Características de superfície de Lactobacillus delbrueckii UFV H2b20 probiótico

• Ano de defesa: 2008
• Autor(a) principal: Floresta, Fernanda Arruda
• Orientador(a): Moraes, Célia Alencar de
• Banca de defesa: Andrade, Nélio José de , Pinto, Cláudia Lúcia de Oliveira
• 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: Lactobacillus delbrueckii; Probióticos; Superfície celular
• Palavras-chave em Inglês: Lactobacillus delbrueckii; Probiotic; Celular surface
• Área do conhecimento CNPq: CNPQ::CIENCIAS AGRARIAS::AGRONOMIA::FITOSSANIDADE::MICROBIOLOGIA AGRICOLA
• Link de acesso: http://locus.ufv.br/handle/123456789/1541

Lactobacillus delbrueckii UFV H2b20 has been demonstrating a high potential to colonize bowels of mice free of germs and also for immunostimulation. This bacterium has physiologic and genetic characteristics that qualify it as a probiotic. A spontaneous mutant without the immunostimulation ability was obtained after successive transferences in growth medium. One of the objectives of this work is to investigate the possible causes of this instability. The existence ofidentity between the bacteria was proved by the rDNA 16S sequencing and by Denaturing Gradient Gel Electrophoresis (DGGE), and the sequences showed 99% of identity an the profile of gel bands was similar. The instability is not due to a plasmid curing process, since none of them have plasmidial DNA, as was demonstrated by the conventional extraction of plasmidial DNA and by Pulsed Field Gel Electrophoresis (PFGE). The two bacteria showed the same profile of bands of surface proteins (SDS-PAGE), but differed as to the expression of the proteins with the size corresponding to the S proteins. Nevertheless, because only the amplified fragment corresponding to the anchor part of the codifier gene of the protein of the S layer was obtained, it was inferred that the L. delbrueckii UFV H2b20 does not express these proteins. The micrographies from transmission eletronic microscopy show the non existence of the S layer in L. delbrueckii UFV H2b20, both in the wild type and the mutant. In these bacteria, the eletrophoresis in two dimensions showed 769 proteins in the extract of surface proteins from the wild bacterium and 860 from the mutant one. From these totals, 323 were present only in the wild type and 414 in the mutant bacterium. The mass spectroscopy of the proteins with greater expression in the wild bacterium and the sequencing of the terminal-C in the MALDI-TOF/TOF showed similarity with intracellular proteins, an evidence that damage occurred on the cellular envelope during the extraction process of the surface proteins. Thus, the hypothesis of differences between the two strains, related to the surfaces, could not be proved with methodology used. The adhesion ability of the mutant L. delbrueckii UFV H2b20 to one of the most abundant carbohydrate of the intestinal mucosa, the N-acetylghucosamine (GlcNac), showed to be 27% less than that of the wild bacterium, by messurements in Surface Plasmon Resonance (SPR). The study of the use of L. delbrueckii UFV H2b20 antigen delivery vehicle was proposed as a result of the work of the development of a recombinant plasmid that contains the signal peptide of the S protein fused to the anticancer NY-ESO-1, and to the anchorage region of the S protein. The resulting sequence shows all of the cloned fragments in the same reading frame, which favours the expression of NY-ESO-1 on the L. delbrueckii UFV H2b20 and a broader use of this bacterium.