INHIBITORY MECHANISM OF ACTION FOR COILED-COIL PEPTIDES AGAINST TYPE THREE SECRETION SYSTEM FROM ENTEROPATHOGENIC ESCHERICHIA COLI.

LARZABAL MARIANO,; BALDONI HECTOR A; SUVIRE, FERNANDO D; CURTO LUCRECIA; GOMEZ W.; WANDER MARQUES DA SILVA; GIUDICESSI S.; CAMPERI, J. M.; DELFINO J. M.; A CATALDI; ENRIZ RICARDO DANIEL

Buenos Aires

Congreso; SAIB; 2017

Human pathogenic Gram negative bacteria, such as enteropathogenic Escherichia coli (EPEC), rely on type III secretion systems (T3SS) to translocate virulence factors directly into host cells, interfering and altering their cellular processes. The coiled-coil domains, present in the structural proteins of T3SS, are conformed by amphipathic α-helical structures that play an important role in the protein-protein interaction and are essential for their assembly. In order to inhibit the function of EPEC T3SS, coiled-coil peptides based on the amino acid sequence of the chaperon CesA were synthesized and analyzed. EspA is the major secretory proteins that compose the translocon part of T3SS and which have coiled-coil domains in the C-terminal region (Figure 1). The EspA-EspA interaction through their coiled-coil domain form filaments, which are transiently present. CesA is the EspA chaperone and possesses coiled-coil domains at the N-terminal by which they avoid premature intracellular associations between EspA-EspA. After recognizing the regions of interaction and considering CesA as a potent inhibitor of the polymerization of the EspA filaments, two peptides of 21 aa (CesA1 and CesA2) were designed from the coiled-coil motifs belonging to the region alpha helix I of the N-terminal chaperone, which replicate the interaction between the monomers EspA and CesA (Table 1).Initially, a blind docking study was performed for the different peptide-protein complexes (CesA1/CesA2-EspA), to obtain the structure of the different complexes. These complexes were subjected to energy minimization under PM7 model. The energies of CesA2 were found to be significantly lower indicating that these complexes are more stable than those of CesA1 (Figure 2). Then, two minimal energy complex structures of each of the peptides were plotted, focusing on the participation of the residues. The amino acids PHE12, PHE19, LEU51 and LEU62 of EspA are the main responsible in the formation of the network of hydrophobic interactions (hydrogen bond) (Figure 3). The inhibitory capacity of the peptides on EPEC T3SS was assessed through in vitro hemolysis assays. Peptide CesA2 inhibited RBCs lysis by 60% with a 0,23 mM concentration, whereas CesA1 did not show a significant difference in relation to hemolysis caused by the control of defective or lacking cells of T3SS (Figure 4). A further hemolysis assay was then performed as a function of the concentration of CesA2 peptides (Figure 5).The presence of CesA2 coiled-coil peptide prior to the elongation of the EspA filament could prevent their functioning. Therefore, as the EspA filament begins to assemble in the presence of the peptides, they may block the EspA-EspA interaction by physical competition, causing inhibition of hemolysis dependent on T3SS of EPEC. According to the in vitro tests performed, CesA2 showed a lower energy profile in reference to the interaction energy and exhibits hydrogen bonding interactions unlike CesA1.This demonstrates that compounds targeting T3SS from pathogenic bacteria can inhibit bacterial infection by presenting a higher specificity than broad-spectrum antibiotics, avoiding selective pressure and thus reducing the development of resistance to antimicrobial agents.