Submitted by Anderson Silva (avargas@icict.fiocruz.br) on 2012-10-26T17:48:32Z No. of bitstreams: 1 clarissa_c_menezes_ioc_bcm_0054_2011.pdf: 2530597 bytes, checksum: 62f04a75c90de18a85463693ba14fb9b (MD5) / Made available in DSpace on 2012-10-26T17:48:32Z (GMT). No. of bitstreams: 1 clarissa_c_menezes_ioc_bcm_0054_2011.pdf: 2530597 bytes, checksum: 62f04a75c90de18a85463693ba14fb9b (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / A bactéria Pseudomonas aeruginosa (PA) é um dos principais agentes etiológicos de pneumonias nosocomiais, cujo tratamento é dificultado por sua resistência a antibióticos e pela secreção de fatores de virulência. Novas alternativas para o tratamento destas infecções incluem manipular o sistema de comunicação bacteriano conhecido como quorum sensing (QS), reduzindo a expressão destes fatores de virulência e prevenindo seus efeitos deletérios em células e sistemas eucariotos. Os efeitos do componente de QS de PA 3-oxo dodecanoil homoserina lactona (3-oxo C12 HSL) nos pulmões não são conhecidos; portanto, a primeira etapa de nosso trabalho consistiu na caracterização dos efeitos desta molécula no ambiente pulmonar. Para isso, camundongos swiss desafiados com 3-oxo C12 HSL por via intratraqueal tiveram amostras de sangue, lavado bronco-alveolar (BAL) e tecido pulmonar e coletadas seis horas após o procedimento. Nossos resultados demonstram que inflamação pulmonar causada por 3-oxo C12 HSL se caracteriza pela migração de células mononucleares e neutrófilos para o espaço alveolar, níveis elevados de atividade mieloperoxidase no tecido pulmonar e formação de edema. A resposta inflamatória causada por 3-oxo C12 HSL não alterou os níveis de TNF-α, MIF, IL- 10 e IL-12 no tempo analisado. Foram observados aumentos nos níveis de IL-6, CCL2/MCP-1, CXCL1/KC e LTB4 no BAL de animais desafiados, sendo o aumento deste eicosanóide acompanhado por uma indução de corpúsculos lipídicos. Evidências in vitro relatam o envolvimento do receptor nuclear PPARγ nos efeitos pró-inflamatórios atribuídos a 3-oxo C12 HSL; portanto, decidimos estudar os efeitos do tratamento com o agonista de PPARγ rosiglitazona no modelo de inflamação pulmonar causado por este componente de QS. Nós observamos que o tratamento com rosiglitazona (0,5 mg/kg) uma hora após o estímulo diminuiu a formação de corpúsculos lipídicos e de edema pulmonar, causando também uma redução na migração de células mononucleares e neutrófilos e uma menor atividade mieloperoxidase no pulmão dos animais tratados. A redução da migração de células mononucleares parece estar associada à uma redução dos níveis de CCL2/MCP-1, enquanto o decréscimo nos neutrófilos parece envolver a modulação de CXCL1/KC. A instilação com 3-oxo C12 HSL diminuiu a expressão da enzima paraoxonase (PON) no tecido pulmonar, fenômeno que não foi revertido pelo tratamento com rosiglitazona nesta dose. Estudos demonstram que o PPARγ está envolvido na expressão de PON e que a superexpressão desta enzima é capaz de proteger animais da mortalidade por PA em função de sua atividade lactonase; por este motivo, decidimos testar uma dose maior de rosiglitazona (5 mg/kg) para alcançar mais um benefício nesta proposta terapêutica. O tratamento com rosiglitazona nesta dose foi capaz de reverter a redução da expressão de paraoxonase causada por 3-oxo C12 HSL, diminuindo a formação de edema, a migração de neutrófilos e os níveis de atividade mieloperoxidase no pulmão de animais desafiados. O número de células mononucleares recuperado no BAL de animais estimulados e tratados com a droga não foi reduzido, bem como os níveis de CCL2/MCP-1. De fato, a droga por si só causou um aumento de CCL2/MCP-1 que parece ter contribuído para a indução de corpúsculos lipídicos observada. Devido ao envolvimento de macrófagos e da quimiocina CCL2/MCP-1 no processo de eliminação bacteriana, estudos em modelos de pneumonia por PA precisam ser conduzidos para melhor avaliar esta dose de rosiglitazona e validar esta droga como uma estratégia terapêutica no combate à esta bactéria. / Pseudomonas aeruginosa (PA) is a major pathogen involved in nosocomial pneumonia. These infections are difficult to manage due bacterial antibiotic resistance and secretion of virulence factors; therefore, current approaches to treat PA infections now focus on manipulating the bacteria communication system known as quorum sensing in order to reduce the production of those virulence factors and mitigate their deleterious effects to the host. Since there are no reports about the effects of the PA main quorum sensing component 3-oxo dodecanoyl homoserine lactone (3-oxo C12 HSL) in the lung the first step of our work consisted in investigating the host pulmonary response to this molecule in a mouse model. In order to do so, swiss mice submitted to intratracheal instillation with 3- oxo C12 HSL had blood, BAL and lung tissue samples collected six hours after the procedure. Our results show that the inflammatory response elicited by 3-oxo C12 HSL is characterized by the migration of both mononuclear cells and neutrophils to the alveolar space, together with high levels of myeloperoxidase activity in lung tissue and development of pulmonary edema. We found that the levels of TNF-α, MIF, IL-10 and IL-12 were not altered, while there was a significant increase in IL-6 CCL2/MCP-1 and CXCL1/KC in BAL. The increase in LTB4 levels in the BAL of defied animal was accompanied by an induction of lipid bodies. In vitro evidences report the involvement of the nuclear receptor PPARγ in the inflammatory events ascribed to 3-oxo C12 HSL; therefore, we decided to study the effects of PPARγ agonist rosiglitazone in the pulmonary inflammation caused by this quorum sensing component. We observed that rosiglitazone administration (0,5 mg/kg) one hour after stimulation was able to reduce the formation of lipid bodies and pulmonary edema, besides decreasing the migration of mononuclear cells, neutrophils and myeloperoxidase activity in the lung of defied animals. This reduction in the migration of mononuclear cells seems to be due to a decrease in the levels of CCL2/MCP-1, while the impairment in neutrophil migration seems to involve the modulation of CXCL1/KC. 3-oxo C12 HSL instillation also decreased paraoxonase (PON) expression in lung tissue, which was not reverted by rosiglitazone treatment at this dose. Since it was reported that PPARγ is able to regulate PON expression and that the overexpression of this enzyme seems to protect animals from PA mortality due its lactonase activity we searched to modulate this enzyme with a higher dose of rosiglitazone (5 mg/kg) in order to achieve this extra benefit in our proposal. We observed that this dose was able to revert the decrease in PON expression caused by 3-oxo C12 HSL, reducing the pulmonary edema, neutrophil migration and myeloperoxidase activity in defied animals. There was no reduction in the number of mononuclear cells recovered from the BAL of stimulated animals, which was also observed for CCL2/MCP-1 levels. In fact, the drug itself at his dose caused an increase in CCL2/MCP-1 that possibly accounted for the induction of lipid bodies. Because macrophages and CCL2/MCP-1 have a crucial role in the clearance of bacteria, this dose of rosiglitazone has to be tested in a PA pneumonia model so the effects observed in our investigation can be validate rosiglitazone as an therapeutic approach to the treatment of these infections.

Staphylococcus aureus é reconhecidamente causador de infecções humanas e um dos principais patógenos alimentares. O regulador gene acessório (Agr) é um sistema central que controla a expressão de diversos fatores de virulência. O polimorfismo do locus agr divide S.aureus em grupos genéticos distintos associados a determinados perfis patogênicos. O objetivo deste estudo foi comparar fenotipicamente e genotipicamente isolados clínicos e alimentares de S.aureus. Um total de 63 S.aureus (34 isolados de cateter venoso central (CVC) e 29 de carne de frango) foram selecionados para o estudo. O gene sea foi encontrado em 44,1% (15/34), seb em 5,9% (2/34) e sec em 8,8% (3/34) dos isolados de CVC. Em isolados de frango, apenas o gene sea foi detectado e foi observado em 31% (9/29). Somente 6% (2/34) dos isolados de CVC formaram biofilme. Já, em isolados de frango, 93% (27/29) foram formadores de biofilme (P<0,01). Nos isolados de CVC, os genes icaA, atlA e sasG envolvidos com a formação de biofilme estiveram presentes em 100% (34/34), 97% (33/34) e 100% (34/34), das amostras respectivamente, enquanto que, em isolados de carne de frango os mesmos genes foram detectados em 93% (27/29), 52% (15/29) e 100% (29/29), respectivamente. Cepas resistentes à penicilina foram detectas em 86,2% dos isolados de carne de frangos e em 88,2%, dos isolados de CVC. Isolados de CVC foram resistentes a oxacilina e multirresistentes em 17,6% (6/34). Com relação ao polimorfismo do locus agr, os S.aureus isolados de CVC foram dividos em 3 grupos, onde 44% (15/34) tiveram o polimorfismo I, 38% (13/34) o II e 18% (6/34) o III. Os S.aureus isolados de carne de frangos, também foram divididos em 3 grupos, onde 86% (25/29) dos isolados tiveram o polimorfismo I, 10% (3/29) o II e 4%o III (1/29) (P<0,05). Entre os isolados de CVC, 88,2% (30/34) produziram a δ-hemolisina, ou seja, expressavam o sistema Agr, já entre os isolados de frangos, apenas 3,3% eram δ-hemoliticos (P<0,05). Conclui-se que os isolados diferiram quanto à capacidade de formação de biofilme, polimorfismo Agr e produção da δ-hemolisina. No entanto, resultados similares foram observados quanto à detecção dos genes das enterotoxinas e os envolvidos com formação de biofilme. A análise molecular mostrou uma elevada similaridade entre os grupos o que pode indicar uma mesma origem destes isolados. / Staphylococcus aureus is recognized as cause of human infections and a one of the important foodborne pathogens. The accessory gene regulator (Agr) is a central system that controls the expression of several virulence factors. The polymorphism of the agr locus divided S. aureus into distinct genetic group associated with determinats of pathogenic profiles. The aim of this study was to the phenotypic and genotypic patterns of S.aureus isolated from clinical and food samples. A total of 63 S. aureus (34 from central venous catheter (CVC) and 29 from poultry meat) were selected for the study. The sea gene was found in 44.1% (15/34), seb in 5.9% (2/34) and sec in 8.8% (3/34) of CVC isolates. In poultry meat isolates, only sea gene was detected and it was observed in 31% (9/29). Only 6% (2/34) of CVC isolates formed biofilm. Further, 93% (27/29) of poultry meat isolates were biofilm formers (P<0.01). In CVC isolates, the icaA, atlA and sasG genes involved in biofilm formation were present in 100% (34/34), 97% (33/34) and 100% (34/34) of the isolates respectively, while in poultry meat isolates the same genes were detected in 93% (27/29), 52% (15/29) and 100% (29/29), respectively.Strains penicillin resistant was detected in 86.2% of poultry meat and 88.2 % CVC isolates. CVC isolates were also oxacillin resistant and multiresistants in 17.6% (6/34). In regard to the polymorphism of agr locus, the CVC isolates were divided into 3 groups, where 44% (15/34) belong to polymorphism I, 38% (13/34) to II and 18% (6/34) to III. The S. aureus strains isolated from poutry meat were also divided into 3 groups, where 86% (25/29) showed the polymorphism I, 10% (3/29) the II and 4% (1/29) the III (P<0.05). Among the CVC isolates 88.2% (30/34) produced δ-hemolysin, i.e., expressed the Agr system, in addition among the poultry meat isolates, only 3.3% were δ-hemolytic (P<0.05). In conclusion, S.aures isolated from CVC and poultry meat showed differences in their ability to form biofilm, Agr polymorphism and production of δ-hemolysin. In conclusion, S.aures isolated from CVC and poultry meat showed differences in their ability to form biofilm, Agr polymorphism and production of δ-hemolysin. However, similar results were observed in relation to enterotoxin genes and those involved in biofilm formation.The molecular analysis show high genotypic similarity to S.aures isolated from CVC and poultry meat, suggesting a common origin of these isolates, possible the human microbiota.

Natural products have long been known for their broad range of useful therapeutic properties, and have been widely utilised in the field of medicine. This dissertation describes work towards the total synthesis of natural products possessing biological activity in two important areas. The first section concerns the total synthesis of six 4-quinolone natural products, four of which had never been synthesised before. These compounds were originally isolated from a soil bacterium of the genus Pseudonocardia, and bear intriguing structural resemblance to the Pseudomonas Quinolone Signal. This signalling molecule is vital to the quorum sensing activity of the human pathogen Pseudomonas aeruginosa, which is a phenomenon by which it regulates many of its virulence factors. These natural products possess the potential to disrupt this system, hence attenuating the pathogenicity of the bacteria. The routes that were developed are highly divergent, efficiently giving access to multiple natural products from mutual late stage intermediates. Key steps included regioselective epoxidation, palladium-catalysed heterocylisation and acid catalysed 1,3-transposition of an allylic alcohol. In the second section, attention turns towards the total synthesis of the complex sesquiterpene lactone 1β-Hydroxyalantolactone. The compound possesses five stereogenic centres, one of which is quaternary, alongside a challenging tricyclic core scaffold. Previous biological studies have revealed a range of intriguing properties, including anti-inflammatory and anti-tumour activity. The chosen route utilises as its key step the catalytic desymmetrisation of a diene which was itself accessed by Birch reduction chemistry. Whilst the synthesis is as yet incomplete, access was granted to a key intermediate encompassing around half of the stereocentres present in the natural product.

Staphylococcus aureus é reconhecidamente causador de infecções humanas e um dos principais patógenos alimentares. O regulador gene acessório (Agr) é um sistema central que controla a expressão de diversos fatores de virulência. O polimorfismo do locus agr divide S.aureus em grupos genéticos distintos associados a determinados perfis patogênicos. O objetivo deste estudo foi comparar fenotipicamente e genotipicamente isolados clínicos e alimentares de S.aureus. Um total de 63 S.aureus (34 isolados de cateter venoso central (CVC) e 29 de carne de frango) foram selecionados para o estudo. O gene sea foi encontrado em 44,1% (15/34), seb em 5,9% (2/34) e sec em 8,8% (3/34) dos isolados de CVC. Em isolados de frango, apenas o gene sea foi detectado e foi observado em 31% (9/29). Somente 6% (2/34) dos isolados de CVC formaram biofilme. Já, em isolados de frango, 93% (27/29) foram formadores de biofilme (P<0,01). Nos isolados de CVC, os genes icaA, atlA e sasG envolvidos com a formação de biofilme estiveram presentes em 100% (34/34), 97% (33/34) e 100% (34/34), das amostras respectivamente, enquanto que, em isolados de carne de frango os mesmos genes foram detectados em 93% (27/29), 52% (15/29) e 100% (29/29), respectivamente. Cepas resistentes à penicilina foram detectas em 86,2% dos isolados de carne de frangos e em 88,2%, dos isolados de CVC. Isolados de CVC foram resistentes a oxacilina e multirresistentes em 17,6% (6/34). Com relação ao polimorfismo do locus agr, os S.aureus isolados de CVC foram dividos em 3 grupos, onde 44% (15/34) tiveram o polimorfismo I, 38% (13/34) o II e 18% (6/34) o III. Os S.aureus isolados de carne de frangos, também foram divididos em 3 grupos, onde 86% (25/29) dos isolados tiveram o polimorfismo I, 10% (3/29) o II e 4%o III (1/29) (P<0,05). Entre os isolados de CVC, 88,2% (30/34) produziram a δ-hemolisina, ou seja, expressavam o sistema Agr, já entre os isolados de frangos, apenas 3,3% eram δ-hemoliticos (P<0,05). Conclui-se que os isolados diferiram quanto à capacidade de formação de biofilme, polimorfismo Agr e produção da δ-hemolisina. No entanto, resultados similares foram observados quanto à detecção dos genes das enterotoxinas e os envolvidos com formação de biofilme. A análise molecular mostrou uma elevada similaridade entre os grupos o que pode indicar uma mesma origem destes isolados. / Staphylococcus aureus is recognized as cause of human infections and a one of the important foodborne pathogens. The accessory gene regulator (Agr) is a central system that controls the expression of several virulence factors. The polymorphism of the agr locus divided S. aureus into distinct genetic group associated with determinats of pathogenic profiles. The aim of this study was to the phenotypic and genotypic patterns of S.aureus isolated from clinical and food samples. A total of 63 S. aureus (34 from central venous catheter (CVC) and 29 from poultry meat) were selected for the study. The sea gene was found in 44.1% (15/34), seb in 5.9% (2/34) and sec in 8.8% (3/34) of CVC isolates. In poultry meat isolates, only sea gene was detected and it was observed in 31% (9/29). Only 6% (2/34) of CVC isolates formed biofilm. Further, 93% (27/29) of poultry meat isolates were biofilm formers (P<0.01). In CVC isolates, the icaA, atlA and sasG genes involved in biofilm formation were present in 100% (34/34), 97% (33/34) and 100% (34/34) of the isolates respectively, while in poultry meat isolates the same genes were detected in 93% (27/29), 52% (15/29) and 100% (29/29), respectively.Strains penicillin resistant was detected in 86.2% of poultry meat and 88.2 % CVC isolates. CVC isolates were also oxacillin resistant and multiresistants in 17.6% (6/34). In regard to the polymorphism of agr locus, the CVC isolates were divided into 3 groups, where 44% (15/34) belong to polymorphism I, 38% (13/34) to II and 18% (6/34) to III. The S. aureus strains isolated from poutry meat were also divided into 3 groups, where 86% (25/29) showed the polymorphism I, 10% (3/29) the II and 4% (1/29) the III (P<0.05). Among the CVC isolates 88.2% (30/34) produced δ-hemolysin, i.e., expressed the Agr system, in addition among the poultry meat isolates, only 3.3% were δ-hemolytic (P<0.05). In conclusion, S.aures isolated from CVC and poultry meat showed differences in their ability to form biofilm, Agr polymorphism and production of δ-hemolysin. In conclusion, S.aures isolated from CVC and poultry meat showed differences in their ability to form biofilm, Agr polymorphism and production of δ-hemolysin. However, similar results were observed in relation to enterotoxin genes and those involved in biofilm formation.The molecular analysis show high genotypic similarity to S.aures isolated from CVC and poultry meat, suggesting a common origin of these isolates, possible the human microbiota.

Submitted by (edna.saturno@ufrpe.br) on 2016-12-14T18:24:04Z No. of bitstreams: 1 Flaviana Goncalves da Silva.pdf: 1354445 bytes, checksum: 5abe875dd282d6291759e442aef4933a (MD5) / Made available in DSpace on 2016-12-14T18:24:04Z (GMT). No. of bitstreams: 1 Flaviana Goncalves da Silva.pdf: 1354445 bytes, checksum: 5abe875dd282d6291759e442aef4933a (MD5) Previous issue date: 2014-07-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Salinity is a limiting factor for agriculture and a frequent problem in arid and semi-arid region which rainfall is low and poorly distributed. In these areas most of plants can not grow due soils unprotected and become degraded with time. The use of bacteria with plant growth promoting and tolerance to salt stress may indicate biotechnological alternative that allows the use of plants associated with these microorganisms as inoculants may provide beneficial effects on soil-plant interaction. The cultivation of Atriplex nummularia L. has been conducted in order revegetate soils, promoting the improvement of their physical and chemical properties as a phytoremediation technique of salinized soils. In order to isolate and select bacteria promoter of plant growth associated with Atriplex nummularia L. plants cultivated in two experiments were carried out in the Pernambuco state and evaluated the effects of its inoculation in Atriplex plants grown in greenhouse. The population density of the bacteria was determined and then the same were tested in respect to plant growth promotion in vitro solubilization of inorganic phosphate (SFI), biological nitrogen fixation (BNF), synthesis of indole acetic acid (IAA), exopolysaccharides production (EPS) and quorum sensing molecule. Some bacteria to plant inoculation Atriplex grown in a protected environment were also selected in order to analyses content of chlorophyll a, b and total; stomatal conductance (gs); leaf temperature; green matter, dry and total fractional parts (root, stem and leaf) of plants; content and levels of sodium, potassium, calcium and magnesium; total nitrogen, crude protein of leaves and total organic carbon. Through the insulation, it was obtained 107 halotolerant bacterial isolates with positive results to plant growth promotion. Regarding the content of chlorophyll a, b and total, stomatal conductance and crude protein in plants, there was no effect of treatments. Inoculation with halotolerant bacteria and plant growth promoters influenced total nitrogen and total organic carbon in plants of Atriplex. Therefore, there are halotolerant bacteria associated with Atriplex plants able to solubilize inorganic phosphate, N2 fixation, IAA production, EPS and quorum sensing molecule, with the possibility of these micro-organisms contribute positively to plant growth. Bacterial isolates are promising on vegetative and nutritional development of Atriplex. However, it requires further explore the effect of bacterial inoculants associated with halophytes, giving improved conditions for phytoremediation process of salinized soils. / A salinidade constitui um fator limitante à agricultura e tem se tornado um problema frequente em áreas sob clima árido e semiárido, onde as precipitações são reduzidas e mal distribuídas. Nessas áreas, a maioria das plantas não consegue se desenvolver, por isso, os solos ficam desprotegidos e tornam-se degradados com o tempo. A utilização de bactérias promotoras de crescimento vegetal com tolerância ao estresse salino pode indicar alternativa biotecnológica que possibilite o uso de plantas associadas a esses micro-organismos como inoculantes, podendo proporcionar efeitos benéficos na interação solo-planta. O cultivo da Atriplex nummularia L. tem sido realizado com o objetivo de revegetar estes solos, promovendo a melhoria de suas propriedades físicas e químicas, como técnica de fitorremediação de solos afetados por sais. Com isso, objetivou-se isolar e selecionar bactérias promotoras de crescimento vegetal associadas às plantas de Atriplex nummularia L. cultivadas em dois experimentos instalados no estado de Pernambuco e avaliar os efeitos da inoculação destas bactérias em plantas de Atriplex cultivadas em ambiente protegido. Foi determinada a densidade populacional das bactérias e em seguida as mesmas foram testadas quanto às características de promoção de crescimento vegetal in vitro: solubilização de fosfato inorgânico (SFI), fixação biológica de nitrogênio (FBN), síntese de ácido indol acético (AIA), produção de exopolissacarídeo (EPS) e molécula quorum sensing. Foram também selecionadas algumas bactérias para inoculação em plantas de Atriplex cultivadas em ambiente protegido, analisando-se nas plantas, aspectos como teor de clorofila a, b e total; condutância estomática (gs); temperatura foliar; fitomassa verde, seca e total das partes fracionadas (raiz, caule e folha) das plantas; conteúdos e teores de sódio, potássio, cálcio e magnésio; nitrogênio total, proteína bruta de folhas e carbono orgânico total. Por meio do isolamento, foi possível obter 107 isolados bacterianos halotolerantes, com resultados positivos quanto às características de promoção de crescimento vegetal. Em relação ao teor de clorofila a, b e total, condutância estomática e proteína bruta nas plantas, não houve efeito dos tratamentos aplicados. A inoculação com bactérias halotolerantes e promotoras de crescimento vegetal influenciou o nitrogênio total e carbono orgânico total em plantas de Atriplex. Portanto, é possível afirmar que existem bactérias halotolerantes associadas às plantas de Atriplex, capazes de solubilizar fosfato inorgânico; fixar N2; produzir AIA, EPS e molécula quorum sensing, havendo a possibilidade destes micro-organismos, quando associados às plantas, contribuírem de forma positiva em relação à promoção de crescimento vegetal. Os isolados bacterianos são promissores no desenvolvimento vegetativo e nutritivo da Atriplex. No entanto, necessita-se explorar melhor o efeito dos inoculantes bacterianos associados às plantas halófitas, dando condições para melhoria no processo de fitorremediação de solos salinos.

As plantas quando colonizadas produzem diversas enzimas de defesa que podem impedir o estabelecimento de microrganismos. Esta condição adversa na planta hospedeira gera uma resposta do microrganismo, a qual está associada à síntese de proteínas e outras moléculas que atuam na sua interação com a planta e alteram a comunidade microbiana associada. Todos os organismos respondem a essa condição, estabelecendo biofilmes, ou sintetizando um grupo de moléculas e proteínas que os protegem de danos e facilita a recuperação. Estas são chamadas proteínas de choque térmico (HSPs), as quais não foram ainda estudadas na interação Methylobacterium - planta. As bactérias do gênero Methylobacterium são metilotróficas facultativas da classe Alfa-proteobactéria, encontradas em relações epifíticas e endofíticas com diferentes espécies vegetais. Assim, o presente trabalho teve como objetivos avaliar o efeito da deficiência da produção de biofilme e de Acil-Homoserina-Lactonas (AHLs), e do estresse térmico de M. extorquens na colonização da planta hospedeira (Saccharum sp.). Para isso, foram usadas linhagens defectivas para produção de biofilme e AHLs, juntamente com a linhagem selvagem submetida ou não ao estresse térmico. Os resultados obtidos mostram a complexidade dos mecanismos envolvidos na produção de biofilme e moléculas AHLs. O estresse térmico não afetou a colonização das raízes nem de colmos após 5 dias de inóculo, porém causou uma diminuição da colonizacão do colmo após 15 dias. A mutação para produção de AHL não causou nenhuma diferença significativa na colonização da planta hospedeira, no entanto o mutante para biofilme apresentou uma diminuição significativa da colonização tanto de raízes quanto de colmos, mostrando a grande importância da formação de biofilme na colonização da planta hospedeira. Entretanto, quando esta linhagem foi coinoculada com uma linhagem transformada (ARGFP) este resultado não foi observado. Os demais tratamentos após co-inoculação com a linhagem ARGFP apresentaram o mesmo padrão de colonização. O estudo proteômico de M. extorquens detectou um grande número de proteínas induzidas ou suprimidas nos tratamentos avaliados que podem estar associadas às alterações no padrão de interação desta bactéria com a planta hospedeira. A identificação dessas proteínas auxiliará a compreensão e o maior entendimento dos fatores envolvidos na interação bactéria-planta. / Plants produce a variety of defense enzymes when colonized that can impair the microorganisms\' establishment. This adverse condition in the host plant lead to a response from the microorganism, which is associated with the synthesis of a set of proteins and other molecules that affect the interaction with the host and shift the bacterial community associated. All organisms respond to this condition by establishing biofilms, or synthesizing a group of proteins and molecules that protect them from injuries and help them recover from damages. They are called heat shock proteins (HSPs), which is still not studied in Methylobacterium-plant interaction. The methylotrophic bacteria of the genus Methylobacterium are found in epiphytic and endophytic association with different plant species. So, in this present work the effect of a deficiency in biofilm and acyl-homoserine-lactones (AHLs) production and the effect of heat shock of M. extorquens on the host colonization (Saccharum sp.) was assessed. Defective strains for biofilm and AHLs production were used. Also the wild type AR 1.6/2 and this strain submitted to heat shock was evaluated. The results demonstrated that the mechanisms associated to biofilm and AHLs production are very complex. The heat stress has no effect on roots or stems colonization after 5 days of the inoculum, but was associated to reduction of bacterial density in stems after 15 days. The strain defective for AHL production showed similar colonization profile with wild type strain, while the biofilm mutant colonized the host plant in low density, suggesting the role of this process in plant colonization. Likewise, coinoculation of this strain with ARGFP target strain, which is AHL producer, the low density of this biofilm mutant was observed. The other treatments after coinoculation with ARGFP strain showed similar result when inoculated alone. A proteomic study of the strains showed that synthesis of many proteins were induced or suppressed in evaluated treatments, which proteins could be associated to shift in the bacteria-plant interaction. The identification of these proteins will contribute to a better understanding of the factors related to bacteria-plant interaction.

Les biomatériaux insérés dans la cavité orale, tout particulièrement les prothèses dentaires amovibles en résine, constituent des surfaces propices à la formation de biofilms incorporant des levures du genre Candida, à l’origine de candidose. Les levures, comme d’autres micro-organismes, adaptent leur prolifération à l’environnement par des molécules dites du quorum sensing. D’autre part, la contamination du milieu oral est contrôlée par de nombreuses protéines sécrétées par les glandes salivaires, mais leur utilisation en hygiène est peu documentée. Le but de ce travail est d’étudier l’effet anti-biofilm de molécules du quorum sensing et de protéines exocrines, le lysozyme et la lactoperoxydase, dans la perspective de réduire la colonisation des surfaces de prothèses dentaires par des levures du genre Candida et par conséquent de prévenir une stomatite prothétique. A cette fin, des biofilms à Candida ont été produits dans des plaques multi-puits à fond plat en polystyrène et quantifiés par coloration au cristal violet. L’effet de deux molécules du quorum sensing - tyrosol et farnésol - sur la formation des biofilms à Candida a été investigué in vitro sur la souche de référence ATCC 10231 et sur des souches cliniques isolées à partir de prothèses dentaires. Le tyrosol et le farnésol à hautes concentrations (> 6 mM et > 1 mM respectivement) entraînent une limitation de la formation de biofilms sans altérer la croissance fongique tandis que des concentrations plus faibles (~ 1 mM et ~ 1 µM respectivement) favorisent la formation de biofilms. Le lysozyme présente aussi un effet dépendant de la dose sur la formation de biofilms par Candida: promoteur de la formation de biofilms à une concentration de 1000 µg/ml et inhibiteur de la formation de biofilms à des concentrations plus faibles (3 et 10 µg/ml). Les composés hypohalogéneux (> 500 µM) produits par un système peroxydase limitent la croissance des levures et par conséquent leur capacité à former des biofilms in vitro. Un seul trempage ex vivo de prothèses amovibles pendant 5 minutes dans une solution d’ions hypohalogéneux (2000 µM) entraîne une diminution d’au moins 1 unité logarithmique du nombre de Candida dans 58,3 % des cas (N = 23) alors qu’un trempage dans de l’eau n’a aucun effet sur la colonisation de la prothèse :cet effet est statistiquement significatif (Chi carré, p = 0,0006, test de Fisher, p = 0,0009). / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Cooperation is a trait that is found at all levels of biological organization. Interestingly, cooperation appears to occur in bacteria that produce small, easily diffusible molecules called autoinducers. To understand why bacteria produce these autoinducers, the scientific community has focused on one predominant theory called quorum sensing. Under this theory, bacteria produce autoinducers so they can sense the density of the population. Once a sufficiently high population density is reached, autoinducers initiate the production of a costly gene product that serves to benefit the population. In contrast, a competing theory called diffusion sensing suggests that autoinducers are used by the individual cells and are not used for cooperation. Here, the production of the autoinducer serves as a mechanism to sense environmental conditions. If the environmental conditions are favorable, a costly gene product is produced. To what extent, and under what conditions, are each of these opposing theories valid remains to be identified. In this thesis, an engineered strain of Escherichia coli was used to identify the conditions under which quorum sensing and diffusing sensing can be observed. It was discovered that, depending upon the frequency at which the spatial distribution of the autoinducer and bacteria was disrupted, the population of engineered bacteria displayed hallmarks of either quorum sensing or diffusion sensing. Specifically, when the spatial distribution was disturbed at high or low frequency, quorum sensing was observed. However, when spatial distribution was disturbed at an intermediate frequency, diffusion sensing was observed. Understanding how these disturbances affect survival in bacteria may result in novel treatments for bacterial infections. In more general applications, it may be exploited in the development of alternative mechanisms for controlling invasive species or aid in species reintroduction.

The black band disease (BBD) microbial consortium often causes mortality of reef-building corals. Microbial chemical interactions (i.e., quorum sensing (QS) and antimicrobial production) may be involved in the BBD disease process. Culture filtrates (CFs) from over 150 bacterial isolates from BBD and the surface mucopolysaccharide layer (SML) of healthy and diseased corals were screened for acyl homoserine lactone (AHL) and Autoinducer-2 (AI-2) QS signals using bacterial reporter strains. AHLs were detected in all BBD mat samples and nine CFs. More than half of the CFs (~55%) tested positive for AI-2. Approximately 27% of growth challenges conducted among 19 isolates showed significant growth inhibition. These findings demonstrate that QS is actively occurring within the BBD microbial mat and that culturable bacteria from BBD and the coral SML are able to produce QS signals and antimicrobial compounds. This is the first study to identify AHL production in association with active coral disease.

This thesis examined the antimicrobial activity of select neotropical plants from Costa Rica and traditional Q’eqchi Maya medicines from Belize. In particular the potential for interference with bacterial quorum sensing (QS) and biofilm formation as well as fungal growth were assessed. Of one hundred and twenty six extracts collected from Costa Rica, one third showed significant QS inhibition while 13 species displayed more biofilm inhibitory activities than the positive control allicin. The active species belonged to the Lepidobotryaceae, Melastomataceae, Meliaceae, Sapindaceae, and Simaroubaceae. Twelve Marcgraviaceae species were tested for the same biological activities; of these, three showed similar QS inhibition to that of the positive control Delisea pulchra (Greville) Montagne and five with at least 30% biofilm inhibition. Only one species inhibited fungal growth – Marcgravia nervosa Triana & Planch. Bioassay-guided isolation of this plant resulted in the identification of the active principle as a naphthoquinone, with a minimum inhibitory concentration (MIC) ranging from 85 to 100 μM against Saccharomyces cerevisiae. Similarly, sixty one Q’eqchi’ Maya medicinal plant species were evaluated for their antimicrobial activities. Of these, four species showed more QS inhibition than D. pulchra, seven with comparable biofilm inhibitory activities that of allicin, and two with similarly antifungal activity to berberine. Two spirostanol saponins were isolated from Cestrum schlechtendahlii G.Don, an active antifungal plant. The major saponin showed growth inhibition against Saccharomyces cerevisiae and Fusarium graminearum, with MICs of 16.5 μM and 132 μM, respectively. Further analyses of this compound using chemical genomics suggested that its antifungal mechanism of action is pleiotropic, affecting multiple targets. Taken together, these findings showed that neotropical plants and traditional Q’eqchi’ Maya medicines contain phytochemicals that interfere with bacterial biofilm formation and quorum sensing as well as fungal growth.