Estudo da aderência localizada de uma amostra de Escherichia coli enteropatogênica atípica
Arquivos
Data
2006
Autores
Hernandes, Rodrigo Tavanelli 
Orientadores
Gomes, Tania Aparecida Tardelli
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Escherichia coli enteropatogênica (EPEC) compreende uma das seis categorias
diarreiogênicas da espécie. Atualmente, ela é dividida em duas subcategorias, EPEC
típica (tEPEC) e EPEC atípica (aEPEC), tendo por base a presença do plasmídio EAF
(EPEC adherence factor), que só ocorre entre as tEPEC.
As EPEC promovem, no epitélio intestinal, a lesão attaching and effacing (A/E),
cujos determinantes genéticos estão localizados em uma ilha de patogenicidade
denominada região LEE (locus of enterocyte effacement). Um dos genes presentes nessa
região, o gene eae, codifica uma proteína de membrana externa (intimina) responsável
pela aderência íntima às células intestinais, observada na lesão A/E.
Uma característica fenotípica marcante das tEPEC é a produção do padrão de
adesão localizada (AL), no qual são observadas microcolônias bacterianas compactas
sobre células HeLa e HEp-2, após 3 horas de contato. O padrão AL está associado com
a expressão da fímbria denominada bundle-forming pilus (BFP), que é codificada pelo
plasmídio EAF e promove agregação bactéria-bactéria, dentro das microcolônias, bem
como a interação inicial, que precede a aderência íntima mediada por intimina. Por
outro lado, a maioria das aEPEC promove a formação de microcolônias mais frouxas,
após ensaios mais prolongados (ensaios de 6 horas), o que caracteriza a chamada adesão
semelhante à AL (AL-like).
Durante um estudo anterior, sobre o potencial de virulência de 59 amostras de
aEPEC, foi observado que 9 delas produziam AL típica (em 6 horas), mesmo na
ausência de BFP. Após um amplo estudo das suas características fenotipicas e
genotipicas, uma amostra (1551-2) foi selecionada para a identificação da estrutura
responsável pela formação de microcolônias compactas. Ensaios de microscopia
eletrônica revelaram que o padrão AL da amostra 1551-2 refletia bactérias
internalizadas e que um mutante não-polar em eae (1551-2:eae-
), perdia essa
propriedade, embora continuasse aderindo de forma difusa (AD). O seqüenciamento da
região variável do gene eae revelou que a intimina da amostra 1551-2 pertence ao
subtipo omicron (ο).
Para caracterizar a adesina responsável pela AD no mutante 1551-2:eae-
, foram
obtidos mutantes não aderentes utilizando-se o transposon Mini-Tn10. Um desses
mutantes foi empregado para a absorção de um soro produzido com a amostra 1551-
2:eae-
Esse soro foi capaz de inibir a adesão da amostra 1551-2:eae-
a células HeLa e,
em ensaios de immunoblot, revelou uma banda de aproximadamente 25 kDa.
Podemos concluir que o padrão AL da amostra 1551-2 reflete um processo de
internalização provavelmente mediado por intimina, e que novos fatores de virulência,
possivelmente uma proteína de ~25 kDa, poderiam desempenhar importante papel na
interação de amostras de aEPEC in vitro.
Enteropathogenic Escherichia coli (EPEC) comprise one of the 6 diarrheagenic categories of the species. Presently, it is sub-grouped into two sub-categories, typical EPEC (tEPEC) and atypical EPEC (aEPEC), based on the presence of the EAF (EPEC adherence factor) plasmid, which occurs only in tEPEC. EPEC promote, in the intestinal epithelium, an attaching and effacing lesion (A/E) whose genetic determinants are located in a pathogenicity island named LEE (locus of enterocyte effacement). One of the LEE genes, the eae gene, encodes an outer membrane protein (intimin), which is responsible for the intimate adherence to the intestinal cells, observed in the A/E lesions. A marked phenotypic characteristic of tEPEC is the production of the localized pattern of adherence (LA), where compact bacterial microcolonies are detected on HeLa and HEp-2 cells, after 3 hours of contact. The LA pattern is associated with the expression of the bundle-forming pilus (BFP), which is encoded on the EAF plasmid and promotes bacterium-bacterium aggregation within the microcolonies as well as the initial interaction that precedes the intimate adherence by intimin. On the other hand, most aEPEC form microcolonies looser than those seen in LA, after more prolonged assays (6 h assays), a phenotype that characterizes the so called LA-like pattern of adherence. During a previous study on the virulence potential of 59 strains of aEPEC, it was observed that 9 of them produced typical LA, even in the absence of BFP. After an ample analysis of their phenotypic and genotypic characteristics, one strain (1551-2) was selected for the identification of the structure responsible for compact microcolony formation in this strain. Electron microscopy assays have revealed that the LA pattern of strain 1551-2 reflected, in fact, internalized bacteria, and that a non-polar mutant in eae (1551-2:eae- ), had lost this property but maintained a strong diffuse adherence. Sequencing experiments of the variable region of the 1551-2 intimin molecule revealed that it corresponds to intimin subtype omicron (ο). To further characterize the adhesin responsible for the diffuse adherence phenotype in strain 1551-2:eae- , non-adherent mutants were obtained by transposon mutagenesis with Mini-Tn10. One of these non-adherent mutants was then used to absorb an antiserum produced against strain 1551-2:eae- . This absorbed antiserum inhibited the diffuse adherence of strain 1551-2:eae- to HeLa cells and, in immunoblot experiments, it has revealed a protein band of approximately 25kDa. From the data presented in this study, it is possible to conclude that the LA phenotype of aEPEC strain 1551-2 comprises, rather, an internalization process probably mediated by intimin, and that novel virulence factors, possibly a ~25 kDa protein, could play an important role in the interaction of aEPEC strains in vitro.
Enteropathogenic Escherichia coli (EPEC) comprise one of the 6 diarrheagenic categories of the species. Presently, it is sub-grouped into two sub-categories, typical EPEC (tEPEC) and atypical EPEC (aEPEC), based on the presence of the EAF (EPEC adherence factor) plasmid, which occurs only in tEPEC. EPEC promote, in the intestinal epithelium, an attaching and effacing lesion (A/E) whose genetic determinants are located in a pathogenicity island named LEE (locus of enterocyte effacement). One of the LEE genes, the eae gene, encodes an outer membrane protein (intimin), which is responsible for the intimate adherence to the intestinal cells, observed in the A/E lesions. A marked phenotypic characteristic of tEPEC is the production of the localized pattern of adherence (LA), where compact bacterial microcolonies are detected on HeLa and HEp-2 cells, after 3 hours of contact. The LA pattern is associated with the expression of the bundle-forming pilus (BFP), which is encoded on the EAF plasmid and promotes bacterium-bacterium aggregation within the microcolonies as well as the initial interaction that precedes the intimate adherence by intimin. On the other hand, most aEPEC form microcolonies looser than those seen in LA, after more prolonged assays (6 h assays), a phenotype that characterizes the so called LA-like pattern of adherence. During a previous study on the virulence potential of 59 strains of aEPEC, it was observed that 9 of them produced typical LA, even in the absence of BFP. After an ample analysis of their phenotypic and genotypic characteristics, one strain (1551-2) was selected for the identification of the structure responsible for compact microcolony formation in this strain. Electron microscopy assays have revealed that the LA pattern of strain 1551-2 reflected, in fact, internalized bacteria, and that a non-polar mutant in eae (1551-2:eae- ), had lost this property but maintained a strong diffuse adherence. Sequencing experiments of the variable region of the 1551-2 intimin molecule revealed that it corresponds to intimin subtype omicron (ο). To further characterize the adhesin responsible for the diffuse adherence phenotype in strain 1551-2:eae- , non-adherent mutants were obtained by transposon mutagenesis with Mini-Tn10. One of these non-adherent mutants was then used to absorb an antiserum produced against strain 1551-2:eae- . This absorbed antiserum inhibited the diffuse adherence of strain 1551-2:eae- to HeLa cells and, in immunoblot experiments, it has revealed a protein band of approximately 25kDa. From the data presented in this study, it is possible to conclude that the LA phenotype of aEPEC strain 1551-2 comprises, rather, an internalization process probably mediated by intimin, and that novel virulence factors, possibly a ~25 kDa protein, could play an important role in the interaction of aEPEC strains in vitro.
Descrição
Citação
HERNANDES, Rodrigo Tavanelli. Estudo da aderência localizada de uma amostra de Escherichia coli enteropatogênica atípica. 2006. 129 f. Tese (Doutorado em Ciências) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2006.