Inhibición del sistema de secreción tipo III de Escherichia coli enteropatógena usando péptidos coiled-coil de EspA

Abstract

Enteropathogenic Escherichia coli (EPEC), is a leading cause of infant diarrhea. This pathogen utilizes the translocon-type three secretion system (translocon-TTSS) to inject its virulence factors directly into the eukaryotic cell cytoplasm, where they alter the cytoskeleton and produce a pathology called attaching and effacing lesion (A/E lesion). Such lesions lead to an electrolyte imbalance that causes diarrhea. Therefore the formation of the translocon-TTSS is an essential step in the pathogenesis caused by EPEC. An important component of the translocon-TTSS is the EspA filament, which contributes to the initial adhesion of bacteria to the enterocyte and also allows the translocation of virulence factors to the cytoplasm of the cell. The filament is composed of EspA protein monomers, which self-polymerize due to coiled-coil domains. It has been shown that disruption of these domains affects the assembly of the EspA filaments and prevents the formation of A/E lesions, which consequently reduces the pathogenesis caused by EPEC. Recently, it has been found that synthetic coiled-coil peptides of 15 amino acids (aa) (CoilA and CoilB), synthesized based on the C-terminal region of EspA, are able to block the translocon-TTSS of EPEC. And it was found that the block is done by inhibiting the assembly of de EspA filaments. These peptides could be a possible therapeutic alternative against diarrhea caused by EPEC. So the aim of this study was to find the minimum core of coiled-coil peptides that could block the translocon-TTSS of EPEC. Therefore three peptides of 8 aa based on the sequence of CoilA (CoilA1, CoilA2 y CoilA3) were designed and three more based on the sequence of CoilB (CoilB1, CoilB2 y CoilB3). However, it was not possible to purify the CoilA3 and CoilB1 peptides, due to their insolubility. It is known that when EPEC infects erythrocytes, it causes lysis of cells by generating pores in the membrane when the translocon-TTSS is formed. We found that by infecting cells with EPEC pre-incubated with the peptides, CoilA1 reduces hemolysis caused by EPEC even more efficiently than CoilA; while CoilA2 reduce in a similar way to CoilA. We found that CoilB2 and CoilB3 are not able to inhibit hemolysis, contrary to what was observed for the peptide from which they were derived (CoilB). The CoilA1 and CoilA2 peptides are able to efficiently reduce the adherence of EPEC and the formation of A/E lesions, when we infected epithelial cells with EPEC cultures, which during the formation of translocon-TTSS are in the presence of the peptides. The CoilB2 and CoilB3 peptides do not reduce the adhesion nor the formation of A/E lesions. We could conclude that the reduction of the adhesion and the formation of A/E lesions are due to that CoilA1 and CoilA2 peptides are able to block the polymerization of the EspA filaments, while CoilB2 and CoilB3 are not. We found clear evidence that the block of the polymerization owe to the fact that when EPEC is in the presence of CoilA1 and CoilA2 peptides, the secretion of translocator proteins EspA, EspB and EspD are blocked; these proteins form the translocon and without which the TTSS of EPEC is not functional. The CoilB2 and CoilB3 peptides are unable to block the secretion of translocator proteins, which explains their ineffectiveness in reducing the pathogenesis generated by EPEC. Therefore, in this paper we show the existence of a minimum region of 8aa (CoilA1) that is more effective than its original peptide of 15 aa (CoilA) in blocking the translocon-TTSS of EPEC.