Detecção do gene ppk e correlação com a produção de biofilme em Corynebacterium diphtheriae

• Autor(a) principal: Lydio, Renata Lourenço
• Data de Publicação: 2016
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da UERJ
• Texto Completo: http://www.bdtd.uerj.br/handle/1/14393
• topic: Corynebacterium diphtheriae; Polyphosphate; Polyphosphatekinase; PPK; Biofilm; Corynebacterium diphtheriae; Polifosfato; Polifosfatoquinase; PPK; Biofilme; Corynebacterium diphtheriae; Biofilme; CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA::MICROBIOLOGIA APLICADA::MICROBIOLOGIA MEDICA

Diphtheria is a bacterial toxemic disease of acute evolution whose etiologic agent is the Gram-positive rod Corynebacterium diphtheriae. Among its virulence factors, we may cite the production of diphtheria toxin and the ability to produce pili and to form biofilm. Biofilms are microbial communities of high organization attached to biotic or abiotic surfaces and embedded by complex extracellular matrix consisting of polymeric substances. Polyphosphate (poliP) has a key role in virulence and resistance to environmental stresses in various bacterial species. It has already been demonstrated the involvement of ppk gene (responsible for the synthesis of polyphosphatekinase) on biofilm formation in Pseudomonas aeruginosa: mutant strains had their motility and biofilm forming capacity reduced. The aim of this study was to detect the ppk gene and to correlate it with the production of biofilm in C. diphtheriae. So, biofilm formation was investigated in different media and substrates which stimulate or not poliP synthesis. In addition, the presence of the ppk gene was analyzed in silico and in vitro. The three-dimensional structure of the PPK proteins was also predicted. This study also intended to clone the C. diphtheriae ppk sequence in the TOPO system. As results, it was observed that all samples were able to form biofilms on polystyrene and glass surfaces, but at different intensities. The cultivation in King B broth did not modify the bacterial adherence profile to polystyrene of most of the strains. Strains classified as non-adherent to glass when grown in TSB, began to strongly adhere to this substrate when cultivated in King B broth. Two genes responsible for poliP synthesis were detected in C. diphtheriae: ppk2a and ppk2b. PPK2A and PPK2B proteins share only 40% of identity, displaying distinct three-dimensional structures. The construction of a ppk2a mutant in C. diphtheriae was initiated with the cloning of the coding sequence in the TOPO system. The presence of the ppk2a gene seemed to stimulate the adherence to glass by the E. coli mutant strain containing the plasmid TOPO::ppk2a. Additional studies are required for the construction of a ppk mutant in C. diphtheriae, in order to understand the influence of this gene on virulence and on stress response.