif et verrouillé par les protéines IpaB et IpaD formant le complexe d’extrémité.<p>Chez Shigella, le gatekeeper MxiC séquestre les effecteurs au niveau du cytoplasme bactérien avant la transmission par l’aiguille du signal d’activation de la sécrétion mais les composants intermédiaires liant l’aiguille à MxiC restaient inconnus. Au cours de ce travail, nous avons montré que MxiC forme un complexe avec la sous-unité de la tige interne, MxiI, afin de bloquer l’entrée du canal de sécrétion et que cette interaction est conservée chez Yersinia et Salmonella. Nous démontrons que, suite au contact cellulaire, la dissociation de ce complexe facilite le switch de sécrétion des translocateurs aux effecteurs. Nos résultats révèlent également que MxiC est capable de s’associer au chaperon IpgC afin de réguler la sécrétion des translocateurs. De plus, nous avons identifié les domaines de MxiC engagés dans la régulation du SST3 et rapporté un nouveau rôle de MxiC dans l’échappement aux macrophage impliquant une possible inhibition de la voie apoptotique classique afin de promouvoir une pyroptose. Chez Shigella, IpaD gouverne la composition du complexe d’extrémité et est impliqué dans la régulation de la sécrétion. Nous avons développé une étude phénotypique de ses régions coiled-coil et centrale et montré que la composition du complexe d’extrémité permet de définir à la fois l’état d’inductibilité de l’AST3 et la sécrétion des effecteurs tardifs. Par ailleurs, notre étude fonctionnelle des domaines de MxiC et IpaD suggère que les capacités de Shigella à échapper au macrophage et à insérer un pore de translocation ne sont pas strictement couplées. <p>La dernière partie de ce travail s’est focalisée sur la caractérisation de la protéine Spa13 de Shigella. Nous avons découvert que le défaut de sécrétion du mutant spa13 est dû à l’instabilité de la sous-unité MxiH de l’aiguille et que Spa13 n’est pas sécrété par le SST3. Nos résultats indiquent également un rôle de Spa13 dans l’escorte de chaperons et l’activation de l’appareil d’exportation afin de promouvoir la sécrétion des substrats./Shigella is the causative agent of shigellosis, also known as bacillary dysentery, an invasive disease of the human colonic epithelium. During infection, Shigella uses a type III secretion system (T3SS) to penetrate enterocytes and to disseminate into the colonic epithelium, leading to destruction of the mucosal lining and shigellosis symptoms. Most of the virulence factors of Shigella are encoded by a large plasmid harboring a 30-kb region that is sufficient to promote bacterial entry into host cells. This entry region is organized in two loci, one corresponding to the the ipa-ipg genes encoding the secreted proteins and their cognate chaperones while the other encodes Mxi-Spa proteins that form the type III secretion apparatus (T3SA), consisting of a cytoplasmic bulb, a basal body spanning the bacterial envelope and a hollow needle. The T3SS allows the ordered and hierarchical secretion of effectors by inserting a translocation pore in the host cell membrane through which effector proteins are injected into the cytosol. Before host cell contact, the T3SA is inactive and plugged by the tip complex proteins IpaB and IpaD. <p>In Shigella, the gatekeeper MxiC is known to sequester effectors within the cytoplasm prior to receiving the activation signal from the needle but the molecules involved in linking the needle and MxiC are unknown. We demonstrated that MxiC and the predicted inner-rod component MxiI form a complex plugging the T3SA entry gate and showed that this interaction is conserved in Yersinia and Salmonella. Dissociation of this complex seems to facilitate the switch in secretion from translocators to effectors upon host cell contact. Our results also revealed that MxiC binds to the chaperone IpgC to regulate translocators secretion. Moreover, we identified the domains of MxiC involved in the T3S regulation and reported a new role in macrophage escape by potential inhibition of the classical apoptosis to promote pro-inflammatory pyroptosis. <p>In Shigella, IpaD rules the composition of the tip complex and is involved in secretion control and translocon insertion. We therefore undertook a phenotypic analysis of its coiled-coil and central regions and showed that the composition of the tip complex defines both the T3SA inducibility state and late effectors secretion. Besides, our functional study on MxiC and IpaD domains suggests that Shigella abilities to escape macrophage vacuole and to insert the translocation pore are uncoupled.<p>The last part of this work is related to the characterization of the Spa13 protein of Shigella. We found that the secretion defect of the spa13 mutant is due to the instability of the needle component MxiH and that Spa13 is not a secreted substrat. Our results also support a dual role of Spa13 as a chaperone escort and as an export gate-activator switch to promote substrates secretion. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

La synthèse d’oligosides complexes reste difficilement réalisable par voie chimique. Le recours aux catalyseurs enzymatiques permettrait de pallier aux contraintes de la chimie mais les enzymes naturelles ne présentent pas toujours les propriétés adéquates et nécessitent d’être optimisées par ingénierie moléculaire. Le couplage de la chimie et de biocatalyseurs conçus « sur mesure », peut offrir une alternative prometteuse pour explorer de nouvelles voies de synthèse des sucres, notamment pour la mise au point de glycovaccins. L’objectif de cette thèse a ainsi visé à mettre en œuvre des stratégies d’ingénierie semi-rationnelles de l’amylosaccharase de Neisseria polysaccharea (ASNp), une α-transglucosylase utilisant le saccharose comme substrat, afin de concevoir de nouvelles spécificités de substrats et d’étendre le potentiel de cette enzyme à catalyser de nouvelles réactions, permettant ainsi d’aller bien au-delà de ce que la Nature peut offrir. Dans une première étude, une approche assistée par ordinateur a été suivie afin de remodeler le site actif de l’enzyme (sous-sites +1, +2 et +3) pour la reconnaissance et la glucosylation en α-1,4 d’un accepteur disaccharidique non-naturel (l’allyl 2-deoxy-2-N-trichloroacetyl-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranose). Le produit attendu, un trisaccharide, est un précurseur dans la synthèse chimio-enzymatique des oligosaccharides mimant les unités répétitives des lipopolysaccharides de Shigella flexneri, dont l’utilisation ultime est le développement de vaccins contre la Shigellose. Une approche computationnelle faisant appel à des outils dédiés au design automatisé de protéines et à une analyse des séquences a conduit au design d’une librairie d’environ 2.7x104 séquences, qui a ensuite été construite expérimentalement puis criblée. Au final, 55 variants actifs sur saccharose (le substrat donneur) ont été identifiés, et un mutant, appelé F3, a révélé sa capacité à glucosyler en α-1,4 le disaccharide cible. De manière étonnante, ce mutant possède 7 mutations au sein de son site actif, nécessaires au déploiement de sa nouvelle spécificité tout en maintenant son aptitude à utiliser le saccharose comme donneur d'unité glucosyle. Dans une deuxième étude, trois variants ont été identifiés lors du criblage de la librairie semi-rationnelle sur saccharose comme présentant de nouvelles spécificités de produits. Ces mutants ont été caractérisés plus en détails, ainsi que leurs produits, sur un plan biochimique et structural. Ces mutants, appelés 37G4, 39A8 et 47A10, contiennent entre 7 et 11 mutations dans leur site actif. Il a été montré qu’ils étaient capables de reconnaitre le saccharose et le maltose (un produit de la réaction avec le saccharose) comme donneur et accepteur pour synthétiser en quantités variables de l’erlose (α-D-Glucopyranosyl-(1→4)-α-D-Glucopyranosyl-(1→2)-β-D-Fructose) et du panose (α-D-Glucopyranosyl-(1→6)-α-D-Glucopyranosyl-(1→4)-α-D-glucose), des molécules non produites par l’enzyme sauvage. Des taux de production relativement élevés ont été obtenus pour ces molécules, dont les propriétés acariogènes et le pouvoir sucrant pourraient présenter un intérêt applicatif pour l’industrie alimentaire. Dans une dernière partie, un autre mutant, appelé 30H3, a été isolé lors du criblage primaire de la librairie de par son activité élevée sur saccharose (une amélioration d’un facteur 6.5 comparé à l’enzyme sauvage). Après caractérisation, le mutant s’est avéré synthétiser un profil unique de produits en comparaison de l’enzyme sauvage ASNp. Il s’est ainsi montré très efficace pour la synthèse de maltooligosaccharides solubles, de taille de chaînes contrôlée allant d’un DP 3 à 21, et de faible polydispersité. Aucun polymère insoluble n’a été identifié. La structure 3D du mutant résolue par cristallographie des rayons X a révélé un agrandissement de la poche catalytique en raison de la présence de 9 mutations introduites dans la première sphère.... / Chemical synthesis of complex oligosaccharides still remains critical. Enzymes have emerged as powerful tools to circumvent chemical boundaries of glycochemistry. However, natural enzymes do not necessarily display the required properties and need to be optimized by molecular engineering. Combined use of chemistry and tailored biocatalysts may thus be attractive for exploring novel synthetic routes, especially for glyco-based vaccines development. The objective of this thesis was thus to apply semi-rational engineering strategies to Neisseria polysaccharea amylosucrase (NpAS), a sucrose-utilizing α-transglucosylase, in order to conceive novel substrate specificities and extend the potential of this enzyme to catalyze novel reactions, going beyond what nature has to offer. In a first study, a computer aided-approach was followed to reshape the active site of the enzyme (subsites +1, +2 and +3) for the recognition and α-1,4 glucosylation of a non-natural disaccharide acceptor molecule (allyl 2-deoxy-2-N-trichloroacetyl-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranose). The trisaccharide product is a building block for the chemo-enzymatic synthesis of oligosaccharides mimicking the repetitive units of the Shigella flexneri lipopolysaccharides, and ultimately, for the production of a vaccine against Shigellosis disease. Using computational tools dedicated to the automated protein design, combined with sequence analysis, a library of about 2.7x104 sequences was designed and experimentally constructed and screened. Altogether, 55 mutants were identified to be active on sucrose (the donor substrate), and one, called mutant F3, was subsequently found able to catalyze the α-1,4 glucosylation of the target disaccharide. Impressively, this mutant contained seven mutations in the first shell of the active site leading to a drastic reshaping of the catalytic pocket without significantly perturbing the original specificity for sucrose donor substrate. In a second study, three variants were identified from the screening of the semi-rational library on sole sucrose as displaying totally novel product specificities. They were further characterized, as well as their products, at both biochemical and structural level. These mutants, called 37G4, 39A8 and 47A10, contained between 7 and 11 mutations into their active site. They were found able to use sucrose and maltose (a reaction product from sucrose) as both donor and acceptor substrates to produce in varying amounts erlose (α-D-Glucopyranosyl-(1→4)-α-D-Glucopyranosyl-(1→2)-β-D-Fructose) and panose (α-D-Glucopyranosyl-(1→6)-α-D-Glucopyranosyl-(1→4)-α-D-glucose) trisaccharides, which are not produced at all by parental wild-type enzyme. Relatively high yields were obtained for the production of these molecules, which are known to have acariogenic and sweetening properties and could be of interest for food applications. In a last part, another mutant 30H3 was isolated due to its high activity on sucrose (6.5-fold improvement compared to wild-type activity) from primary screening of the library. When characterized, the mutant revealed a singular product profile compared to that of wild-type NpAS. It appeared highly efficient for the synthesis of soluble maltooligosaccharides of controlled size chains, from DP 3 to 21, and with a low polydispersity. No formation of insoluble polymer was found. The X-ray structure of the mutant was determined and revealed the opening of the catalytic pocket due to the presence of 9 mutations in the first sphere. Molecular dynamics simulations suggested a role of mutations onto flexibility of domain B’ that might interfere with oligosaccharide binding and explain product specificity of the mutant.

Abstract not available

Shigella flexneri attacks cells of the intestinal tract, causing over 1 million deaths annually from bacterial dysentery. A type III secretion system (T3SS) initiates the host-pathogen interaction and transports virulence factors directly into host cells via a needle complex (NC) comprising an extracellular needle and membrane-spanning basal body. Rings formed by the single-pass membrane proteins MxiG and MxiJ are arranged concentrically within the inner membrane ring (IMR) of the NC. The Neterminal domain of MxiG (MxiG-N) is the predominant IMR cytoplasmic structure, however it was structurally and functionally uncharacterised. Determination of the solution structure of MxiG-N in this study revealed it to be a forkhead associated (FHA) domain, although subsequent analyses of conserved residues suggested it does not have the canonical role in cell-signalling via phospho-threonine recognition. Subsequent positioning of the structure in the electron microscopy (EM) density for the S. flexneri NC supported models with 24-fold symmetry in the IMR. Both MxiG and MxiJ also have significant periplasmic domains, which were purified to homogeneity in this study, facilitating preliminary characterisation of their structures and intermolecular interactions. In addition, the entire IMR within the context of intact basal bodies was isolated and visualised in vitro by EM. The essential function of MxiG-N could be to localise the putative cytoplasmic ring (Cering) at the base of the T3SS. Although absolutely required for secretion, the Csring component, Spa33, was structurally uncharacterised. The crystal structure of the Cvterminal domain of Spa33 (Spa33-C) was determined in this study, showing an intertwined dimer that aligned with homologous structures and exhibited a novel interaction with the N-terminus of the ATPase regulator, MxiN. Subsequently, Spa33-C was identified as an altemative translation product of spa33 that formed a 2: 1 complex with Spa33 in vitro. This complex oligomerised further, demonstrating for the first time that Spa33 has the propensity to form the ordered, high molecular weight assemblies that would be required for C-ring formation in S. flexneri.