Biophysical mechanisms of endotoxin neutralization by cationic amphiphilic peptides

Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS

Diseño racional de péptidos derivados de la lactoferricina humana con actividad permeabilizante mejorada y capacidad de producir Permeabilidad Asociada al Efecto Postantibiótico

Para la realización de este estudio, se establecieron los siguientes objetivos: 1. Desarrollar un modelo cuantitativo que relacione la estructura química con la actividad permeabilizante de los péptidos disponibles actualmente y diseñar una nueva generación de péptidos con capacidad permeabilizante mejorada. 2. Determinar la actividad permeabilizante y antimicrobiana de los nuevos péptidos frente a P. aeruginosa y caracterizar su espectro de acción frente a otras especies bacterianas. 3. Caracterizar la capacidad neutralizante de endotoxina de los nuevos péptidos y estudiar posibles asociaciones entre parámetros con interés terapéutico. 4. Determinar la toxicidad y actividad hemolítica de los nuevos péptidos. 5. Estudiar las relaciones estructura-actividad y establecer las bases estructurales que determinan la actividad permeabilizante de péptidos catiónicos sobre P. aeruginosa. 6. Seleccionar los mejores péptidos permeabilizantes y evaluar su capacidad para potenciar antibióticos in vitro y en un modelo animal de sepsis por P. aeruginosa. 7. Determinar si los péptidos pueden producir efecto postantibiótico y si la presencia simultánea de antibióticos en las terapias combinadas puede potenciar el efecto post-antibiótico de los péptidos

A synthetic peptide sensitizes multi-drug resistant pseudomonas aeruginosa to antibiotics for more than two hours and permeabilizes its envelope for twenty hours

Background: Pseudomonas aeruginosa is a Gram-negative pathogen that frequently causes life-threatening infections in immunocompromised patients. We previously showed that subinhibitory concentrations of short synthetic peptides permeabilize P. aeruginosa and enhance the lethal action of co-administered antibiotics. Methods: Long-term permeabilization caused by exposure of multidrug-resistant P. aeruginosa strains to peptide P4–9 was investigated by measuring the uptake of several antibiotics and fluorescent probes and by using confocal imaging and atomic force microscopy. Results: We demonstrated that P4–9, a 13-amino acid peptide, induces a growth delay (i.e. post-antibiotic effect) of 1.3 h on a multidrug-resistant P. aeruginosa clinical isolate. Remarkably, when an independently P4–9-treated culture was allowed to grow in the absence of the peptide, cells remained sensitive to subinhibitory concentrations of antibiotics such as ceftazidime, fosfomycin and erythromycin for at least 2 h. We designated this persistent sensitization to antibiotics occurring in the absence of the sensitizing agent as Post-Antibiotic Effect associated Permeabilization (PAEP). Using atomic force microscopy, we showed that exposure to P4–9 induces profound alterations on the bacterial surface and that treated cells need at least 2 h of growth to repair those lesions. During PAEP, P. aeruginosa mutants overexpressing either the efflux pump MexAB-OprM system or the AmpC β-lactamase were rendered sensitive to antibiotics that are known substrates of those mechanisms of resistance. Finally, we showed for the first time that the descendants of bacteria surviving exposure to a membrane disturbing peptide retain a significant level of permeability to hydrophobic compounds, including propidium iodide, even after 20 h of growth in the absence of the peptide. Conclusions: The phenomenon of long-term sensitization to antibiotics shown here may have important therapeutic implications for a combined peptide-antibiotic treatment because the peptide would not need to be present to exert its antibiotic enhancing activity as long as the target organism retains sensitization to the antibiotic

A synthetic peptide sensitizes multi-drug resistant pseudomonas aeruginosa to antibiotics for more than two hours and permeabilizes its envelope for twenty hours

Background: Pseudomonas aeruginosa is a Gram-negative pathogen that frequently causes life-threatening infections in immunocompromised patients. We previously showed that subinhibitory concentrations of short synthetic peptides permeabilize P. aeruginosa and enhance the lethal action of co-administered antibiotics. Methods: Long-term permeabilization caused by exposure of multidrug-resistant P. aeruginosa strains to peptide P4–9 was investigated by measuring the uptake of several antibiotics and fluorescent probes and by using confocal imaging and atomic force microscopy. Results: We demonstrated that P4–9, a 13-amino acid peptide, induces a growth delay (i.e. post-antibiotic effect) of 1.3 h on a multidrug-resistant P. aeruginosa clinical isolate. Remarkably, when an independently P4–9-treated culture was allowed to grow in the absence of the peptide, cells remained sensitive to subinhibitory concentrations of antibiotics such as ceftazidime, fosfomycin and erythromycin for at least 2 h. We designated this persistent sensitization to antibiotics occurring in the absence of the sensitizing agent as Post-Antibiotic Effect associated Permeabilization (PAEP). Using atomic force microscopy, we showed that exposure to P4–9 induces profound alterations on the bacterial surface and that treated cells need at least 2 h of growth to repair those lesions. During PAEP, P. aeruginosa mutants overexpressing either the efflux pump MexAB-OprM system or the AmpC β-lactamase were rendered sensitive to antibiotics that are known substrates of those mechanisms of resistance. Finally, we showed for the first time that the descendants of bacteria surviving exposure to a membrane disturbing peptide retain a significant level of permeability to hydrophobic compounds, including propidium iodide, even after 20 h of growth in the absence of the peptide. Conclusions: The phenomenon of long-term sensitization to antibiotics shown here may have important therapeutic implications for a combined peptide-antibiotic treatment because the peptide would not need to be present to exert its antibiotic enhancing activity as long as the target organism retains sensitization to the antibiotic

Biophysical mechanisms of endotoxin neutralization by cationic amphiphilic peptides

Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS

New antiseptic peptides to protect against endotoxin-mediated shock

Systemic bacterial infections are associated with high mortality. The access of bacteria or constituents thereof to systemic circulation induces the massive release of immunomodulatory mediators, ultimately causing tissue hypoperfusion and multiple-organ failure despite adequate antibiotic treatment. Lipid A, the "endotoxic principle" of bacterial lipopolysaccharide (LPS), is one of the major bacterial immunostimuli. Here we demonstrate the biological efficacy of rationally designed new synthetic antilipopolysaccharide peptides (SALPs) based on the Limulus anti-LPS factor for systemic application. We show efficient inhibition of LPS-induced cytokine release and protection from lethal septic shock in vivo, whereas cytotoxicity was not observed under physiologically relevant conditions and concentrations. The molecular mechanism of LPS neutralization was elucidated by biophysical techniques. The lipid A part of LPS is converted from its "endotoxic conformation," the cubic aggregate structure, into an inactive multilamellar structure, and the binding affinity of the peptide to LPS exceeds those of known LPS-binding proteins, such as LPS-binding protein (LBP). Our results thus delineate a novel therapeutic strategy for the clinical management of patients with septic shock