Utilize este identificador para referenciar este registo: http://hdl.handle.net/10400.5/1133
Título: O stress ambiental e a secreção de proteinas em Listeria monocytogenes
Autor: Fonseca, Catarina Isabel Freitas
Orientador: Brito, Maria Luísa de Castro
Ferreira, Ricardo Boavida
Palavras-chave: listeria monocytogenes
carvão activado
NaCl
proteinas secretadas
stress ambiental
activated charcoal
environmental stress
ph
secreted proteins
Data de Defesa: 2009
Resumo: Listeria monocytogenes is recognized as a foodborne pathogen for humans and animals, causing gastroenteritis, and more severe manifestations including septicemia, central nervous system infections, and maternofetal infections leading to stillbirths and abortions. Excluding the host risk factors, there is well documented evidence for the heterogeneity in the virulence of L. monocytogenes. Although rare, listeriosis is of public health concern because of its high case-fatality (20-30%). L. monocytogenes cells are exposed to different types of stresse, in the food industry, related with food processing or with cleaning and sanitizing of equipments. Because of its versatility, Listeria is able to persist in the environment. This pathogenic bacterium has developed strategies to efficiently avoid both host defense mechanisms (as a parasite), and to survive and grow (as a saprophyte) under environmental conditions that are lethal or inhibitory to many other nonsporing foodborne bacteria. The application of proteomics to biological research is increasing at an exponential rate. Microbiology represents one of the major disciplines which are opening up to proteomics-based approaches. Secretory proteins are the frontline in host-pathogen interactions and protein expression is characterized by variations and adjustments to different environmental conditions. This work aimed the optimization of the conditions for the separation and SDS-PAGE analysis of L. monocytogenes proteins, secreted under favorable and unfavorable(environmental stresseful) conditions. Four strains, belonging to serovars 1/2a, 4b, 4c and 4d/4e were used. These strains were from different origins (raw milk, animal case, cheese and other food product) and presented different levels of pathogenicity (two were virulent and two exhibited low virulence). The effects of acid stresse (pH 5.5 and pH 6; control, pH 7), cold stresse (10 ºC; control, 37 ºC) nutritional stresse (growth in minimal medium MWB) and saline stresse (11% (w/v) NaCl) on protein secretion were investigated. The effect of the supplementation of both the minimal medium (MWB) and the rich medium (BHI) with activated charcoal on protein expression was also studied. The technique used to fractionate the proteins was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This technique has emerged, throughout the last decades, as the method of choice to analyze the polypeptide composition of biological materials. The SDS-PAGE is a type of electrophoresis that uses polyacrylamide gels in the presence of sodium dodecyl sulfate, allowing polypeptide separation according to their molecular masses. After electrophoresis, the polypeptides were stainned with Coomassie Brilliant Blue G-250, a stain generally used for protein detection after electrophoresis in this type of gels.The strain from serovar 1/2a (3049), in acidic minimal medium (pH 5.5 and pH 6), presented at least four over-expressed proteins and the sub-expression of two, when compared with the results obtained in neutral medium (pH 7). In acidic rich medium (BHI), only one over-expressed protein band was detected. In the presence of salt, the same strain showed over-expression of proteins (at least two) in minimal medium, when compared to rich medium. In the presence of activated charcoal, in minimal medium, no growth of this strain was detected, suggesting the sequestration, by charcoal, of medium components essential for growth. In rich medium, and in the presence of charcoal, at least one protein appeared to be over-expressed, confirming results from other authors. In minimal medium, the four strains showed differences in the secretion of proteins, according to the grow temperature, with sub-expression of some proteins and over-expression of others at 10 ºC, when compared to 37 ºC. The cluster analysis based on the secreted protein profiles of the four strains, in minimal medium at 37 °C, grouped them according to the three genetic lineages described for this species. The preliminary results obtained in this work may help to identify protein markers for use in the effective control of the presence of this bacterium in food. RESUMO De modo a avaliar o efeito do stresse ambiental na secreção de proteínas em Listeria monocytogenes, foram utilizadas quatro estirpes com níveis de patogenicidade distintos. Utilizaram-se dois meios de cultura (meio mínimo, MWB e meio rico, BHI). Em MWB a 10 ºC, as quatro estirpes apresentaram sobre-expressão de umas proteínas e sub-expressão de outras, relativamente a 37 ºC. A estirpe 3049, em MWB a pH 5,5 e 6, e relativamente ao valor de pH 7, apresentou, pelo menos, sobre-expressão de 4 proteínas e sub-expressão de duas. Na presença de 11 % (m/v) de NaCl, a mesma estirpe apresentou sobre-expressão de proteínas (pelo menos duas) em MWB, relativamente ao meio BHI. Em MWB com carvão activado, não houve crescimento, sugerindo o sequestro pelo carvão de componentes essenciais. Em BHI com carvão, pelo menos uma proteína parece ter sido sobre-expressa, confirmando resultados de outros autores relativamente a proteínas virulentas. A análise de grupos efectuada aos perfis de proteínas das quatro estirpes, em MWB a 37 ºC, permitiu identificar as três linhagens genéticas descritas para esta espécie. Os resultados obtidos poderão contribuir para a identificação de marcadores proteicos a utilizar no controlo efectivo da presença desta bactéria nos alimentos.
Descrição: Mestrado em Engenharia Alimentar - Qualidade e Segurança Alimentar - Instituto Superior de Agronomia
URI: http://hdl.handle.net/10400.5/1133
Aparece nas colecções:BISA - Dissertações de Mestrado / Master Thesis



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