Improvement of outer membrane-permeabilizing and lipopolysaccharide-binding activities of an antimicrobial cationic peptide by C-terminal modification

Abstract

Antimicrobial cationic peptides have been discovered in many different organisms and often possess a broad range of activity. In this study, we investigated the mechanisms of actions of melittin and two synthetic peptides, CEME (a cecropin-melittin hybrid) and CEMA, against gram-negative bacteria. CEMA was produced by recombinant DNA procedures and is an analog of CEME with a modified C terminus resulting in two additional positive charges. All three peptides showed good antimicrobial activity against four different gram-negative bacteria, but only CEMA was able to somewhat augment the activity of some conventional antibiotics in synergy studies. Studies using the bacteria Pseudomonas aeruginosa and Enterobacter cloacae showed that the peptides all possessed the ability to permeabilize bacterial outer membranes to the hydrophobic fluorophor 1-N-phenylnaphthylamine and the protein lysozyme, with CEMA being the most active. CEMA also had the strongest relative binding affinity for bacterial endotoxin (lipopolysaccharide).These data collectively indicated that these peptides all cross the outer membrane by the self-promoted uptake pathway and that CEMA is the peptide most effective at accessing this pathway.The past few decades have witnessed the emergence of many different peptide antibiotics, including defensins (19), insect cecropins (5), magainins (42), and melittin (12). Cecropins and melittin belong to the group of antimicrobial peptides that exist in a random-coil configuration in aqueous solutions but adopt a helix-turn-helix structure upon interaction with membranes (9, 34). Both of these peptides have one amphipathic a-helix and one hydrophobic a-helix, but the order of these helices in the two peptides is inverted. Many synthetic peptides have been created in attempts to improve antibacterial activity. Included in these are cecropin-melittin hybrid peptides which possess the amphipathic N-terminal a-helix of cecropin A followed by the hydrophobic N-terminal a-helix of melittin (40). These peptides have been shown to have a broad range of antibacterial activity against both gram-negative and grampositive bacteria (40). Cecropins (6), melittin (36), and the hybrid peptides (41) have all been shown to form ion-permeable channels in lipid membranes.Gram-negative bacteria pose an additional challenge to these peptides in the form of an outer membrane which constitutes a permeability barrier to antibiotics (14,24,37

The Human Antimicrobial Peptide LL-37 Is a Multifunctional Modulator of Innate Immune Responses

“…They also have an affinity for LPS (3)(4)(5)(6). Such cationic peptides can suppress cytokine production in response to endotoxic LPS and to varying extents can prevent lethal endotoxemia (7)(8)(9).…”

Use of steroids to monitor alterations in the outer membrane of Pseudomonas aeruginosa

“…In P. aeruginosa, the permeabilization of the lipid bilayer was studied in assays involving either the crypticity of the periplasmic ␤-lactamase, the partition of NPN into the membrane interior, or the sensitization to noxious products normally excluded from the bacterial cells (hydrophobic antibiotics, anionic detergents, or lysozyme) (18,26,39,53). However, these methods do not provide a direct and accurate measurement of outer membrane permeability.…”

Antimicrobial peptides: mode of action and perspectives of practical application

“…Пептиды связываются с молекулами отрицательно заряженных липополисахаридов (ЛПС) -одного из компонентов внешней мембраны грамотрицательных бактерий [27]. Возмущения, возникающие при этом в ЛПС-слое, образуют так называемый "вертикальный саморегулирующийся канал", облегчающий пептиду проникновение к плазматической мембране [21,28]. Полагают, что при этом АМП создают трещины в слое ЛПС (рис 2А), или связываются с участками в ЛПС (рис 2В), отвечающими за взаимодействие с двухвалентными катионами Ca 2+ и Mg 2+ , необходимыми для стабилизации поверхности клетки и перекрестного связывания отрицательных зарядов ЛПС [29].…”