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Please use this identifier to cite or link to this item: http://hdl.handle.net/10400.5/1991

Title: Análise comparativa de proteínas secretadas em Listeria monocytogenes
Authors: Batista, Sandra Isabel Esteves
Advisor: Brito, Maria Luísa de Castro e
Ferreira, Ricardo Boavida
Keywords: Listeria monocytogenes
secreted proteins
stress response
proteínas secretadas
resposta ao stress
Issue Date: 2009
Abstract: Listeria monocytogenes is a Gram-positive bacteria present in a diversity of environments, including soil, vegetation, and water, where it survive as saprophyte. When consumed via contaminated foods by susceptible mammalian hosts, such as pregnant women, the elderly, or immunosuppressed individuals, L. monocytogenes moves into intracellular environments, where it multiplies, evades the host's immunity, and spreads to neighboring cells and other organs. The average mortality rate of human listeriosis is 30%, which makes this pathogen a serious health issue and of great concern to the food industry. The virulence potencial of L. monocytogenes is linked to its ability to survive harsh environmental conditions. The identification and characterization of a large number of key proteins involved in L. monocytogenes cold-, acid-, saline-, and nutritional responses have undoubtedly contributed to our appreciation of the resistance and survival of this bacterium in adverse environments. This work aimed to investigated whether unique or common proteins, are up- or down-regulated under nutritional stresse (minimal medium (MWB)), low temperature (11 ºC), acid stresse (pH 5,5) and saline stresse (11% (w/v) NaCl). The four L. monocytogenes strains used in this study, belonging to the serotypes 1/2a, 4b, 4c and 4d/4e, were selected according to differences in virulence. Secreted proteins present in the supernatants of the cell cultures, in late exponential phase, were precipitated and separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), using equivalent amounts of total secreted protein in the wells. The detergent SDS (sodium dodecyl sulfate) is used to denature proteins with multiple subunits and to cover the protein molecules with negative charges. Thus, the proteins are separated according to their molecular masses. After electrophoresis, the proteins were stainned with Coomassie Brilliant Blue G-250, a stain often used for the detection of proteins in such gels. For each bacterial strain, at least three independent culture assays were set. The most abundant polypeptide bands and those suggesting the widest range in differential expression among strains were identified by MALDI-TOF-MS and LC-ESI-MS/MS. The protein identification was performed to correlate differences on protein expression patterns among strains. The preliminary results obtained in this work shows different expressed proteins. In MWB at 37 ºC, two particular bands over-expressed were identified as the virulence factors p60 and listeriolysin O, but their intensities were not consistent among isolates. The internalin C was present only in the more virulent strains. In MWB at 11 ºC, the p60 virulence protein was still expressed in all strains, but the listeriolysin O and the internalin C were not detected in any of the strains. In the serotype1/2a strain (3049), flagellin was the major protein identified. The serotypes 4c (3006) and 4d/4e (3993) showed lower levels of flagellin whereas for the serotype 4b strain (3077) no flagellin was detected. In general, in acidic minimal medium (pH 5,5) as well as in saline (11% (w/v) of NaCl) minimal medium, the strains presented, at least, two over-expressed proteins. The cluster analysis based on the secreted protein profiles of the four strains, in MWB at 37 ºC and at 11 ºC was not consistent. At 37 ºC, the strains were gathered, according to their level of pathogenicity, in two clusters. At 11 ºC the clustering of the strains band on their secreted protein profiles was not in accordance with virulence level or genetic lineage. These results may contribute to the development of effective strategies to control this food pathogen.-----------------------------------Neste estudo foram investigadas várias condições de stresse (nutritivo, frio, ácido e salino) na secreção de proteínas por L. monocytogenes. As proteínas secretadas por quatro estirpes, pertencentes a quatro serótipos diferentes e, com diferentes níveis de patogenicidade, foram separadas através de SDS-PAGE. A partir de meio mínimo (MWB), a 37 ºC, identificaram-se, por MALDI-TOF-MS, os factores de virulência, p60, internalina C e listeriolisina O. A 11 ºC, a proteína p60 foi identificada, por LC-ESI-MS/MS, em todas as estirpes, enquanto a listeriolisina O e a internalina C não foram detectadas em nenhuma estirpe. A flagelina foi identificada nas estirpes 4c e 4d/4e e, com particular abundância, na estirpe 1/2a, mas não o foi na estirpe 4b. Na presença de 11% (m/v) de NaCl ou a pH 5,5 foram visualizadas, pelo menos, duas proteínas sobre-expressas. Quando se procedeu ao agrupamento das estirpes por UPGMA, de acordo com os perfis de proteínas secretadas, a 37 ºC e a 11 ºC, verificou-se que a 37 ºC, as estirpes formaram dois grupos relacionados com a virulência, o mesmo não ocorrendo a 11 ºC. A identificação de proteínas diferencialmente expressas por L. monocytogenes poderá contribuir para o controlo efectivo deste patogénico alimentar.
Description: Mestrado em Engenharia Alimentar - Qualidade e Segurança Alimentar - Instituto Superior de Agronomia
URI: http://hdl.handle.net/10400.5/1991
Appears in Collections:BISA - Dissertações de Mestrado / Master Thesis

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