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Construção de mutantes para os genes ychO, luxS e qseC presentes em uma linhagem de Escherichia coli patogênica para aves (APEC) : análises in vivo e in vitro / Mutant construction for ychO, luxS and qseC genes present in an Avian Pathogenic Escherichia coli (APEC) : in vivo and in vitro analysisDa Silva, Livia Pilatti Mendes, 1989- 24 August 2018 (has links)
Orientador: Wanderley Dias da Silveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T14:02:14Z (GMT). No. of bitstreams: 1
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Previous issue date: 2014 / Resumo: Linhagens de Escherichia coli patogênica para aves causam doenças extraintestinais em aves, levando a perdas econômicas consideráveis para a indústria avícola em todo o mundo. Fatores de virulência ainda não conhecidos podem apresentar papéis importantes na patogenicidade, os quais podem ser significativos para o desenvolvimento de medidas de controle de processos infecciosos dessas linhagens. Invasinas permitem que o patógeno invada células hospedeiras, sobrepujando as defesas do hospedeiro, por interagir com receptores específicos na superfície celular. Baseando-se em análises do genoma da linhagem APEC SEPT362, detectou-se a presença de uma provável invasina, homóloga ao gene ychO, ainda não caracterizado, da linhagem E. coli str. K-12 substr. MG1655, sendo seu efeito na patogenicidade estudado. Neste trabalho, a linhagem SEPT362 deficiente para ychO reduziu a taxa de mortalidade em pintos, a adesão a células de fibroblasto de embrião de galinha (FEG), a invasão em células fibroblasto de embrião de galinha (CEC-32), a sobrevivência em macrófagos de aves (HD11), a formação de biofilme e a motilidade. Esses resultados sugerem, pela primeira vez, que o gene ychO codifica para uma invasina e que tem papel na patogenicidade da linhagem APEC SEPT362. Além dos diversos fatores de virulência, o estudo de diferentes tipos de sinais extracelulares que medeiam a comunicação entre bactérias e regulam a expressão gênica abre grandes possibilidades no estudo de mecanismos de patogenicidade e possíveis meios de interferir nos mesmos, transformando organismos patogênicos em organismos não virulentos. Quorum sensing (QS) é um mecanismo dependente da densidade celular bacteriana que as permitem agir como complexos multicelulares, aumentando suas chances de sobrevivência no meio ambiente. O sistema QS autoindutor 2 (AI-2) é um sistema de comunicação universal de bactérias mediado pelo AI-2, produzido pela ação da enzima LuxS. O sistema QS autoindutor 3 (AI-3) é um sistema que pode também mediar a comunicação entre reinos, sendo regulado pela hisitidina quinase QseC. Neste contexto, mutantes para os genes luxS e qseC foram construídos na linhagem SEPT362. A linhagem deficiente para luxS apresentou uma redução significativa na adesão a células FEG, devido a um papel do gene na regulação da fímbria do tipo 1. A linhagem deficiente para qseC teve uma menor taxa de mortalidade em pintos e uma redução significativa na adesão a células FEG, pela falta da fímbria do tipo 1 no mutante / Abstract: Avian pathogenic Escherichia coli strains cause extraintestinal diseases in poultry, leading to substantial economic losses to the poultry industry worldwide. Virulence factors not yet known may play important roles in pathogenicity, which may be significant for measures control development of infectious processes of these strains. Invasins allow the pathogen to invade host cells, overcoming host defenses by interacting with specific cell surface receptors. Based on analysis of APEC SEPT362 strain genome detected the presence of a putative invasin gene homologous to ychO, not yet characterized, of E. coli str. K-12 substr. MG1655, and its effect on pathogenicity was studied. In this work, the SEPT362 strain deficient for ychO reduced the mortality rate in chicks, the adherence to chicken embryo fibroblast (CEF) cells, the invasion of chicken embryo cells (CEC-32), the survival in poultry macrophages (HD11), the motility and biofilm formation. These results suggest, for the first time, that ychO gene encodes for an invasin and plays a role in APEC SEPT362 pathogenicity. In addition to the many virulence factors, the study of different types of extracellular signals that mediate communication between bacteria and regulate gene expression opens up great possibilities to study pathogenic mechanisms and possible ways to interfere in it, transforming pathogenic organisms into non-virulent organisms. Quorum sensing (QS) is a mechanism dependent on the bacterial cell density that enables them to act as multicellular complexes, increasing their chances of survival in the environment. The QS system autoinducer 2 (AI- 2) is a universal communication system of bacteria mediated by AI- 2 produced by the action of the enzyme LuxS. The QS system autoinducer 3 (AI- 3) is a system that can also mediate communication between realms, being regulated by kinase QseC. In this context, mutants for the luxS and qseC genes were built in SEPT362 strain. The strain deficient for luxS showed a significant reduction in CEF cell adhesion due to a role in the regulation of gene type 1 fimbriae. The deficient strain for qseC had a lower rate of mortality in chicks and a significant reduction in cell adhesion to CEF, due to a lack of type 1 fimbriae in the mutant / Mestrado / Genetica de Microorganismos / Mestra em Genética e Biologia Molecular
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Caractérisation de deux systèmes de sécrétion de type VI de Pseudomonas aeruginosa : Cross-régulation et rôle dans l'invasion microtubules-dépendante de cellules non-phagocytairesSana, Thibault 20 June 2013 (has links)
Pseudomonas aeruginosa est une bactérie pathogène opportuniste extracellulaire. La souche PAO1 de P. aeruginosa possède trois loci codant des Systèmes de Sécrétion de Type VI (T6SS) indépendants, nommés H1 à H3-T6SS. Le premier d'entre eux, H1-T6SS, a été largement décrit dans la littérature scientifique et injecte des toxines à activité antiprocaryote dans d'autres bactéries permettant une défense active contre l'attaque d'un T6SS d'une autre bactérie. Les deux autres T6SS de P. aeruginosa sont probablement des facteurs de virulence de cette bactérie, mais aucun effecteur sécrété, ni cible intracellulaire ou rôle n'ont été proposés jusqu'ici. Dans ce travail de thèse, nous avons mis en évidence que la machinerie H2-T6SS permet l'invasion de cellules non-phagocytaires et participe à la virulence de P. aeruginosa. Cette entrée requière la dynamique du réseau de microtubules, via VgrG2b injectée par la machinerie H2-T6SS dans les cellules épithéliales, et capable de cibler les complexes de gamma-tubulines, centre nucléateur des microtubules. Il s'agit d'un mécanisme d'internalisation tout à fait original et jamais encore décrit. Nous avons également exploré la régulation croisée entre H2 et H3-T6SS. Enfin par une analyse de « RNAseq », nous avons observé entre les souches PAO1 et PA14 de P. aeruginosa une expression différentielle de nombreux gènes, certains codant des facteurs de virulence. Pour près de 30% d'entre eux, l'origine de cette variation reposerait sur une quasi-absence d'expression dans la souche PA14 de qslA, codant un anti-activateur du Quorum-Sensing. Ceci serait une nouvelle explication du phénotype hyper-virulent de la souche PA14 de P. aeruginosa. / Pseudomonas aeruginosa is an opportunistic extracellular pathogen. The PAO1 strain of P. aeruginosa harbors three loci encoding independent Type VI Secretion Systems (T6SS), named H1 to H3-T6SS. H1-T6SS has been widely described in the literature and injects toxins with antiprokaryotic activity in target bacteria, allowing an active defense against the attack of a T6SS from another bacterium. The other two T6SS of P. aeruginosa are likely virulence factors, but no secreted effector or intracellular target or role have been proposed so far.In this work, we demonstrated that the H2-T6SS machinery triggered the invasion of non-phagocytic epithelial cells and is involved in the virulence of P. aeruginosa. This uptake is mediated by the microtubule network dynamics, and via VgrG2b, injected by the H2-T6SS machinery in epithelial cells, to target the gamma-tubulin complex, the microtubule nucleating center. This is an original mechanism of internalization never described before. We also studied the cross-regulation between H2 and H3-T6SS. Finally through a « RNAseq » analysis, we observed differential expression of many genes, including some encoding virulence factors, between PAO1 and PA14 strains of P. aeruginosa. For nearly 30% of them, the origin of this variation is an almost lack of expression in the PA14 strain of qslA, encoding an anti-Quorum Sensing activator. This can be a new explanation of hyper-virulent phenotype of the PA14 strain of P. aeruginosa.
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Les régulateurs transcriptionnels Rgg de Streptococcus thermophilus LMG18311 : étude du rôle de la protéine Rgg0182 / Rgg transcriptional regulators of Streptococcus thermophilus LMG18311 : Study of the Rgg0182 protein roleBruneau, Emmanuelle 24 September 2013 (has links)
Cette thèse a pour objectif la caractérisation des gènes rgg de S. thermophilus LMG18311 codant des régulateurs transcriptionnels et l'étude de leur implication dans l'adaptation à l'environnement. Ce travail montre que le gène rgg0182 code un régulateur transcriptionnel activant la transcription de ses gènes adjacents. Par ailleurs, le couple Rgg0182/Shp0182 participerait à un mécanisme de quorum sensing. De plus, la protéine Rgg0182 participe à la tolérance au stress chaud. En outre, les cellules du mutant [delta]rgg0182 présentent un phénotype d'adhésion thermo-induite via des interactions de types hydrophobes. L'analyse par microscopie à force atomique des cellules de la souche LMG18311 et du mutant [delta]rgg0182 révèle la présence de polymères de surface uniquement chez la souche sauvage, suggérant que la protéine Rgg0182 régulerait l'expression de protéines de surface et de la division cellulaire. Une étude protéomique couplée à une analyse transcriptomique ont permis d'identifier plusieurs cibles de Rgg0182 qui participeraient à diverses fonctions biologiques. L'ensemble des données obtenues démontre que la protéine Rgg0182 de S. thermophilus LMG18311 est un régulateur global de l'expression génique. Par ailleurs, la transcription des 7 gènes rgg présents au sein du génome de S. thermophilus LMG18311 est modulée par les conditions environnementales. Les profils de transcription des 7 gènes rgg diffèrent les uns par rapport aux autres, suggérant que chacun d'eux seraient requis dans des conditions de croissances différentes. Ces données posent l'hypothèse que les protéines Rgg participeraient à la régulation fine et complexe de l'expression génique de S. thermophilus / This thesis aims to characterize the rgg genes of S. thermophilus LMG18311 coding transcriptional regulator and their involvement in environmental adaptation. This work shows that rgg0182 gene encodes a transcriptional regulator controlling the transcription of its flanking genes. The Rgg0182/Shp0182 pair could be involved in a quorum sensing mechanism. This work also demonstrates that the Rgg0182 protein is involved in S. thermophilus tolerance to heat stresses. In addition, the mutant delta rgg0182 cells exhibit a thermo-induced adhesion phenotype via hydrophobic interactions. Analyses by atomic force microscopy of LMG18311 cells of the wildtype and its derivative rgg0182 mutant reveal the presence of polymers only on the surface of the wild-type strain, suggesting that the protein Rgg0182 would regulate the expression of surface proteins and proteins of cell division. A proteomic study coupled with transcriptomic analysis led to the identification of several targets of Rgg0182 involving in various biological functions. The data obtained in this work have shown that the S. thermophilus LMG18311 rgg0182 genes encodes a global regulator of gene expression. Furthermore, transcriptional analyses, in different growth conditions, of the 7 rgg genes present in the genome of S. thermophilus LMG18311 showed that they display different expression profiles that are modulated by environmental conditions. This suggests that these genes would be required in distinct growth conditions. These data raise the hypothesis that Rgg proteins participate in the fine and complex regulation of S. thermophilus gene expression
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Envolvimento dos genes qseC e sdiA na formação de biofilme por Escherichia coli enteropatogênica atípica / Influence of qseC and sdiA gene in biofilm formation by atypical enteropathogenic Escherichia coliCuller, Hebert Fabricio, 1984- 27 August 2018 (has links)
Orientador: Marcelo Palma Sircili / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T01:00:20Z (GMT). No. of bitstreams: 1
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Previous issue date: 2015 / Resumo: As Escherichia coli enteropatogênicas atípicas são capazes de formar biofilme em superfícies abióticas e bióticas. Diversos mecanismos em E. coli são regulados por Quorum Sensing, incluindo a expressão de fatores de virulência e formação de biofilme. Quorum Sensing é um sistema de sinalização que confere às bactérias a habilidade de responder à moléculas químicas denominadas autoindutores (AI). SdiA e QseC são receptores de quorum sensing encontrados em diversas bactérias, entre elas EPECa. SdiA detecta moléculas autoindutoras do tipo 1 (AI-1) denominadas N-acil homoserina lactonas (AHLs). Entretanto as Escherichia coli não possuem a sintase para estas moléculas e, deste modo, SdiA detecta AHLs produzidas por outras bactérias. O receptor QseC detecta moléculas autoindutoras do tipo 3, além dos hormônios humanos adrenalina e noradrenalina. Neste estudo verificamos a influência da deleção de sdiA e qseC na formação e arquitetura do biofilme, formação de película, anel e também na transcrição de alguns dos genes envolvidos nestes fenótipos (bcsA, csgA, csgD, fliC, fimA e rpoS) em duas amostras de EPECa, sendo uma do sorotipo O55:H7 e outra do sorotipo ONT:H25. Os resultados das análises nas duas amostras de EPECa foram distintos, confirmando a grande heterogeneidade reportada em outros estudos em amostras deste patótipo. A amostra ONT:H25?sdiA formou espesso biofilme em placas de 96 poços e espessa estrutura (em anel) como anéis na parede do tubo de ensaio em relação às amostras selvagem e complementada. Além disso, o mutante sdiA desta amostra foi capaz de formar película na superfície do meio enquanto as amostras selvagem e complementada foram negativas. A amostra O55:H7?sdiA não apresentou diferença significativa na formação (das estruturas analisadas) destas estruturas em relação às amostras selvagem e complementada. Análises de qRT-PCR demonstraram (maiores níveis de transcrição de) um aumento na transcrição de csgA, csgD e fimA na amostra ONT:H25 deletada em sdiA, possivelmente indicando que o aumento na formação de biofilme por esta amostra esteja relacionado ao aumento da expressão das fímbrias tipo 1 e curli. A adição de AHLs à amostra selvagem ONT:H25 diminuiu a formação de biofilme e os níveis transcricionais de csgD e fimA, enquanto na amostra deletada não houve diferença, indicando que sdiA participa da regulação da formação de biofilme em EPECa e que as AHLs aumentam os efeitos repressores deste receptor nos genes relacionados à formação de biofilme. Quanto às amostras deletadas em qseC foi possível verificar uma diminuição da formação de biofilme em relação às amostras selvagens e complementadas, mas não houve diferença na formação de película na interface ar-líquido e também na formação de anel na parede dos tubos. A amostra ONT:H25?qseC foi negativa para expressão de fímbria curli em placas contendo vermelho congo e apresentou aumento da transcrição de bcsA e fimA, enquanto teve a transcrição de csgA, csgD e fliC diminuída em relação à amostra selvagem. A adição de adrenalina aumentou a formação de biofilme e motilidade nas duas amostras mutantes indicando a possível presença de outro receptor em EPECa responsável pela detecção destes hormônios na ausência de QseC. Portanto, conclui-se que estes dois receptores de quorum sensing estão relacionados, direta ou indiretamente, à formação de biofilme em EPECa / Abstract: Atypical enteropathogenic Escherichia coli are capable to form biofilm on biotic and abiotic surfaces. Several E. coli mechanisms are regulated by Quorum Sensing, including expression of virulence factors and biofilm formation. Quorum Sensing is a signaling system that confers bacteria the ability to respond to chemical molecules known as autoinducers. SdiA and QseC are Quorum Sensing receptors found in several bacteria, including aEPEC. SdiA detects type 1 autoinducer molecules (AI-1) known as N-acil homoserin lactones. However, Escherichia coli do not produce this kind of molecules and SdiA detects AHLs produced by other bacteria. QseC receptor detects type 3 autoinducer molecules and the human hormones adrenalin and noradrenaline. In this study the influence of the sdiA and qseC deletion in the biofilm formation and architecture, pellicle and ring-like structure formation and transcription of some genes (bcsA, csgA, csgD, fliC, fimA and rpoS) in two strains of aEPEC (O55:H7 and ONT:H25) was verified. The results of the analysis of the two aEPEC strains were distinct, confirming the heterogeneity reported by other studies in the same patotypes. The strain ONT:H25?sdiA formed a thick biofilm in 96-well plates and thick ring-like structure on the tube wall compared with the wild type and complemented strains. Furthermore, the sdiA mutant strain was capable to form pellicle on both surfaces, while the wild type and complemented strains were negative. The strain O55:H7?sdiA did not show a significant difference on the formation of these structures compared to the wild type and complemented strains. qRT-PCR analysis demonstrated an enhance on the transcription of csgA, csgD and fimA in the deleted sdiA ONT:H25 strain, suggesting that a relative increase of biofilm formation in ONT:H25 strain could be related to the increase of the expression of type 1 and curli fimbriae. The addition of AHLs to the wild type strain ONT:H25 decreased the biofilm formation and the transcriptional levels of csgD and fimA, but not in the mutant strain, indicating that sdiA plays a role in the regulation of biofilm formation in aEPEC. AHLs also increased the repressor efects of this receptor in biofilm related genes. The mutant qseC strains showed a decrease in biofilm formation when compared to wild type and complemented strains, but there was no difference in the pellicle formation in the air-liquid interface, as in the ring-like structure formation in the tube wall. The ONT:H25?qseC strain was negative to curli fimbriae expression in congo red plates and displayed an increase in the transcription of bcsA and fimA genes, while a decrease was verified in the csgA, csgD and fliC genes compared to the wild type strain. The addition of adrenalin increased the biofilm formation and motility in both mutant strains, possibly indicating the presence of other receptor in aEPEC involved in the detection of these hormones in the absence of QseC. In conclusion, these two Quorum Sensing receptors are related with biofilm formation in aEPEC / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular
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Inibição do sistema quorum sensing AI-1 por Capsicum frutescens e Capsicum annuum em bactérias Gram-negativas / Inhibition of AI-1 quorum-sensing system by Capsicum frutescens and Capsicum annuum in Gram-negative bacteriaMilagros Liseth Castillo Rivera 22 March 2018 (has links)
A inibição do quorum sensing (QS) altera a comunicação bacteriana, reduzindo a expressão de fatores de virulência e a formação de biofilmes, o que pode conferir menor pressão seletiva em comparação aos antibióticos tradicionais. As frutas e hortaliças constituem uma fonte rica em compostos com propriedades potenciais de inibição do QS. Entretanto, há pouca referência sobre o potencial de pimentas do gênero Capsicum e de seus compostos isolados como inibidores do QS. Esse trabalho teve como objetivo avaliar o efeito de extratos orgânicos obtidos das variedades de pimenta-malagueta e pimentão vermelho sobre o sistema QS dependente do sinalizador AI-1 (acil homoserina lactona - AHL) em bactérias Gram-negativas. Os extratos foram obtidos por extração em fase sólida e separados em uma fração metanólica e outra amônica; sendo os compostos característicos identificados e quantificados por cromatografia líquida de alta eficiência (CLAE). A atividade antimicrobiana dos extratos foi avaliada pela determinação da concentração inibitória mínima (MIC) e pela curva de crescimento de Chromobacterium violaceum ATCC 12472, Serratia liquefaciens MG1 e Pseudomonas aeruginosa PAO1. O efeito anti-QS dos extratos foi avaliado pelos testes de difusão em ágar e quantificação da produção de violaceína em meio líquido por C. violaceum e sobre a formação de biofilme, avaliado pelo ensaio de cristal violeta e microscopia confocal, em S. liquefaciens e P. aeruginosa nas temperaturas 30 ºC e 37 ºC. Os resultados obtidos pela CLAE indicaram que o extrato metanólico de pimenta-malagueta (EMPM) continha capsaicinoides como a capsaicina e dihidrocapsaicina, luteolina e outros compostos não identificados; já o extrato amônico desta não continha os compostos capsaicinoides. Ambos os extratos de pimentão vermelho continham luteolina e compostos não identificados, mas não apresentaram capsaicinoides. Como o EMPM era representativo dos demais extratos, por conter tanto capsaicinóides quanto luteolina, o foco deste trabalho foi avaliar os efeitos do EMPM sobre fenótipos microbianos nas concentrações 5; 2,5; 1,25 e 0,625 mg/ml, além de utilizar a capsaicina como controle comparativo em concentrações equivalentes às do extrato (25, 50 e 100 µg/ml). Os resultados da atividade antimicrobiana mostraram inibição parcial do crescimento das bactérias nas concentrações sub-MIC (MIC >5 mg/ml) de 5 e 2,5 mg/ml de EMPM. A capsaicina também inibiu parcialmente o crescimento das bactérias a 100 µg/ml, com exceção de S. liquefaciens a 37 ºC, cujo crescimento foi induzido em 50 e 25 µg/ml. A produção de violaceína foi reduzida pelo EMPM a 1,25 e 0,625 mg/ml, sem afetar o crescimento de C. violaceum. Ensaios com C. violaceum CV026, estirpe biosensora capaz de produzir o pigmento na presença de AI-1 exógeno, sugerem que o possível mecanismo de atuação do extrato sobre o sistema QS em C. violaceum 12472 é sobre a síntese do sinalizador, já que não foi observada inibição da produção de violaceína em CV026 pelo extrato. Contrariamente, a capsaicina incrementou a produção do pigmento na estirpe 12472, mas ensaios com a estirpe CV026 indicaram que a capsaicina não atua como sinalizador do QS, uma vez que esta não induziu a produção de violaceína nesta estirpe. Já a formação de biofilme foi incrementada na presença do EMPM, sendo consideravelmente maior em P. aeruginosa a 30 ºC. Igualmente, observou-se indução da formação de biofilme por capsaicina em S. liquefaciens (37 ºC) e P. aeruginosa (30 ºC). Porém, a capsaicina não teve efeito sobre a formação de biofilme de S. liquefaciens quando cultivada a 30 ºC, nem P. aeruginosa a 37 ºC. Os resultados revelam que a produção de violaceína em C. violaceum ATCC 12472 é inibida pelo EMPM, mas não pela capsaicina. Já, o EMPM e a capsaicina, de forma geral, não inibem a formação de biofilme de S. liquefaciens MG1 nem P. aeruginosa PAO1. Outros estudos são necessários para elucidar os mecanismos pelos quais o EMPM e a capsaicina agem sobre os fenótipos avaliados neste trabalho. / Quorum sensing inhibition alters bacterial communication by reducing virulence factors expression and biofilm formation, exerting less selective pressure compared to antibiotics. Fruits and vegetables are rich sources of compounds with potential QS-inhibition properties. However, there are few references about the potential of peppers belonging to the genus Capsicum and its isolated compounds as QS inhibitors. This study aimed to assess the effect of organic extracts obtained from Capsicum varieties, pimenta-malagueta (red chili) and pimentão vermelho (red bell pepper), on the AI-1 dependent QS system. The extracts were obtained by solid phase extraction and split into a methanolic and an ammonic fraction. Characteristic compounds were identified and quantified by high performance liquid chromatography (HPLC). The antimicrobial activity of the extracts was assessed by determining the minimal inhibitory concentration (MIC) and the growth curve of Chromobacterium violaceum ATCC 12472, Serratia liquefaciens MG1 and Pseudomonas aeruginosa PAO1. The anti-QS effect of the extracts was evaluated by the agar diffusion assay and the quantification of violacein production was assessed in liquid medium by C. violaceum, as well as in the biofilm formation test determined by the crystal violet assay and confocal microscopy with S. liquefaciens and P. aeruginosa at 30 ºC and 37 ºC. HPLC results showed that the methanolic extract of pimenta-malagueta (EMPM) contained capsaicinoids such as capsaicin and dihidrocapsaicin, luteolin and other unidentified compounds in lower concentrations; while its ammonic extract did not have capsaicinoids. Both pimentão vermelho extracts contained luteolin and other unidentified compounds in low concentrations, but they did not contain capsaicinoids. As EMPM was representative among the extracts because it contained capsaicinoids and luteolin, the focus of this work was to assess the effect of EMPM over microbial phenotypes at concentrations of 5, 2.5, 1.25 and 0.625 mg/ml, using capsaicin as a comparative control at equivalent concentrations to those in EMPM (25, 50 and 100 µg/ml). Antimicrobial activity assays showed a partial inhibition growth of bacteria at sub-MIC concentrations (MIC >5 mg/ml) of EMPM at 5 and 2.5 mg/ml. Similarly, capsaicin partially inhibited bacterial growth at 100 µg/ml, except for S. liquefaciens at 37 ºC in which growth was induced at 50 and 25 µg/ml. Violacein production was reduced by EMPM at 1,25 and 0,625 mg/ml without affecting C. violaceum growth. Assays with C. violaceum CV026, a biosensor strain that produces violacein in the presence of exogenous AI-1, suggest that EMPM reduced violacein production in C. violaceum 12472 by interfering with the AI-1 synthesis. In contrast, capsaicin incremented violacein synthesis in strain 12472, but experiments with strain CV026 revealed that capsaicin does not function as an analog of AI-1. Biofilm formation was increased in EMPM presence, being remarkably superior in P. aeruginosa cultivated at 30 ºC, as opposed to cultivation at 37 ºC. Similarly, capsaicin induced biofilm formation in S. liquefaciens (37 ºC) and P. aeruginosa (30 ºC). However, capsaicin did not affect biofilm formation on S. liquefaciens cultured at 30 ºC, neither on P. aeruginosa at 37 ºC. These results show that violacein production in C. violaceum ATCC 12472 is inhibited by EMPM, but not by capsaicin. In general, EMPM and capsaicin did not inhibit biofilm formation in S. liquefaciens MG1 neither in P. aeruginosa PAO1. More studies are necessary to elucidate the mechanisms by which EMPM and capsaicin affect the studied phenotypes in this work.
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Caractérisation d'interactions moléculaires entre P. aeruginosa et S. aureus co-isolés d'infections respiratoires chroniques chez les patients adultes atteints de fibrose kystiqueFugère, Alexandre January 2014 (has links)
La fibrose kystique est une maladie autosomale récessive multisystémique se manifestant principalement au niveau du système respiratoire et digestif. En raison d’une altération de la protéine régulatrice CFTR, l’équilibre ionique chez les cellules de l’hôte est déficient. Il en résulte, entre autres, une sécrétion et une accumulation d’un mucus pulmonaire très consistant. L’évacuation du mucus étant très fastidieuse chez les patients atteints de fibrose kystique, l’élimination des particules et bactéries des voies respiratoires est inadéquate. Cet aspect de la maladie participe grandement à sa progression, car l’établissement d’infections bactériennes chroniques contribue principalement à l’effet délétère sur la santé respiratoire des patients atteints de fibrose kystique.
La colonisation du mucus pulmonaire par les bactéries pathogènes est le sujet de nombreuses recherches extensives dans le contexte de la fibrose kystique. La présence d’une diversité microbienne dans les voies respiratoires est connue depuis longtemps, mais avec les techniques récentes d’analyse moléculaire, il a été constaté que celle-ci peut présenter une très grande richesse qui était préalablement non détectée par les méthodes d’isolement classiques. Les interactions et comportements adoptés par les populations polymicrobiennes du microbiome FK dans la progression de la maladie sont maintenant de grand intérêt. La virulence des principaux pathogènes en FK a préalablement et exhaustivement été décrite de façon spécifique. Parmi les plus notoires, comme Staphylococcus aureus, Pseudomonas aeruginosa et Burkholderia cepacia, certains utilisent des systèmes de communication cellulaire élaborés («quorum sensing») dans le but de réguler leur virulence.
De nouvelles voies de recherche sont construites sur la suggestion qu’une population polymicrobienne peut être perçue comme un seul agent infectieux. Notre étude a été conduite afin de documenter les possibles réponses interespèces entre S. aureus et P. aeruginosa suite à l’échange de molécules sécrétées dans le cadre du «quorum sensing». Pour ce faire, nous avons caractérisé un éventail d’interactions existantes entre des isolats cliniques des deux pathogènes d’intérêt. Nous avons également développé un modèle nous permettant de mieux comprendre l’impact de la coexistence sur leur pathogenèse. Inévitablement, l’étude vise à définir l’instance de la présence simultanée des deux bactéries dans l’établissement d’infections chroniques chez les patients atteints de fibrose kystique ainsi que sur leur santé respiratoire.
Cette étude tente ultimement de contribuer à l’effort visant à comprendre l’importance des interactions polymicrobiennes dans un contexte infectieux dans le but de trouver de nouveaux indices quant à l’élaboration de stratégies thérapeutiques.
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Structural studies of Vibrio cholerae quorum sensing proteinsJahan, Nasrin January 2011 (has links)
The spread of cholera is always associated with contaminated food or water and this is the reason this disease has been endemic in developing countries for centuries due to their lack of proper sanitation facilities and poor or no infrastructure for sewage systems. Cholera can spread quickly and sporadically after any natural disaster that destroys the sewage system or safe drinking water supply of both developed and undeveloped countries. In Southeast Asia in December 2004 and in Pakistan and Haiti 2010, cholera outbreaks followed the natural disasters; with most of the cholera victims being children. Although it is known that the best way to prevent cholera outbreak is the development of the infrastructure, provision of a safe drinking water supply and proper sanitation, this is a very long-term process, and most of the developing countries cannot afford such improvements. These situations can be made worse by natural disasters. Therefore there is a pressing need for the development of a cholera vaccine and there have been numerous research projects working towards this end for several decades. A few of them have been successful to date but because of the severe side effects and narrow range of protection, more effective and wider range vaccine development is still ongoing. In this study, crystallographic and enzymatic studies have been carried out on several novel proteins involved in the control of the production of the factors required for quorum sensing. Quorum sensing is a process in which bacterial cells communicate among themselves by the synthesis, release and detection of small chemical compounds called autoinducers. In this work, structural analysis was carried out on proteins involved in the synthesis and detection of the major autoinducer of Vibrio cholerae, named CAI-1. The crystal structure of CqsA involved in CAI-1 synthesis has been successfully solved and its enzymatic properties have been characterized. The structure of one domain of the cytoplasmic region of the CAI-1 receptor CqsS was also elucidated, and other domains were expressed. The crystal structure of another enzyme (VCA0859, an aldo-keto reductase) thought to have been involved in the synthesis of CAI-1 was also determined. Another protein named VCA0939 was also studied, due to its importance in biofilm development, and its ability to control quorum-sensing in an alternative pathway in the mutated version of pathogenic strains of V. cholerae that were responsible for the seventh cholera pandemic. The aim of this project was to understand the three dimensional structure of some proteins that are involved in quorum sensing and control of the expression of virulence genes for the pathogenesis of V. cholerae. Understanding the three dimensional structure of the proteins and the mode of autoinducer binding to its specific receptor could be highly valuable in the development of a chemical compound that could lead to the discovery of a novel drug with the ability to target cross species specification.
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Exclusion mutuelle de groupes dans les systèmes distribuésThiare, Ousmane 25 June 2007 (has links) (PDF)
L'exclusion mutuelle de groupe est une généralisation de l'exclusion mutuelle. Ce problème a été présenté par Joung et quelques algorithmes pour le problème ont été proposés en incorporant ds algorithmes d'exclusion mutuelle. L'exclusion mutuelle de groupe se produit naturellement dans une situation ou une ressource peut être partagée par des processus du même groupe, mais pas par des processus de différents groupes. Il est aussi appelé "problème des philosophes parlant d'une même voix". <br />Un exemple d'application intéressant est un serveur CD (documentation, films...) sur Internet: plusieurs utilisateurs désirant accéder au même CD pourront le faire en même temps au lieu d'attendre la fin de la requête des autres utilisateurs. Des solutions efficaces, écrites dans le modèle à passage de messages et basées sur les quorums d'une part et sur la circulation de jeton d'autre part sont présentées dans cette thèse.<br />Nous abordons aussi le problème de l'exclusion mutuelle de groupe sur les réseaux mobiles ad hoc et nous proposons un algorithme qui est sensible aux formations et coupures de liens et est ainsi approprié pour les réseaux mobiles ad hoc.
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Growth, Vibriosis, and Streptococcosis Management in Shrimp-Tilapia Polyculture Systems, and the Role of Quorum Sensing Gene cqsS in Vibrio harveyi VirulenceNaim, Sidrotun January 2012 (has links)
Tilapia culture in Indonesia was started with the Mozambique Tilapia (Oreochromis mossambicus) in the 1930’s, and the Nile Tilapia (Oreochromis niloticus) the 1960’s. The genetic improvement program of the Nile Tilapia, has led Indonesia to be one of the main tilapia producers in the world. On the other hand, shrimp aquaculture in the country was not started until the 1960’s, it became more popular after the eye ablation technology for broodstock maturation was developed in the early 1980’s. The first experimental study was conducted to investigate the feasibility of low salinity shrimp farming in a polyculture system with tilapia. Polyculture increased the survival for shrimp (77% compared to 62%), but at the same time decreased the survival of tilapia (87% compared to 97%). Together, the data on survival, specific growth rates, and feed conversion ratios showed that the shrimp performed well at low salinity. The second experimental study investigated the feasibility of brackishwater shrimp farming in a polyculture system with tilapia. Polyculture increased the survival for shrimp (82% compared to 65%), and had higher survival for the tilapia (60% compared to 43%). The Red hybrid Tilapia strain used in the study experienced mortalities after one month, suggesting the need for a salt tolerant strain. The presence of tilapia stimulated the growth of microalgae (Chlorella dominance), promoted higher numbers of heterotrophic bacteria in the water, and had lower presumptive vibrios on TCBS agar. A challenge study was conducted by mixing pathogenic luminescent Vibrio harveyi UAZ-651 into shrimp and tilapia feed. The survival of shrimp in monoculture were significantly lower (20%) compared to in polyculture systems (75 - 95%). Mortality was not found in tilapia. Based on 16S rRNA gene sequence, shrimp monoculture water was dominated by marine Vibrio spp., while the polyculture system had Bacillus spp. and Vibrio spp. with high homology to V. cholerae. The presence of Bacillus spp. which produce a lactonase enzyme AiiA, seems to inhibit vibrio growth. While providing advantages, shrimp-tilapia polyculture might also contribute to streptococcosis transmission. Injecting shrimp with Streptococcus iniae and S. agalactiae resulted in mortalities. S. iniae caused higher mortality in the shrimp cultured in 20 ppt (40%) compared to 10 ppt (20%), and no mortality in 5 ppt. S. agalactiae caused higher mortality in 5 ppt (40%) compared to 10 ppt (20%) and 20 ppt (20%). Quorum sensing (QS) is a density dependent cell to cell communication process in bacteria. Based on challenge studies in shrimp, the luminescent Vibrio harveyi BB120 wild-type strain caused 75-90 % mortality through injection of 106 CFU/shrimp. The mortality patterns in the QS mutants suggest that QS defined, when specific virulence genes were expressed or repressed. As QS in V. harveyi consists of three different circuits, further experiments deployed six mutants lacking either a synthase or a receptor for each circuit. The highest survival in the CqsS (a receptor for CAI-1 circuit) mutant group indicates that the CAI-1 circuit is the most crucial for virulence, followed by the AI-2 and HAI-1 cascades. Chitin acquisition and oxygen scavenging may be two reasons for luminescence in V. harveyi evolution and why they infect shrimp.
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La réponse au Farnésol de Candida albicans : production de biofilms et Parenté génétiqueRoss, Jean-françois 03 1900 (has links)
C. albicans est une levure pathogène opportuniste. Elle est un agent causal fréquent des infections muco-cutanées. C. albicans peut alterner entre la forme blastospore et mycélium. Cette dernière forme est impliquée dans la formation des biofilms. Le dimorphisme de C. albicans est contrôlé en partie par le phénomène de perception du quorum (quorum sensing) qui en autre, est associé à la molécule farnésol, produit par cette levure. La présence de cette molécule, inhibe la formation d’hyphe par C. albicans et par conséquent limite la formation de biofilms. Certaines souches ne répondent pas au farnésol et nous avons vérifié les hypothèses que : 1) la proportion des Non-Répondeurs (NR) au farnésol est similaire entre les souches de provenance orale et vaginale; 2) la capacité de formation de biofilm varie d’une souche à une autre mais les Non-Répondeurs en produisent en plus grande quantité; 3) la technique RAPD-PCR permettra de regrouper les souches de cette levure suivant leur provenance, leur capacité de formation de biofilm et leur aptitude à répondre au farnésol. La découverte d’une souche vaginale Non-Répondeur nous permet de croire que la proportion de ces souches est similaire entre les deux types de provenance. Les souches caractérisées Non-Répondeurs produisent 30 % plus de biofilm que les Répondeurs en absence de farnésol exogène dans le milieu. En présence de farnésol exogène, les Non-Répondeurs produisent 70 % plus de biomasse que les Répondeurs. De plus, la technique RAPD ne nous a pas permis de classer nos souches d’après les caractéristiques proposées.
En conclusion, les souches orales de C. albicans semblent produire en moyenne plus de biomasse que les souches vaginales. Aussi, les NR semblent moins affectés par la présence du farnésol, ce qui pourrait causer la présence de biofilms tenaces. Les amorces utilisées pour la technique RAPD n’ont pas été efficace à la classification des souches dépendamment de leur provenance, de leur capacité à former un biofilm et à répondre au farnésol.
Mots clés : Candida albicans, biofilm, perception du quorum (quorum sensing), farnésol, Non-Répondeur / The opportunistic dimorphic yeast Candida albicans is frequently associated with mucosal infections in humans. It can colonize both oral and vaginal mucous membranes. One of its virulence factors is the capacity to form or participate in biofilms formation. Biofilms are a community of bacteria embedded in a matrix of exopolysaccharide and provide protection against environmental stress and antibiotics. The dimorphic characteristic of this yeast is involved in biofilm formation and is controlled in part by the quorum sensing system. Recently, it has been shown that farnesol, a quorum sensing molecule, can control the formation of biofilms through the inhibition formation of hyphae. However, we have shown that some oral and vaginal strains can still form biofilms under the influence of exogenous farnesol and these strains were named Non-Responders (NR). We hypothesized that: 1) The proportion of Non-Responders is similar between oral and vaginal strains; 2) biofilm formation differs between strains but Non-Responders produce more of it; 3) The technique of RAPD-PCR will allow us to group strains according to their origin, biofilm formation and response to farnesol. The discovery of a NR vaginal strain leads us to believe that the proportion of NR is similar between oral and vaginal strains. The NR strains seem to produce 30 % more biofilm than Responders in media without exogenous farnesol. This percentage increased to 70 % when exogenous farnesol was added to the media. The RAPD technique did not allow us to group strains according to their proposed characteristic.
In conclusion, oral strains seem to produce more biofilm than vaginal strains. The NR’s are less affected by farnesol and may cause persistent biofilms. The primers used for RAPD technique were not effective to discriminate C. albicans strains according to origin, capacity to form biofilms and farnesol responding.
Key words: Candida albicans, biofilm, quorum sensing, farnesol, Non-Responders
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