RESUMO
Dispersin is a 10.2 kDa-immunogenic protein secreted by enteroaggregative Escherichia coli (EAEC). In the prototypical EAEC strain 042, dispersin is non-covalently bound to the outer membrane, assisting dispersion across the intestinal mucosa by overcoming electrostatic attraction between the AAF/II fimbriae and the bacterial surface. Also, dispersin facilitates penetration of the intestinal mucus layer. Initially characterized in EAEC, dispersin has been detected in other E. coli pathotypes, including those isolated from extraintestinal sites. In this study we investigated the binding capacity of purified dispersin to extracellular matrix (ECM), since dispersin is exposed on the bacterial surface and is involved in intestinal colonization. Binding to plasminogen was also investigated due to the presence of conserved carboxy-terminal lysine residues in dispersin sequences, which are involved in plasminogen binding in several bacterial proteins. Moreover, some E. coli components can interact with this host protease, as well as with tissue plasminogen activator, leading to plasmin production. Recombinant dispersin was produced and used in binding assays with ECM molecules and coagulation cascade compounds. Purified dispersin bound specifically to laminin and plasminogen. Interaction with plasminogen occurred in a dose-dependent and saturable manner. In the presence of plasminogen activator, bound plasminogen was converted into plasmin, its active form, leading to fibrinogen and vitronectin cleavage. A collection of E. coli strains isolated from human bacteremia was screened for the presence of aap, the dispersin-encoding gene. Eight aap-positive strains were detected and dispersin production could be observed in four of them. Our data describe new attributes for dispersin and points out to possible roles in mechanisms of tissue adhesion and dissemination, considering the binding capacity to laminin, and the generation of dispersin-bound plasmin(ogen), which may facilitate E. coli spread from the colonization site to other tissues and organs. The cleavage of fibrinogen in the bloodstream, may also contribute to the pathogenesis of sepsis caused by dispersin-producing E. coli.
RESUMO
Dispersin is a 10.2 kDa-immunogenic protein secreted by enteroaggregative Escherichia coli (EAEC). In the prototypical EAEC strain 042, dispersin is non-covalently bound to the outer membrane, assisting dispersion across the intestinal mucosa by overcoming electrostatic attraction between the AAF/II fimbriae and the bacterial surface. Also, dispersin facilitates penetration of the intestinal mucus layer. Initially characterized in EAEC, dispersin has been detected in other E. coli pathotypes, including those isolated from extraintestinal sites. In this study we investigated the binding capacity of purified dispersin to extracellular matrix (ECM), since dispersin is exposed on the bacterial surface and is involved in intestinal colonization. Binding to plasminogen was also investigated due to the presence of conserved carboxy-terminal lysine residues in dispersin sequences, which are involved in plasminogen binding in several bacterial proteins. Moreover, some E. coli components can interact with this host protease, as well as with tissue plasminogen activator, leading to plasmin production. Recombinant dispersin was produced and used in binding assays with ECM molecules and coagulation cascade compounds. Purified dispersin bound specifically to laminin and plasminogen. Interaction with plasminogen occurred in a dose-dependent and saturable manner. In the presence of plasminogen activator, bound plasminogen was converted into plasmin, its active form, leading to fibrinogen and vitronectin cleavage. A collection of E. coli strains isolated from human bacteremia was screened for the presence of aap, the dispersin-encoding gene. Eight aap-positive strains were detected and dispersin production could be observed in four of them. Our data describe new attributes for dispersin and points out to possible roles in mechanisms of tissue adhesion and dissemination, considering the binding capacity to laminin, and the generation of dispersin-bound plasmin(ogen), which may facilitate E. coli spread from the colonization site to other tissues and organs. The cleavage of fibrinogen in the bloodstream, may also contribute to the pathogenesis of sepsis caused by dispersin-producing E. coli.
RESUMO
O câncer é uma das maiores causas de morte no mundo, sendo o câncer de mama o segundo mais comum no mundo e o mais comum entre as mulheres. O câncer de mama é mais frequentemente diagnosticado em países desenvolvidos, porém é nos países em desenvolvimentos que ocorre a maioria das mortes. Muitos estudos usam produtos derivados de bactérias para desenvolver drogas anticâncer, buscando drogas que tem preferência pela célula tumoral ao invés da célula normal. Escherichia coli são bacilos Gram- negativos, que podem pertencer a microbiota e serem importantes reguladores da função intestinal, quanto também podem causar doenças. Existem vários patotipos de E. coli, entre eles as Escherichia coli enteroagregativa (EAEC). A dispersina é uma proteína de 10.7 kDa e carga positiva secretada para a membrana externa da bacteria. Ao revestir o LPS das bactérias, que tem carga negativa, a proteína repele a fimbria AAF, que possui também carga positiva. A dispersão dessas fimbrias tem como consequência o aumento na dispersão dessas bactérias. A proteína Pet é uma toxina de 104 kDa que pertence ao grupo das proteínas autotransportadoras (SPATEs) do tipo 1. Essa toxina entra em contato com citoqueratina 8 das células epiteliais e é internalizada por endocitose, podendo degradar a α-fodrina e causar alterações no citoesqueleto, causando arredondamento e destacamento das células. Como as proteínas dispersina e Pet são proteínas secretadas para fora da bacteria e podem entrar em contato com a célula do hospedeiro. Sendo assim, foram realizados testes comparativos, em relação à ação dessas proteínas, entre células de mama normais e tumorais. Para tal, foram utilizadas células de mama, MCF10A, MCF7 e MDA-MB-231 e o ensaio com MTT. Os resultados não foram totalmente conclusivos, pois o teste de citotoxicidade utilizando MTT necessita da complementação com outros ensaios não metabólicos e genômicos para a obtenção de respostas mais acuradas.
