Características fenotípicas reguladas por quorum sensing em Aeromonas hydrophila

• Ano de defesa: 2011
• Autor(a) principal: Ponce, Adriana dos Reis
• Orientador(a): Vanetti, Maria Cristina Dantas
• Banca de defesa: Bazzolli, Denise Mara Soares , Oliveira, Leandro Licursi de , Pinto, Uelinton Manoel
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Doutorado em Microbiologia Agrícola
• Departamento: Associações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesse
• País: BR
• Palavras-chave em Português: Comunicação celular; Mutante AHL; Alterações fenotípicas; Proteases; Biofilme bacteriano
• Palavras-chave em Inglês: Cell communication; Mutant AHL; phenotypic changes; Proteases, Bacterial biofilm
• Área do conhecimento CNPq: CNPQ::CIENCIAS AGRARIAS::CIENCIA E TECNOLOGIA DE ALIMENTOS::CIENCIA DE ALIMENTOS
• Link de acesso: http://locus.ufv.br/handle/123456789/1557

Bacteria have a communication system among cells, dependent on cellular density, called quorum sensing. Knowledge of phenotypes controlled by this system, such as production of hydrolytic enzymes which result in food spoilage, biofilm formation and mechanisms of pathogenicity will contribute to elucidate important processes in food microbiology and development of control strategies for pathogenic and spoilage microorganisms. The aim of this study was to determine the phenotypes regulated by quorum sensing in Aeromonas hydrophila isolated from cooled raw milk. Phenotypic characteristics, such as production of the hydrolytic enzymes, motility, biofilm formation and protein profile of A. hydrophila ATCC 7966 and Embrapa 029 wild-type strains, and acyl homoserine lactones (AHLs)-deficient mutant were compared. A. hydrophila QS mutants were constructed using a site-directed mutagenesis strategy. The ahyI gene was interrupted by gentamicin 3-acetyltransferase gene using the suicide vector pKNG101. Mutant bacteria 029-8, 029-14 and 029-21 showed the quorum sensing system compromised and they were not able to produce detectable AHL by the Chromobacterium violaceum CV026. However, A. hydrophila mutants showed intact ahyI and ahyR when analyzed by PCR. By RT-PCR technique, A. hydrophila mutant strain QS 029-14 was unable to transcribe ahyI and ahyR genes, which proved the inability to produce the regulatory protein response (AhyR) and AHL synthase (AhyI). These mutants lost the capacity to hydrolyze gelatin and showed losses or reduction of proteolytic activity. Swimming motility and β-hemolysis activity were observed in wild-type strain while these characteristics were absent in A. hydrophila QS 029-14. This leads to the indication that these features are under regulation of quorum sensing system. Both wild-type and mutant strains showed amylase and lipase activities. A. hydrophila wild-type and its mutant strain QS 029-14 were able to adhere on stainless steel coupons immersed in TYEP broth. The mutant strain QS 029-14 showed a lower adhesion which was verified by both the viable sessile cells counts and microscopic analysis of the coupons. This result strongly indicates the role of quorum sensing mechanism in the regulation of biofilm formation in this species. Polyacrylamide gel analysis of cell-free supernatants and intracellular proteins showed a differential expression of intracellular and extracellular proteins produced by A. hydrophila ATCC 7966 and Embrapa 029 wild-type strains and A. hydrophila 029-14 mutant strain. These data leads to the indication that quorum sensing system might be involved in the regulation of some extracellular and intracellular proteins. The mass spectrometry analysis and sequencing of the amino-terminal region of the protein band present in the extracellular supernatant of the mutant strain QS 029-14 suggest an increased expression of ABC-type transporter. These results suggest that the quorum sensing system or the factor or factors related to the transcription of genes ahyI and ahyR regulate the expression of genes responsible for the proteolytic activity, β-hemolysis, motility and biofilm formation in A. hydrophila.