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Studies on the involvement of quorum sensing in the regulation of exopolysaccharide biosynthesis by Burkholderia cepacia complex isolates

By 1988- Vitor Hugo Jorge de Oliveira

Abstract

Tese de mestrado. Biologia (Microbiologia Aplicada). Universidade de Lisboa, Faculdade de Ciências, 2012Quorum sensing (QS) is a cell-to-cell communication mechanism used by bacteria that produce and recognize signaling molecules named autoinducers. It is known to be involved in the regulation of different virulence factors including exopolysaccharide (EPS) biosynthesis in several pathogens. Bacteria belonging to Burkholderia cepacia complex (Bcc) are a group of related species that had emerged as important opportunistic pathogens mainly in cystic fibrosis (CF) patients. EPS produced by these bacteria plays an important role in the development of Bcc infections. Like in other bacteria, Bcc possess several QS-regulated phenotypes but little is known about the effect of this regulation mechanism in the Bcc EPS biosynthesis. Therefore, to evaluate the role of QS in such regulation, we tested the effects of acyl-homoserine lactones (AHLs) degradation by AiiA lactonase and the presence of a quorum sensing inhibitor (QSI) 4-nitro-pyridine-1-oxide (4NPO) in EPS biosynthesis inhibition. The results indicate that indeed QS regulates EPS biosynthesis in Bcc. However the signaling molecules involved are not dependent on the CepI/R QS system since an insertion mutant in the cepI gene did not inhibit the EPS production. Thus, bioinformatics analyses led to the identification of protein CbsI as another putative AHL synthase of Burkholderia. Bcc bacteria are resistant to most of the conventional antibiotic treatments making its eradication a challenge. Thus, it has become essential to identify and develop alternative therapies to deal with these infections. The use of QSI seems a promising field to be explored. Since 4NPO was able to inhibit EPS production in Burkholderia, it was decided test its effect in several other Bcc QS-dependent phenotypes, such as motility, production of extracellular proteases and siderophores, and biofilm formation. Furthermore, its potential use as co-adjuvant of antibiotics in different Bcc species was also evaluated. The results confirmed that 4NPO is indeed affecting the tested phenotypes and increases the susceptibility to the antibiotics tested in both Bcc planktonic and sessile cells. The results also showed that 4NPO potentiates the activity of detergents such as Triton X100 and Tween 20. Since the clinical application of this compound is limited, its usage to clean and disinfect abiotic surfaces could be a possibility.As bactérias pertences ao complexo Burkholderia cepacia (Bcc) estão divididas em 17 espécies e são consideradas patogénicas oportunistas infectando sobretudo doentes com fibrose quística (FQ). Apesar de representarem apenas uma pequena percentagem da totalidade das infecções em doentes com FQ, os danos causadas por estas bactérias apresentam uma grande heterogeneidade, uma vez que as infecções que causam podem ser infecções crónicas ou podem estar associadas a quadros clínicos mais graves, levando muitas vezes à falência das funções pulmonares, desenvolvimento de pneumonia e de septicémia, designada síndrome da cepacia. A severidade destas infecções é agravada pela possibilidade de transmissão cruzada entre pacientes e pela partilha de equipamentos de terapêutica inalatória. Consequentemente, a maioria dos centros de FQ em todo o mundo teve que implementar regras especiais de higiene hospitalar e medidas severas de isolamento de indivíduos colonizados e indivíduos não colonizados com Bcc. Adicionalmente, estas bactérias são intrinsecamente resistentes à maioria dos antibióticos clinicamente usados e à maioria dos desinfectantes. Os compostos antibacterianos, com acção bacteriostática ou bacteriocida, têm-se assim revelado pouco eficazes no tratamento destas infecções, sendo necessário recorrer a elevadas concentrações de antibióticos e ao uso combinado desses mesmos antibióticos. Torna-se, portanto, fundamental o desenvolvimento de novas estratégias que permitam um tratamento mais rápido e eficaz desta infecções. Umas das estratégias que tem sido fortemente estudada e que se tem revelado promissora é a interferência dos mecanismos de quorum sensing (QS). Este mecanismo é dependente de uma densidade populacional elevada, permitindo a comunicação entre as bactérias de uma população através da libertação de sinais químicos, sendo os mais comuns em bactérias Gram-negativa as acil-homoserina lactonas (AHLs). Quando esses sinais se acumulam no meio e atingem um determinado nível, ocorre a activação de proteínas reguladoras que vão activar ou reprimir a expressão de genes regulados por QS, levando a comportamentos sincronizados dentro dessa população. Tem sido demonstrado que o QS está envolvido na regulação da expressão de diversos genes relacionados com a virulência. No caso das bactérias do complexo Bcc, o sistema mais conservado é constituído pela sintase cepI, que produz C6-HSL e C8-HSL, e pelo regulador de transcrição cepR, que está envolvido na expressão de genes codificantes para proteases e sideróforos, motilidade e formação de biofilmes, entre outros. Ao contrário do que ocorre em outras espécies, pouco se sabe sobre os efeitos do QS, e em concreto do sistema CepI/R ao nível da regulação da síntese de exopolissacárido, um importante factor de virulência em Bcc. Trabalhos feitos anteriormente no nosso laboratório sugerem que a biossíntese deste polímero deverá ser regulada por QS. Assim, um dos objectivos deste trabalho foi compreender qual o papel do mecanismo de QS na regulação do exopolissacárido em diferentes espécies de Burkholderia. Para tal, estudaram-se os efeitos da degradação de AHLs pela lactonase AiiA e do inibidor de QS, 1-óxido- 4-nitro-piridina (4NPO) na síntese de exopolissacárido. Os resultados obtidos, sugerem que o QS está efectivamente a regular a produção de exopolissacárido em Bcc mas as moléculas sinalizadoras não são as dependentes do sistema CepI/R. Isto porque a construção de um mutante de inserção no gene cepI não aboliu a síntese do exopolissacárido. Assim sendo, utilizaram-se ferramentas bioinformáticas para identifcar outro possível sistema de QS em Burkholderia. O segundo objectivo deste trabalho foi determinar os efeitos de um inibidor de QS descrito em Pseudomas aeruginosa, o 4NPO, em diferentes espécies do complexo Bcc, ao nível de diversos fenótipos regulados por QS, nomeadamente a mobilidade, produção de proteases extracelulares e de sideróforos e a formação de biofilmes. Uma vez que se confirmou que efectivamente o 4NPO inibe o QS em Burkholderia, decidiu-se estudar a potencial utilização deste composto como co-adjuvante de antibióticos. Para tal, testaram-se diferentes antibióticos e os resultados mostraram que o 4NPO aumenta a capacidade antibacteriana desses antimicrobioanos, tanto ao nível da inibição do crescimento das células planctónicas como ao nível da inibição da formação de biofilmes. Embora o uso clínico deste produto esteja limitado devido às suas propriedades mutagénicas, o uso de 4NPO em superfícies abióticas poderá ser uma potencial aplicação deste composto, uma vez que, também se verificou potenciar a actividade de detergentes como o Triton X100 e o Tween 20, ao nível do crescimento planctónico e do desenvolvimento de biofilmes. De acordo com estes resultados, os inibidores de QS, e o 4NPO em particular, poderão de facto ter aplicações a nível da desinfecção de superfícies e de material clínico, prevenindo o aparecimento de casos de contaminações hospitalares, que já levaram a graves surtos de Bcc em doentes com FQ

Topics: Bactérias patogénicas, Virulência, Burkholderia cepacia, Resistência a antibióticos, Expressão génica, Teses de mestrado - 2012
Year: 2012
OAI identifier: oai:repositorio.ul.pt:10451/7957

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