O stress ambiental e a secreção de proteinas em Listeria monocytogenes

Detalhes bibliográficos
Autor(a) principal: Fonseca, Catarina Isabel Freitas
Data de Publicação: 2009
Tipo de documento: Dissertação
Idioma: por
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10400.5/1133
Resumo: Mestrado em Engenharia Alimentar - Qualidade e Segurança Alimentar - Instituto Superior de Agronomia
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spelling O stress ambiental e a secreção de proteinas em Listeria monocytogeneslisteria monocytogenescarvão activadoNaClproteinas secretadasstress ambientalactivated charcoalenvironmental stressphsecreted proteinsMestrado em Engenharia Alimentar - Qualidade e Segurança Alimentar - Instituto Superior de AgronomiaListeria 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.Brito, Maria Luísa de CastroFerreira, Ricardo BoavidaRepositório da Universidade de LisboaFonseca, Catarina Isabel Freitas2009-07-13T13:52:30Z20092009-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10400.5/1133porinfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-03-06T14:32:24Zoai:www.repository.utl.pt:10400.5/1133Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T16:49:20.057412Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv O stress ambiental e a secreção de proteinas em Listeria monocytogenes
title O stress ambiental e a secreção de proteinas em Listeria monocytogenes
spellingShingle O stress ambiental e a secreção de proteinas em Listeria monocytogenes
Fonseca, Catarina Isabel Freitas
listeria monocytogenes
carvão activado
NaCl
proteinas secretadas
stress ambiental
activated charcoal
environmental stress
ph
secreted proteins
title_short O stress ambiental e a secreção de proteinas em Listeria monocytogenes
title_full O stress ambiental e a secreção de proteinas em Listeria monocytogenes
title_fullStr O stress ambiental e a secreção de proteinas em Listeria monocytogenes
title_full_unstemmed O stress ambiental e a secreção de proteinas em Listeria monocytogenes
title_sort O stress ambiental e a secreção de proteinas em Listeria monocytogenes
author Fonseca, Catarina Isabel Freitas
author_facet Fonseca, Catarina Isabel Freitas
author_role author
dc.contributor.none.fl_str_mv Brito, Maria Luísa de Castro
Ferreira, Ricardo Boavida
Repositório da Universidade de Lisboa
dc.contributor.author.fl_str_mv Fonseca, Catarina Isabel Freitas
dc.subject.por.fl_str_mv listeria monocytogenes
carvão activado
NaCl
proteinas secretadas
stress ambiental
activated charcoal
environmental stress
ph
secreted proteins
topic listeria monocytogenes
carvão activado
NaCl
proteinas secretadas
stress ambiental
activated charcoal
environmental stress
ph
secreted proteins
description Mestrado em Engenharia Alimentar - Qualidade e Segurança Alimentar - Instituto Superior de Agronomia
publishDate 2009
dc.date.none.fl_str_mv 2009-07-13T13:52:30Z
2009
2009-01-01T00:00:00Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
format masterThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10400.5/1133
url http://hdl.handle.net/10400.5/1133
dc.language.iso.fl_str_mv por
language por
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron:RCAAP
instname_str Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron_str RCAAP
institution RCAAP
reponame_str Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
collection Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository.name.fl_str_mv Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
repository.mail.fl_str_mv
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