Evaluación del mecanismo de acción antimicrobiana del péptido 35409 sobre E. coli ML 35 (ATCC 43827)

Abstract

Introduction: Natural antimicrobial peptides (AMPs) have emerged as a response to the large increase of antimicrobial resistance from a broad spectrum of microorganisms. However, numerous changes in the physicochemical and structural properties of the AMPs have been introduced to improve their selectivity and decrease its toxicity on mammalian cells. Background: Studies in the Fundación Instituto de Inmunología de Colombia - FIDIC found that by modifying the peptide sequence Pf-Rif 20628 with hemolytic activity the analog peptide 35409 (11KA) is obtained, which has a low hemolytic activity and inhibits the growth of E. coli bacteria to a minimum inhibitory concentration of 22μM. From this information, in this paper, the mechanism of action of synthetic peptide 35409 on Gram-negative bacterial strain ML-35 was determined. Methodology: Initially the bacteriostatic and/or bactericidal effect of the peptide on bacteria by plate count was evaluated. From this information, two possible targets of action were established: the bacterial cell membrane and DNA. By testing calcein release of liposomes of varying lipid composition and scanning electron microscopy with bacteria and spheroplasts action of the peptide on the membrane was determined; meanwhile retardation assays of electrophoretic mobility of DNA and filamentation were used to evaluate the action of the peptide on bacterial DNA. Results: The results show that the peptide 35409 decreases by one logarithm the bacterial concentration, while the rest of the population is recovered in the absence of peptide. This function is performed by the action of the peptide on the bacterial membrane where an interaction occurs with the surface phospholipids, mainly with phosphatidylethanolamine, allowing the insertion of the peptide at the interior of the cell and its interaction with the bacterial DNA inhibiting DNA synthesis and the cell division in the Gram-negative E. coli ML35 strain. Conclusions: The cell membrane and bacterial DNA were established as the main targets of action of the peptide 35409 over the ML35 (ATCC 43827) strain. Cell wall was identified as a barrier to the interaction of the peptide with the inner membrane of the bacterium. It is suggested that the peptide 35409 has a dual bacteriostatic and bactericidal activity, which is a good candidate as a therapeutic agent that could be useful as a template for the synthesis of new shorter and effective peptides. This study establishes themethodological basis for studying and comparing modified peptides quickly and easily, in order to find a logic amino acids sequence.