Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 101-125).

Aeromonas spp. são bactérias Gram negativas capazes de causar doenças intestinais e extra-intestinais. A virulência de algumas espécies do gênero já foi associada a diversos fatores, como a expressão de flagelos e conseqüente mobilidade bacteriana. Aeromonas caviae apresenta dois distintos sistemas flagelares, um deles responsável pela natação em meio líquido (swimming) onde está envolvido o flagelo polar, e outro associado ao deslizamento em superfícies sólidas ou viscosas (swarming), o qual participam flagelos laterais. A fim de avaliar a contribuição de ambos os flagelos no contexto da formação de biofilme em superfície plástica e aderência à linhagem celular HEp-2, foram estudadas 76 cepas de A. caviae isoladas de diferentes origens. Os resultados obtidos em nosso estudo demonstraram que a detecção do gene flaA (flagelina polar) foi mais freqüente (94%) que a do gene lafA (flagelina lateral) (71%) e, aparentemente, a presença deste último parece estar condicionada à do primeiro. Todas as cepas que apresentaram o genótipo flaA-lafA-, foram incapazes de formar biofilme, bem como 76% das cepas flaA+lafA-. As cepas provenientes de água foram as que exibiram menor percentual de positividade para o gene da flagelina lateral (57%) e aquelas onde se observou maior incapacidade de formação de biofilme (71%). Além disso, 95% das cepas flaA+lafA+ formaram biofilme e todas as cepas que apresentaram, quantitativamente, um biofilme forte, albergavam ambos os genes. A aderência à HEp-2 se mostrou eficiente em todos os casos onde ao menos um dos flagelos estava presente e em dois casos onde ambos estavam ausentes, com prevalência do padrão agregativo. Em contrapartida, não foi notado uma interferência exclusiva dos flagelos laterais nesse processo. Todas as três cepas que não aderiram ao modelo celular utilizado, ou o fizeram muito discretamente, não exibiram nem o gene flaA, tampouco lafA. Em conclusão aos dados observados, apontamos que uma plena atividade de ambos os flagelos, geralmente, é requerida nos processos de aderência às superfícies abióticas e bióticas. Contudo, trabalhos adicionais sobre a contribuição dessas estruturas na patogênese de A. caviae necessitam ser realizados. / Aeromonas spp. are bacteria Gram-negative able to cause intestinal and extraintestinal diseases. The virulence of some species of this genus has already been associated to several aspects, such as the expression of flagellum and following bacterium mobility. Aeromonas caviae presents two distinct flagellar systems, one of which is responsible for mobility in liquid environments (swimming) at where the polar flagellum is involved, and the other is associated to sliding in solid or viscous surfaces (swarming), in which lateral flagella take part. In order to evaluate the contribution of both flagella in the biofilm formation in plastic surfaces and its adherence to cell line HEp-2, 76 strains of isolated A. caviae from distinct sources were analyzed. The results from our studies showed that detection of gene flaA (polar flagellin) was more frequent (94%) than gene lafA (lateral flagellin) (71%), and apparently, the presence of the last one seems to be conditioned to the first one (the gene flaA). All the strains that presented the genotype flaA-lafA- were unable to form biofilm, such as 76% of strains flaA+lafA-. The aquatic strains were those who showed lesser percentage of positivity for the lateral flagellin gene (57%), and higher incapacity of biofilm formation (71%). Moreover, 95% of the flaA+lafA+ strains formed biofilm, and all strains that quantitatively showed a strong biofilm, contained both genes. The adherence to HEp-2 proved to be efficient in all cases where at least one of the two flagella was present at, and in two cases where both were absent, with prevalence of the aggregative pattern. However, an exclusive interference from the lateral flagella was not perceived in this process. All of the three strains that did not adhere to the cell model used, or that adhered in a very discrete way, did not show neither gene flaA nor gene lafA. In conclusion, we suggest that the complete function of both flagella usually is a requirement in the adherence processes to biotic and abiotic surfaces. However, additional studies on the contribution of these structures in the pathogenesis of A. caviae need to be carried out.

V. macrocarpon (canneberge) est traditionnellement associé à la prévention des IU, les mécanismes restant mal élucidés. L'effet d'une préparation commerciale de canneberge sur l'adhésion d'E.coli UTI89 aux cellules urothéliales T24, a montré l'importance de pré-incuber les bactéries avec le composé pour obtenir une inhibition dose-dépendante et réversible de l'adhésion. L'étude du transcriptome (E.coli Gene expression microarray, AgilentTechnologies) montre un effet puissant portant sur de nombreux gènes liés aux adhésines (sauf les fimbriae P), au chémotactisme et au flagelle. L'étude en microscopie électronique confirme un effet sur la taille et les structures de surface (adhésines, flagelles), l'étude de la mobilité en microscopie à champ large montre de moindres capacités de déplacement (distances, linéarité). Chez la souris C57BL/6, la pré-incubation n'a pas d'effet significatif sur la colonisation des vessies, ni sur l'adhésion ex vivo aux cellules T24, des bactéries récupérées dans les vessies. Un modèle intégratif d'étude de V.macrocarpon, basé sur des tests simples in vitro (adhésion, swarming, microscopie à champ large) est défini. / V.macrocarpon (cranberry) is traditionally associated with the prevention of urinary tract infections although the mechanisms of action remaining poorly elucidated. Preincubation of E.coli UTI89 strain with commercial extracts of V.macrocarpon inhibited adhesion to T24 human urothelial cell line in a dose-dependent and reversible manner. Transcriptomic assay (E.coli Gene expression microarray, Agilent Technologies) highlighted a strong impact on most genes related to adhesion, but P fimbriae, chemotactism and flagella. Electron microscopy study confirmed V.macrocarpon-induced alterations on UTI89 size and surface structures (fimbriae, flagella). In keeping, broad field microscopy (ImarisTrack) evidenced alterations in E.coli motility (track displacement length, duration, speed & straightness). In C57BL/6 mice, pre-incubation of UTI89 with V.macrocarpon extracts failed to impact bladder colonization after intravesical instillations and adhesion to T24 cells of bacteria recovered 3days after instillation. A simple, in vitro model based on adhesion and swarming assays and broad field microscopy is described to evaluate cranberry activity.

Aeromonas spp. são bactérias Gram negativas capazes de causar doenças intestinais e extra-intestinais. A virulência de algumas espécies do gênero já foi associada a diversos fatores, como a expressão de flagelos e conseqüente mobilidade bacteriana. Aeromonas caviae apresenta dois distintos sistemas flagelares, um deles responsável pela natação em meio líquido (swimming) onde está envolvido o flagelo polar, e outro associado ao deslizamento em superfícies sólidas ou viscosas (swarming), o qual participam flagelos laterais. A fim de avaliar a contribuição de ambos os flagelos no contexto da formação de biofilme em superfície plástica e aderência à linhagem celular HEp-2, foram estudadas 76 cepas de A. caviae isoladas de diferentes origens. Os resultados obtidos em nosso estudo demonstraram que a detecção do gene flaA (flagelina polar) foi mais freqüente (94%) que a do gene lafA (flagelina lateral) (71%) e, aparentemente, a presença deste último parece estar condicionada à do primeiro. Todas as cepas que apresentaram o genótipo flaA-lafA-, foram incapazes de formar biofilme, bem como 76% das cepas flaA+lafA-. As cepas provenientes de água foram as que exibiram menor percentual de positividade para o gene da flagelina lateral (57%) e aquelas onde se observou maior incapacidade de formação de biofilme (71%). Além disso, 95% das cepas flaA+lafA+ formaram biofilme e todas as cepas que apresentaram, quantitativamente, um biofilme forte, albergavam ambos os genes. A aderência à HEp-2 se mostrou eficiente em todos os casos onde ao menos um dos flagelos estava presente e em dois casos onde ambos estavam ausentes, com prevalência do padrão agregativo. Em contrapartida, não foi notado uma interferência exclusiva dos flagelos laterais nesse processo. Todas as três cepas que não aderiram ao modelo celular utilizado, ou o fizeram muito discretamente, não exibiram nem o gene flaA, tampouco lafA. Em conclusão aos dados observados, apontamos que uma plena atividade de ambos os flagelos, geralmente, é requerida nos processos de aderência às superfícies abióticas e bióticas. Contudo, trabalhos adicionais sobre a contribuição dessas estruturas na patogênese de A. caviae necessitam ser realizados. / Aeromonas spp. are bacteria Gram-negative able to cause intestinal and extraintestinal diseases. The virulence of some species of this genus has already been associated to several aspects, such as the expression of flagellum and following bacterium mobility. Aeromonas caviae presents two distinct flagellar systems, one of which is responsible for mobility in liquid environments (swimming) at where the polar flagellum is involved, and the other is associated to sliding in solid or viscous surfaces (swarming), in which lateral flagella take part. In order to evaluate the contribution of both flagella in the biofilm formation in plastic surfaces and its adherence to cell line HEp-2, 76 strains of isolated A. caviae from distinct sources were analyzed. The results from our studies showed that detection of gene flaA (polar flagellin) was more frequent (94%) than gene lafA (lateral flagellin) (71%), and apparently, the presence of the last one seems to be conditioned to the first one (the gene flaA). All the strains that presented the genotype flaA-lafA- were unable to form biofilm, such as 76% of strains flaA+lafA-. The aquatic strains were those who showed lesser percentage of positivity for the lateral flagellin gene (57%), and higher incapacity of biofilm formation (71%). Moreover, 95% of the flaA+lafA+ strains formed biofilm, and all strains that quantitatively showed a strong biofilm, contained both genes. The adherence to HEp-2 proved to be efficient in all cases where at least one of the two flagella was present at, and in two cases where both were absent, with prevalence of the aggregative pattern. However, an exclusive interference from the lateral flagella was not perceived in this process. All of the three strains that did not adhere to the cell model used, or that adhered in a very discrete way, did not show neither gene flaA nor gene lafA. In conclusion, we suggest that the complete function of both flagella usually is a requirement in the adherence processes to biotic and abiotic surfaces. However, additional studies on the contribution of these structures in the pathogenesis of A. caviae need to be carried out.

Orientador: Domingos da Silva Leite / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T10:33:43Z (GMT). No. of bitstreams: 1 Moura_Claudiade_D.pdf: 2791356 bytes, checksum: 7830cb1ece9ec9ac3c34c2794b264c93 (MD5) Previous issue date: 2010 / Resumo: Escherichia coli é um membro comensal da microbiota de animais, porém podem causar doenças desde diarréias até sepses. A caracterização dos seus antígenos de superfície O (somático) e H (flagelar) auxilia na determinação de linhagens patogênicas dentro da espécie. Contudo, algumas bactérias não expressam flagelo in vitro, demonstrado que a amplificação do gene fliC, a análise dos fragmentos de polimorfismo (PCR-RFLP) e sequenciamento podem ser utilizadas para identificação dos antígenos H, em substituição à sorologia convencional. Até meados de 1980, pensava-se que, diferentemente da Salmonella, E. coli possui um único gene para expressão de flagelina (fliC), mas algumas amostras podem conter genes para expressão de flagelina flkA, fllA, flmA, flnA e fljA (repressor de fliC). Em nosso trabalho, analisamos 31 amostras de E. coli isolados de animais e alimentos que apresentavam o fenótipo HNT em ensaios de sorologia. Utilizamos PCR-RFLP e sequenciamento para descrever novos genes para flagelina, da qual foram obtidos antissoros. Identificamos por PCR e sequenciamento os genes responsáveis pela variação de fase fljA, flkA e flmA, realizamos experimentos de motilidade para determinar a variação de fase flagelar e detectar a expressão dos genes através de RT-PCR. Dezessete amostras tiveram seus antígenos H caracterizados, sendo nove caracterizadas por PCR-RFLP: H2 (duas amostras) H16 (duas amostras), H34 (três amostras), H33 (uma amostra) e H38 (uma amostra). Na análise de sequenciamento identificamos duas amostras portadoras do gene fliCh25, duas amostras fliCh7 e uma amostra apresentando fliCh32. Três novos genes para flagelina foram descritos: fliCh2', fliC4c, fliC40c. Identificamos o gene fljA em duas amostras HNT (3C e 4C) e na amostra padrão H35. O gene das amostras HNT apresentaram homologia ao fljA de Salmonella enterica, cuja variação de fase é bem estabelecida. As amostras padrão H11, H35, H40 e H47, bem como as amostras HNT 3C e 4C foram positivas para o gene flmA. As amostras padrão H3 e H53 são portadoras do gene flkA, contudo apenas a amostra H53 apresentou fljA. A amostra H54 é portadora de fljA e flmA. Nenhuma amostra H padrão mostrou variação de fase, diferentemente da literatura, sugerindo a perda da capacidade de variar a fase flagelar. A amostra 4C mostrou variação de fase positiva quando induzida em meios de cultura contendo antissoros anti-H48, anti-H54 e anti-H4C. Do mesmo modo, a detecção dos RNAm em diferentes condições de cultura confirmou a variação de fase. Como resultado um esquema de identificação para detecção de grupos de antígenos H e identificação de fliC foi testado. A técnica de fliC-RFLP provou ser eficiente e rápida, auxiliando a sorologia clássica para detecção de antígenos H de E. coli. Um modelo geral de variação de fase da amostra 4C é expresso por fliCoff + flmAon ? fliCon + flmAoff. Além disso, nós verificamos que a amostra 4C apresenta um gene novo para expressão de flagelina. Este trabalho é pioneiro em relação à variação de fase flagelar, demonstrando uma nova associação entre os antígenos H48 e H54 / Abstract: Escherichia coli are a species of microflora, and characterization of the cell surface lipopolysaccharide O antigen and the flagellar H antigen allow the grouping of pathogenic clones within this species. Moreover, some bacteria in vitro do not obtain to express its flagella, demonstrated that PCR-restriction fragment length polymorphism (PCR-RFLP) and sequencing analysis has been used for the identification of these antigens, in substitution of traditional serology. Moreover, until middle of years 80, are believed, differently of the Salmonella, E. coli possesss an only gene for flagelin expression (fliC), but some s/strains can contain genes for flagellin expression flkA, fllA, flmA, flnA and fljA (repressor of fliC). In this work, we analyzed 31 strains of E. coli isolated from animals and foods that presented HNT phenotype in serology assays. We use PCR-RFLP and sequencing to describe new genes for flagellin, of which antiserum were obtained. We identify for PCR and sequencing the genes for phase variation fljA, flkA and flmA, we carry through motility experiments to determine the flagellar phase variation and to detect the expression of the genes (RNAm) through RT-PCR. Seventeen strains had had its H antigen characterized and nine of then were characterized for PCR-RFLP: H2 (two strains) H16 (two strains), H34 (three strains), H33 (one strain) and H38 (one strain). Through sequencing analysis we identify to two carrying strains of the gene fliCh25, two strains fliCh7 and one strain presenting fliCh32. Three new genes for flagellin had been described: fliCh2', fliC4c, fliC40c. Using PCR and sequencing, we identify fljA gene in two strains HNT (3C and 4C) and in the H35 control strain. The HNT genes showed homology to fljA of Salmonella enterica, whose variation of phase well is established. The control strains H11, H35, H40 and H47, as well as HNT 3C and 4C strains were positive for flmA gene. The control strains H3 and H53 are carrying of flkA gene, however only the H53 strain presented fljA. The H54 control strain is carrying of fljA and flmA. No H control strain showed phase variation, differently of literature, suggesting the loss of the capacity to flagellar phase variation. The 4C strain showed positive phase variation when cultured with antiserum anti-H48, anti-H54 and anti-H4C. In a similar way, the detention of RNAm in different conditions of culture confirmed the phase variation. As a result, an identification scheme was tested to deduce H antigen groups and new genes of fliC. The fliCRFLP technique proved to be faster than classic serotyping for the deduction of the E. coli H antigen, characterizing the antigens with few days and indicating new putative genes. Thus, a general model for flagellar phase variation in 4C strain can be expressed as fliCoff + flmAon ? fliCon + flmAoff. In addition, we found that strains 3C and 4C express unidentified flagellin antigens. This is the first report of flagellar phase variation in wild E. coli strains. We have also provided evidence that strain 4C, identified here for the first time, expresses three flagellar antigens, H48, H54 and a previously unidentified flagellin / Doutorado / Microbiologia / Doutor em Genetica e Biologia Molecular

Orientador: Domingos da Silva Leite / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T15:47:58Z (GMT). No. of bitstreams: 1 Tiba_MoniqueRibeiro_D.pdf: 3934673 bytes, checksum: 211302f7129afd8376ba9096064a21c4 (MD5) Previous issue date: 2009 / Resumo: Escherichia coli tem sido isolada, com certa freqüência, apresentando antígenos flagelares (H) que não são reconhecidos por nenhum dos anti-soros disponibilizado pelo mais importante centro de referência de E. coli, The International Escherichia and Klebsiella Centre (WHO) do Statens Serum Institut, Copenhague, Dinamarca. Atualmente são reconhecidos 53 antígenos "H" e, nos últimos 29 anos, nenhuma modificação ocorreu na lista dos antígenos flagelares associados à Escherichia coli. Isto posto, os objetivos deste trabalho foram identificar os antígenos flagelares das cepas de E. coli que expressam H não tipável (HNT) e que apresentam fatores de virulência associados à diferentes enteropatias. Esta identificação foi realizada inicialmente, pela reação em cadeia da polimerase (PCR) do gene fliC, responsável pela proteína flagelina, das 53 amostras padrões para os antígenos H e das 20 amostras HNT (H não-tipável). Em seguida, os amplicons foram digeridos por enzimas de restrição e daquelas amostras que apresentaram perfis de restrições distintos daqueles observados para as amostras padrões de antígeno H, foram produzidos soros em coelhos. Foram realizados testes de titulação frente aos 53 antígenos padrões, frente ao antígeno homólogo e frente aos antígenos das amostras HNT. As seqüência gênicas das amostras HNT, obtidas na reação de sequenciamento, foram comparadas aos diferentes genes de fliC armazenados no banco de dados do "National Center for Biotecnology Information" (NCBI) através do sistema BLAST, e o programa ClustalW foi utilizado para alinhamento das seqüências. Os resultados demonstraram que estas amostras apresentaram similaridade com antígenos padrões, entretanto, elas não possuem a mesma seqüência nucleotídica e também não reagiram fenotipicamente com o anti-soro esperado. Os dados obtidos permitem concluir que no conjunto de amostras estudado, treze amostras apresentaram antígeno flagelar diferente daqueles já descritos na literatura, quando utilizado as técnicas de PCR e/ou sorologia. / Abstract: Escherichia coli has been isolated frequently, showing flagellar antigens that are not recognized by any of the antisera, provided by the most important reference center of E. coli, The International Escherichia and Klebsiella Centre (WHO) of the Statens Serum Institute, Copenhagen, Denmark. Are currently recognized 53 H antigens and in the last 29 years, no change occurred in the list of flagellar antigens associated with Escherichia coli. The objectives of this study were to identify the flagellar antigens of E. coli that do not express non-typeable H antigens and presenting the virulence factors associated with different diseases. This identification was performed initially by gene amplification of the fliC, (flagellin protein) by the polymerase chain reaction (PCR) in all 53 standards E. coli strains for the H antigens and 20 non-typeable H-antigens E. coli strains, being then, the amplicons were digested by restriction enzymes. Anti-sera were produced in rabbits, those strains that showed different restriction profiles of these patterns observed for the nontypeable H antigens E. coli strains. Agglutination testes were carried out against the 53 antigens standards, against the homologous antigen and H antigens of the non-typeable strains. DNA sequences were compared to different fliC genes stored in the database of the National Center for Biotecnology Information (NCBI) through the BLAST, and ClustalW program was used to align the sequences. The results showed that although these strains have homology with a standard H-antigen, they do not have the same nucleotide sequence and did not phenotypically reacted with the antiserum expected. The data obtained showed that thirteen strains had a different H antigen those already described in the literature when used the techniques of PCR-RFLP and/or serology. / Doutorado / Microbiologia / Doutor em Genetica e Biologia Molecular

Les cils et les flagelles sont des organites présents à la surface cellulaire. Ils sont conservés chez les eucaryotes chez lesquels ils jouent un rôle essentiel dans la régulation de nombreux processus physiologiques. La zone de transition (ZT) est une structure complexe, localisée à la base des cils, qui assure une fonction importante dans l'assemblage et la régulation du trafic des constituants ciliaires. Trois complexes protéiques ont été identifiés à la ZT : MKS-JBTS, NPHP1-4-8 et NPHP5-CEP290. D'autres protéines sont également situées à la ZT telles que CBY et AZI1 mais leur interaction avec ces trois modules reste encore peu connue. Chez l'Homme, des mutations de gènes codant des protéines de la ZT sont associées à des maladies génétiques rares, les ciliopathies. Deux modes d'assemblage des cils ont été décrits : la ciliogenèse compartimentée et la ciliogenèse cytosolique. Alors que la fonction de la ZT au cours de la ciliogenèse compartimentée a été bien étudiée, son rôle dans la ciliogenèse cytosolique reste peu connu. La Drosophile possède deux sortes de cellules ciliées, les neurones sensoriels et les flagelles de spermatozoides dont les cils s'assemblent selon ces deux modes d'assemblage. Au cours de ma thèse, j'ai utilisé ce modèle pour analyser la fonction des protéines de la ZT dans ces deux types cellulaires. Mes résultats montrent que les protéines MKS ne jouent pas un rôle essentiel dans l'assemblage de la ZT dans ces deux types cellulaires. J'ai aussi révélé que CBY et AZI1, coopèrent pour assembler la ZT et qu'elle est nécessaire à l'ancrage du corps basal à la membrane plasmique. De plus, mes travaux ont démontré que KLP59D, une kinésine dépolymérisante des microtubules, est indispensable à la régulation de l'élongation de l'axonème au cours de la ciliogenèse cytosolique. En conclusion, ce travail apporte de nouvelles connaissances sur la dynamique d'assemblage de la ZT des cils et sur les mécanismes qui contrôlent l'élongation de l'axonème / Cilia and flagella are cellular organelles that protrude at the cell surface. They are composed of a microtubular cytoskeleton and they are highly conserved across eukaryotic species from plantae to Human. In mammals, they play essential functions during development and regulate numerous physiological processes in adults. At the ciliary base a complex structure called transition zone (TZ) is necessary for cilia assembly and regulation of ciliary components trafficking inside the cilia. Three protein complexes have been identified at the TZ : MKS-JBTS, NPHP1-4-8 and NPHP5-CEP290. Other TZ proteins such as CBY and AZI1 have been studied but their interaction with these 3 modules is not yet elucidated. In Human, mutations of genes encoding TZ proteins are associated with several genetic diseases called ciliopathies. Two different modes of cilia assembly have been identified: compartimentalized and cytosolic ciliogenesis. While TZ function in compartimentalized ciliogenesis is well studied, its role in cytosolic ciliogenesis remains poorly understood. In Drosophila, there are only two types of ciliated cells, sensory neurons and sperm flagella, representative of these two ciliogenesis pathways. During my PhD, I used Drosophila to study the function of TZ proteins during cilia assembly in these two ciliated cell types. My data show that proteins of the MKS complex do not play an essential role in TZ assembly in the cilia of sensory neurons and in spermatozoon flagella. I also demonstrated that CBY and AZI1 cooperate to assemble the TZ components and that the TZ is necessary to dock the basal bodies to the plasma membrane, one of the first important step in cilia assembly. Finally, I showed that KLP59D, a microtubule-depolymerising kinesin, is required to control axoneme elongation during the cytosolic ciliogenesis. In conclusion, this work brings new insights into the understanding of the dynamic assembly of TZ proteins and the mechanisms that regulate flagella elongation

Clostridium difficile (CD) représente la première cause d'infections digestives nosocomiales dans les pays développés. Les infections à CD (ICD) induisent une inflammation intestinale importante qui se manifeste principalement par des colites pseudomembraneuses ainsi que par un taux de mortalité élevé. Les facteurs majeurs de virulence sont les toxines TcdA et TcdB. Dans la première partie des travaux de cette thèse, nous avons validé le rôle in vivo d'un autre facteur bactérien, les flagelles, dans un modèle murin d'ICD, en montrant que les flagelles induisent une réponse inflammatoire au niveau de la muqueuse caecale en synergie avec les toxines. Nous avons ensuite montré une régulation de de cette réponse par un microARN (miARN) exerçant un rôle anti-inflammatoire en modulant l'activation de la voie de signalisation de NF-KB. Le traitement des souris infectées par ce miARN réduit l'inflammation intestinale apportant la preuve du concept pour une nouvelle approche thérapeutique. / Clostridium difficile (CD) is the leading cause of nosocomial digestive infections in developed countries. CD infections (CDI) induce significant intestinal inflammation that is manifested primarily by pseudomembranous colitis and a high mortality rate. The major virulence factors are TcdA and TcdB toxins. In the first part of the work of this thesis, we validated the in vivo role of another bacterial factor, the flagella, in a mouse model of CDI, showing that the flagella induce an inflammatory response in ceacal mucosa in synergy with toxins. We then showed a regulation of this response by a microRNA (miRNA) exerting an anti-inflammatory role by modulating the activation of the NF-KB signaling pathway. The treatment of mice infected with this miRNA reduces intestinal inflammation providing proof of concept for a new therapeutic approach.

Das Flagellum von Salmonella enterica ist eine komplexe molekulare Nanomaschine, die zur Fortbewegung verwendet wird. Die Synthese erfordert die Sekretion extrazellulärer Bausteine durch die Zellhülle. Der Substratexport erfolgt durch ein hochkonserviertes Typ-III-Sekretionssystem. Der Kern des fT3SS ist eine komplexe Proteinsekretionsmaschine, bestehend aus den Proteinen FliPQR und FlhBA. Ziel dieser Arbeit war die molekularen Mechanismen, die eine korrekte Funktion gewährleisten, tiefergehend zu erforschen. Im ersten Kapitel wurden die molekulare Mechanismen der Qualitätskontrolle während der Synthese des fT3SS untersucht. Es wurde kürzlich gezeigt, dass die korrekte Synthese durch das fT3SS-spezifischen Chaperon FliO gewährleistet wird. Ziel war es, den molekularen Mechanismus, wie FliO an diesem Prozess beteiligt ist, aufzuklären. Die Ergebnisse zeigten, dass mehrere Aminosäuren von FliO während der Assemblierung mit FliP interagieren. Des Weiteren wurde die Relevanz des spaltbaren Signalpeptids am N-Terminus von FliP untersucht. Diese Studie zeigt, dass die Anwesenheit des Signalpeptids und seine korrekte Spaltung entscheidend, aber nicht unerlässlich für die Funktion der Flagellen sind. Das fT3SS ist in der Lage Proteine mit einer bemerkenswerten Geschwindigkeit von mehreren tausend Aminosäuren pro Sekunde zu sekretieren. Das zweite Kapitel konzentrierte sich darauf, wie das fT3SS Proteine mit hoher Geschwindigkeit sekretiert, während das Austreten kleiner Moleküle verhindert wird. Unsere Mutationsanalysen zeigten, dass eine Methioninschleife in FliP, eine sperrige Plug-Domäne in FliR und intermolekulare Salzbrücken zwischen FliQ-Untereinheiten zusammenarbeiten, um die Integrität der Membran aufrechtzuerhalten. Diese Arbeit liefert neue Einblicke in die Synthese des fT3SS Kerns und die Regulation der Substratsekretion. Beide Prozesse werden an mehreren Stellen streng kontrolliert, um eine korrekte Funktion des Flagellums sicherzustellen. / The flagellum of Salmonella enterica is a sophisticated molecular nanomachine, which is used for locomotion. Flagella synthesis requires the translocation of extracellular subunits across the cell envelop, which is mediated by a highly conserved type-III secretion system (fT3SS). The core fT3SS is a complex protein secretion machine consisting of the proteins FliPQR and FlhBA. Productive assembly is crucial for flagella function. The molecular mechanisms which ensure correct function of the fT3SS remain poorly understood. In this thesis, we aimed to gain a profound insight into the molecular mechanisms of fT3SS core assembly and function. The first chapter investigated the molecular mechanisms underlying the quality control during the assembly of the fT3SS. It was recently shown that productive assembly of the core fT3SS relies on the flagella-specific chaperone FliO. We aimed to elucidate the molecular mechanism of how FliO facilitates this process. Our results demonstrated, that several residues of FliO are interacting with FliP during the assembly process. Furthermore, we aimed to identify the relevance of the cleavable signal peptide at the N-terminus of FliP. This study showed, that the presence of the signal peptide and its correct cleavage are crucial but not essential for flagella function. The fT3SS is able to secrete proteins with a remarkable speed of several thousand amino acids per second. The second chapter focused on how the fT3SS secretes proteins at high speed while preventing the leakage of small molecules. Our mutational analyses demonstrated that a methionine loop in FliP, a bulky plug domain in FliR and intermolecular salt bridges between FliQ subunits are acting cooperatively to maintain the membrane barrier. Overall, this work provides new insights into the assembly process of the fT3SS core and the regulation of substrate secretion. Both processes are tightly controlled at multiple stages to ensure the proper functioning of the flagellum.

Eukaryotic cilia are complex, highly conserved microtubule-based organelles with a broad phylogenetic distribution. Cilia were present in the last eukaryotic common ancestor and many proteins involved in cilia function have been conserved through eukaryotic diversification. The evolution of these ciliary functions may be inferred from the distribution of the molecular components from which these organelles are composed. By linking protein distribution in 45 diverse eukaryotes with organismal biology, I define an ancestral ciliary inventory. Analysis of these core proteins allows the inference that the cenancestor of the eukaryotes possessed a cilium for motility and sensory function. I show that the centriolar basal body function is ancestral, whereas the centrosome is specific to the Holozoa, and I use this information to predict a number of roles for proteins based on their phylogenetic profile. I also show that while remarkably conserved, significant divergence in ciliary protein composition has occurred in many lineages, such as the unusual centriole of Caenorhabditis elegans and the transitional changes throughout the land plants. I exemplify this divergence through ultrastructural studies of the fern Ceratopteris richardii and the liverwort Marchantia polymorpha both of which have cilia that exhibit a number of distinctive morphological features, the most conspicuous of which is a general breakdown of canonical microtubule arrangements. Cilia have also been lost multiple times in different lineages: at least twice within the land plants. During these evolutionary transitions proteins with ancestral ciliary functions may be lost or co-opted into different functions. I have interrogated genomic data to identify proteins that I predict had an ancestral ciliary role, but which have been maintained in non-ciliated land plants. I demonstrate that several of these proteins have a flagellar localisation in protozoan trypanosomes and I use expression data correlation to predict potential non-ciliary plant roles.