Evaluación de la actividad antibacteriana y citotóxica de péptidos derivados de la secuencia PfRif (321-340): RYRRKKKMKKALQYIKLLKE

La resistencia de los microorganismos a los antibióticos es un problema creciente de salud a nivel mundial, la búsqueda de nuevos antimicrobianos ha venido cobrando cada vez mayor relevancia y los péptidos antimicrobianos (PAMs), de origen natural o sintético, han surgido como una opción terapéutica promisoria. El péptido sintético [A331]-PfRif (321-340) derivado de una proteína de Plasmodium falciparum, es activo contra bacterias E. coli ML35 (CMI 25 µM) pero causa lisis de los glóbulos rojos desde 1,56 µM. Con el fin de identificar péptidos de secuencia corta y actividad selectiva contra bacterias, en este trabajo se evaluó la actividad antibacteriana de péptidos sintéticos cortos derivados de [A331]-PfRif (321-340). Se observó que los péptidos [A331]-PfRif (324-340), [A331]-PfRif (325-340) y [A331]-PfRif (327-340) de 17,16 y 14 residuos respectivamente, son activos contra bacterias E. coli y no son hemolíticos a concentraciones ≤200 µM. Por esto, su índice terapéutico incrementó significativamente (más de 200, 160 y 40 veces, respectivamente), en comparación con el del péptido original [A331]PfRif (321-340) (IT 0,083). El análisis informático y por dicroísmo circular sugiere que la capacidad de asumir una conformación α-helicoidal en ambientes que mimetizan las condiciones del entorno de membranas cargadas negativamente y la presencia de dos lisinas en la cara no polar de la hélice α anfipática son cruciales para la actividad y selectividad de estas secuencias. Aunque las tres secuencias muestran características interesantes, [A331]-PfRif (324-340): RKKKMKKALQYIKLLKE fue el producto de síntesis de mayor pureza, tiene el mayor IT, fue el único activo contra una cepa de P. aeruginosa y adicionalmente presentó efecto sinérgico con Gentamicina; por esto sugerimos a [A331]-PfRif (324-340) como un candidato potencial para su uso como antimicrobiano.Abstract: Microorganisms resistance to antibiotics is a growing problem of health worldwide, then the search for new antimicrobials has been gaining increasing relevance and antimicrobial peptides (AMPs), of natural or synthetic origin, have emerged as a therapeutic promising option. Synthetic peptide [A331]-PfRif (321-340) which is derived from a Plasmodium falciparum protein, is active against E. coli ML35 bacteria (25 μM MIC) but causes lysis of red blood cells from 1.56 μM. In order to identify peptides having short sequence and selective activity against bacteria, in this work, the antibacterial activity of short synthetic peptides derived from [A331]-PfRif (321-340) was evaluated. It was observed that the peptides [A331]-PfRif (324-340), [A331]-PfRif (325-340) and [A331]-PfRif (327-340) with 17, 16 and 14 residues respectively, are active against bacteria E. coli and are not hemolytic at concentrations ≤200 μM. For this, its therapeutic index increased significantly (more than 200, 160 and 40 times, respectively), compared to that of the original peptide [A331]-PfRif (321-340) (IT 0.083). Bioinformatic analysis and circular dichroism suggest that ability to assume α-helical conformation in environments mimic negatively charged membranes environmental conditions and presence of two lysines in the non-polar face of the amphipathic α-helix are crucial for the activity and selectivity of these sequences. Although the three sequences show interesting characteristics, peptide [A331]-PfRif (324-340): RKKKMKKALQYIKLLKE was the synthesis product having greater purity, highest IT, and the only with activity against a P. aeruginosa stain; additionally presented synergistic effect with Gentamicin. For this, we suggest that the peptide [A331]-PfRif (324-340) is a potential candidate for its use as an antimicrobial.Maestrí

How to Combat Gram-Negative Bacteria Using Antimicrobial Peptides: A Challenge or an Unattainable Goal?

Antimicrobial peptides (AMPs) represent a promising and effective alternative for combating pathogens, having some advantages compared to conventional antibiotics. However, AMPs must also contend with complex and specialised Gram-negative bacteria envelops. The variety of lipopolysaccharide and phospholipid composition in Gram-negative bacteria strains and species are decisive characteristics regarding their susceptibility or resistance to AMPs. Such biological and structural barriers have created delays in tuning AMPs to deal with Gram-negative bacteria. This becomes even more acute because little is known about the interaction AMP–Gram-negative bacteria and/or AMPs’ physicochemical characteristics, which could lead to obtaining selective molecules against Gram-negative bacteria. As a consequence, available AMPs usually have highly associated haemolytic and/or cytotoxic activity. Only one AMP has so far been FDA approved and another two are currently in clinical trials against Gram-negative bacteria. Such a pessimistic panorama suggests that efforts should be concentrated on the search for new molecules, designs and strategies for combating infection caused by this type of microorganism. This review has therefore been aimed at describing the currently available AMPs for combating Gram-negative bacteria, exploring the characteristics of these bacteria’s cell envelop hampering the development of new AMPs, and offers a perspective regarding the challenges for designing new AMPs against Gram-negative bacteria

Evaluation of the antimicrobial action mechanism of the peptide 35409 on E. coli ML 35 (ATCC 43827)

Introducción: Los péptidos antimicrobianos (PAMs) naturales han surgido como una respuesta al gran aumento de resistencia antimicrobiana de un amplio espectro de microorganismos. Sin embargo, diferentes cambios en las propiedades fisicoquímicas y estructurales de los PAMs han sido introducidas para mejorar la selectividad y disminuir la toxicidad sobre células de mamíferos. Antecedentes: En estudios realizados en la Fundación Instituto de Inmunología de Colombia - FIDIC se encontró que al modificar la secuencia del péptido Pf-Rif 20628 con actividad hemolítica, se obtiene el péptido análogo 35409 (11KA) que presenta una baja actividad hemolítica e inhibe el crecimiento de bacterias E. coli a una concentración mínima inhibitoria de 22μM. A partir de ésta información, en este trabajo, se determinó el mecanismo de acción del péptido sintético 35409 sobre la cepa bacteriana Gram-negativa ML-35. Metodología: Inicialmente se evaluó el efecto bacteriostático y/o bactericida del péptido sobre las bacterias mediante conteo de placa. A partir de esta información se establecieron dos posibles blancos de acción: la membrana celular y el ADN bacteriano. Mediante ensayos de liberación de calceína sobre liposomas de composición lipídica variable y microscopía electrónica de barrido con bacterias y esferoplastos se determinó la acción del péptido sobre la membrana, mientras que, ensayos de retraso de movilidad electroforética de ADN y de filamentación fueron usados para evaluar la acción del péptido sobre el ADN bacteriano. Resultados: Los resultados muestran que el péptido 35409 disminuye en un logaritmo la concentración bacteriana, mientras el resto de la población se recupera en ausencia de péptido. Esta función es ejecutada por la acción del péptido sobre la membrana bacteriana donde se da una interacción con los fosfolípidos de superficie, principalmente con la fosfatidiletanolamina, lo que permite la inserción del péptido al interior de la célula y su interacción con el ADN bacteriano inhibiendo la síntesis de ADN y la división celular en la cepa Gram-negativa de E. coli ML35. Conclusiones: La membrana celular y el ADN bacteriano se establecieron como los principales blancos de acción del péptido 35409 sobre la cepa ML35 (ATCC 43827). Se identificó la pared celular como una barrera para la interacción del péptido con la membrana interna de la bacteria. Se sugiere que el péptido 35409 presenta una actividad dual bacteriostática y bactericida, que es un buen candidato como agente terapéutico y que podría ser útil como plantilla para la síntesis de nuevos péptidos más cortos y efectivos. Este estudio permite establecer la base metodológica para estudiar y comparar péptidos modificados de forma rápida y sencilla, con el fin de encontrar una lógica en la secuencia de aminoácidos.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 (11KA) 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.Fundación Instituto de Inmunología FIDI

How to combat gram-negative bacteria using antimicrobial peptides: A challenge or an unattainable goal?

Antimicrobial peptides (AMPs) represent a promising and effective alternative for combating pathogens, having some advantages compared to conventional antibiotics. However, AMPs must also contend with complex and specialised Gram-negative bacteria envelops. The variety of lipopolysaccharide and phospholipid composition in Gram-negative bacteria strains and species are decisive characteristics regarding their susceptibility or resistance to AMPs. Such biological and structural barriers have created delays in tuning AMPs to deal with Gram-negative bacteria. This becomes even more acute because little is known about the interaction AMP–Gram-negative bacteria and/or AMPs’ physicochemical characteristics, which could lead to obtaining selective molecules against Gram-negative bacteria. As a consequence, available AMPs usually have highly associated haemolytic and/or cytotoxic activity. Only one AMP has so far been FDA approved and another two are currently in clinical trials against Gram-negative bacteria. Such a pessimistic panorama suggests that efforts should be concentrated on the search for new molecules, designs and strategies for combating infection caused by this type of microorganism. This review has therefore been aimed at describing the currently available AMPs for combating Gram-negative bacteria, exploring the characteristics of these bacteria’s cell envelop hampering the development of new AMPs, and offers a perspective regarding the challenges for designing new AMPs against Gram-negative bacteriaIncluye referencias bibliográfica

A new synthetic peptide having two target of antibacterial action in E. coli ML35

The increased resistance of microorganisms to the different antimicrobials available to today has highlighted the need to find new therapeutic agents, including natural and/or synthetic antimicrobial peptides. This study has evaluated the antimicrobial activity of synthetic peptide 35409 (RYRRKKKMKKALQYIKLLKE) against S. aureus ATCC 29213, P. aeruginosa ATCC 15442 and E. coli ML 35 (ATCC 43827). The results have shown that peptide 35409 inhibited the growth of these three bacterial strains, having 16 fold greater activity against E. coli and P. aeruginosa, but requiring less concentration regarding E. coli (22 µM). When analyzing this activity against E. coli compared to time taken, it was found that this peptide inhibited bacterial growth during the first 60 min and reduced CFU/mL 1 log after 120 min had elapsed. This antimicrobial peptide permeabilized the E. coli membrane by interaction with membrane phospholipids, mainly phosphatidylethanolamine, inhibited cell division and induced filamentation, suggesting two different targets of action within a bacterial cell. Cytotoxicity studies revealed that peptide 35409 had low hemolytic activity and was not cytotoxic for two human cell lines. We would thus propose, in the light of these findings, that the peptide 35409 sequence should provide a promising template for designing broad-spectrum antimicrobial peptides

A new synthetic peptide having two target of antibacterial action in E. coli ML35

The increased resistance of microorganisms to the different antimicrobials available to today has highlighted the need to find new therapeutic agents, including natural and/or synthetic antimicrobial peptides (AMPs). This study has evaluated the antimicrobial activity of synthetic peptide 35409 (RYRRKKKMKKALQYIKLLKE) against Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 15442 and Escherichia coli ML 35 (ATCC 43827). The results have shown that peptide 35409 inhibited the growth of these three bacterial strains, having 16-fold greater activity against E. coli and P. aeruginosa, but requiring less concentration regarding E. coli (22 μM). When analyzing this activity against E. coli compared to time taken, it was found that this peptide inhibited bacterial growth during the first 60 min and reduced CFU/mL 1 log after 120 min had elapsed. This AMP permeabilized the E. coli membrane by interaction with membrane phospholipids, mainly phosphatidylethanolamine, inhibited cell division and induced filamentation, suggesting two different targets of action within a bacterial cell. Cytotoxicity studies revealed that peptide 35409 had low hemolytic activity and was not cytotoxic for two human cell lines. We would thus propose, in the light of these findings, that the peptide 35409 sequence should provide a promising template for designing broad-spectrum AMPs. © 2016 Barreto-Santamaría, Curtidor, Arévalo-Pinzón, Herrera, Suárez, Pérez and Patarroyo

A new synthetic peptide having two target of antibacterial action in E. coli ML35

The increased resistance of microorganisms to the different antimicrobials available to today has highlighted the need to find new therapeutic agents, including natural and/or synthetic antimicrobial peptides (AMPs). This study has evaluated the antimicrobial activity of synthetic peptide 35409 (RYRRKKKMKKALQYIKLLKE) against Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 15442 and Escherichia coli ML 35 (ATCC 43827). The results have shown that peptide 35409 inhibited the growth of these three bacterial strains, having 16-fold greater activity against E. coli and P. aeruginosa, but requiring less concentration regarding E. coli (22 μM). When analyzing this activity against E. coli compared to time taken, it was found that this peptide inhibited bacterial growth during the first 60 min and reduced CFU/mL 1 log after 120 min had elapsed. This AMP permeabilized the E. coli membrane by interaction with membrane phospholipids, mainly phosphatidylethanolamine, inhibited cell division and induced filamentation, suggesting two different targets of action within a bacterial cell. Cytotoxicity studies revealed that peptide 35409 had low hemolytic activity and was not cytotoxic for two human cell lines. We would thus propose, in the light of these findings, that the peptide 35409 sequence should provide a promising template for designing broad-spectrum AMPs. © 2016 Barreto-Santamaría, Curtidor, Arévalo-Pinzón, Herrera, Suárez, Pérez and Patarroyo

Plasmodium falciparum rhoptry neck protein 4 has conserved regions mediating interactions with receptors on human erythrocytes and hepatocyte membrane

Plasmodium falciparum-related malaria represents a serious worldwide public health problem due to its high mortality rates. P. falciparum expresses rhoptry neck protein 4 (PfRON4) in merozoite and sporozoite rhoptries, it participates in tight junction-TJ formation via the AMA-1/RON complex and is refractory to complete genetic deletion. Despite this, which PfRON4 key regions interact with host cells remain unknown; such information would be useful for combating falciparum malaria. Thirty-two RON4 conserved region-derived peptides were chemically synthesised for determining and characterising PfRON4 regions having high host cell binding affinity (high activity binding peptides or HABPs). Receptor-ligand interaction/binding assays determined their specific binding capability, the nature of their receptors and their ability to inhibit in vitro parasite invasion. Peptides 42477, 42479, 42480, 42505 and 42513 had greater than 2% erythrocyte binding activity, whilst peptides 42477 and 42480 specifically bound to HepG2 membrane, both of them having micromolar and submicromolar range dissociation constants (Kd). Cell-peptide interaction was sensitive to treating erythrocytes with trypsin and/or chymotrypsin and HepG2 with heparinase I and chondroitinase ABC, suggesting protein-type (erythrocyte) and heparin and/or chondroitin sulphate proteoglycan receptors (HepG2) for PfRON4. Erythrocyte invasion inhibition assays confirmed HABPs’ importance during merozoite invasion. PfRON4 800–819 (42477) and 860–879 (42480) regions specifically interacted with host cells, thereby supporting their inclusion in a subunit-based, multi-antigen, multistage anti-malarial vaccine

Shorter antibacterial peptide having high selectivity for e. Coli membranes and low potential for inducing resistance

Antimicrobial peptides (AMPs) have been recognised as a significant therapeutic option for mitigating resistant microbial infections. It has been found recently that Plasmodium falciparum-derived, 20 residue long, peptide 35409 had antibacterial and haemolytic activity, making it an AMP having reduced selectivity, and suggesting that it should be studied more extensively for obtaining new AMPs having activity solely targeting the bacterial membrane. Peptide 35409 was thus used as template for producing short synthetic peptides (<20 residues long) and evaluating their biological activity and relevant physicochemical characteristics for therapeutic use. Four of the sixteen short peptides evaluated here had activity against E. coli without any associated haemolytic effects. The 35409-1 derivative (17 residues long) had the best therapeutic characteristics as it had high selectivity for bacterial cells, stability in the presence of human sera, activity against E. coli multiresistant clinical isolates and was shorter than the original sequence. It had a powerful membranolytic effect and low potential for inducing resistance in bacteria. This peptide’s characteristics highlighted its potential as an alternative for combating infection caused by E. coli multiresistant bacteria and/or for designing new AMPs