Structural features governing the activity of lactoferricin-derived peptides that act in synergy with antibiotics against Pseudomonas aeruginosa in vitro and in vivo

Pseudomonas aeruginosa is naturally resistant to many antibiotics, and infections caused by this organism are a serious threat, especially to hospitalized patients. The intrinsic low permeability of P. aeruginosa to antibiotics results from the coordinated action of several mechanisms, such as the presence of restrictive porins and the expression of multidrug efflux pump systems. Our goal was to develop antimicrobial peptides with an improved bacterial membrane-permeabilizing ability, so that they enhance the antibacterial activity of antibiotics. We carried out a structure activity relationship analysis to investigate the parameters that govern the permeabilizing activity of short (8- to 12-amino-acid) lactoferricin-derived peptides. We used a new class of constitutional and sequence-dependent descriptors called PEDES (peptide descriptors from sequence) that allowed us to predict (Spearman's ρ = 0.74; P < 0.001) the permeabilizing activity of a new peptide generation. To study if peptide-mediated permeabilization could neutralize antibiotic resistance mechanisms, the most potent peptides were combined with antibiotics, and the antimicrobial activities of the combinations were determined on P. aeruginosa strains whose mechanisms of resistance to those antibiotics had been previously characterized. A subinhibitory concentration of compound P2-15 or P2-27 sensitized P. aeruginosa to most classes of antibiotics tested and counteracted several mechanisms of antibiotic resistance, including loss of the OprD porin and overexpression of several multidrug efflux pump systems. Using a mouse model of lethal infection, we demonstrated that whereas P2-15 and erythromycin were unable to protect mice when administered separately, concomitant administration of the compounds afforded long-lasting protection to one-third of the animals

Enhancement of endotoxin neutralization by coupling of a C12-alkyl chain to a lactoferricin-derived peptide

Antibacterial peptide acylation, which mimics the structure of the natural lipopeptide polymyxin B, increases antimicrobial and endotoxin-neutralizing activities. The interaction of the lactoferricin-derived peptide LF11 and its N-terminally acylated analogue, lauryl-LF11, with different chemotypes of bacterial lipopolysaccharide (LPS Re, Ra and smooth S form) was investigated by biophysical means and was related to the peptides' biological activities. Both peptides exhibit high antibacterial activity against the three strains of Salmonella enterica differing in the LPS chemotype. Lauryl-LF11 has one order of magnitude higher activity against Re-type, but activity against Ra- and S-type bacteria is comparable with that of LF11. The alkyl derivative peptide lauryl-LF11 shows a much stronger inhibition of the LPS-induced cytokine induction in human mononuclear cells than LF11. Although peptide-LPS interaction is essentially of electrostatic nature, the lauryl-modified peptide displays a strong hydrophobic component. Such a feature might then explain the fact that saturation of the peptide binding takes place at a much lower peptide/LPS ratio for LF11 than for lauryl-LF11, and that an overcompensation of the negative LPS backbone charges is observed for lauryl-LF11. The influence of LF11 on the gel-to-liquid-crystalline phase-transition of LPS is negligible for LPS Re, but clearly fluidizing for LPS Ra. In contrast, lauryl-LF11 causes a cholesterol-like effect in the two chemotypes, fluidizing in the gel and rigidifying of the hydrocarbon chains in the liquid-crystalline phase. Both peptides convert the mixed unilamellar/non-lamellar aggregate structure of lipid A, the 'endotoxic principle' of LPS, into a multilamellar one. These data contribute to the understanding of the mechanisms of the peptide-mediated neutralization of endotoxin and effect of lipid modification of peptides

Comparative analysis of selected methods for the assessment of antimicrobial and membrane-permeabilizing activity: a case study for lactoferricin derived peptides

Growing concerns about bacterial resistance to antibiotics have prompted the development of alternative therapies like those based on cationic antimicrobial peptides (APs). These compounds not only are bactericidal by themselves but also enhance the activity of antibiotics. Studies focused on the systematic characterization of APs are hampered by the lack of standard guidelines for testing these compounds. We investigated whether the information provided by methods commonly used for the biological characterization of APs is comparable, as it is often assumed. For this purpose, we determined the bacteriostatic, bactericidal, and permeability-increasing activity of synthetic peptides (n = 57; 9-13 amino acid residues in length) analogous to the lipopolysaccharide-binding region of human lactoferricin by a number of the most frequently used methods and carried out a comparative analysis. RESULTS: While the minimum inhibitory concentration determined by an automated turbidimetry-based system (Bioscreen) or by conventional broth microdilution methods did not differ significantly, bactericidal activity measured under static conditions in a low-ionic strength solvent resulted in a vast overestimation of antimicrobial activity. Under these conditions the degree of antagonism between the peptides and the divalent cations differed greatly depending on the bacterial strain tested. In contrast, the bioactivity of peptides was not affected by the type of plasticware (polypropylene vs. polystyrene). Susceptibility testing of APs using cation adjusted Mueller-Hinton was the most stringent screening method, although it may overlook potentially interesting peptides. Permeability assays based on sensitization to hydrophobic antibiotics provided overall information analogous - though not quantitatively comparable- to that of tests based on the uptake of hydrophobic fluorescent probes. CONCLUSION: We demonstrate that subtle changes in methods for testing cationic peptides bring about marked differences in activity. Our results show that careful selection of the test strains for susceptibility testing and for screenings of antibiotic-sensitizing activity is of critical importance. A number of peptides proved to have potent permeability-increasing activity at subinhibitory concentrations and efficiently sensitized Pseudomonas aeruginosa both to hydrophilic and hydrophobic antibiotics

Structural features governing the activity of lactoferricin-derived peptides that act in synergy with antibiotics against Pseudomonas aeruginosa in vitro and in vivo

Pseudomonas aeruginosa is naturally resistant to many antibiotics, and infections caused by this organism are a serious threat, especially to hospitalized patients. The intrinsic low permeability of P. aeruginosa to antibiotics results from the coordinated action of several mechanisms, such as the presence of restrictive porins and the expression of multidrug efflux pump systems. Our goal was to develop antimicrobial peptides with an improved bacterial membrane-permeabilizing ability, so that they enhance the antibacterial activity of antibiotics. We carried out a structure activity relationship analysis to investigate the parameters that govern the permeabilizing activity of short (8- to 12-amino-acid) lactoferricin-derived peptides. We used a new class of constitutional and sequence-dependent descriptors called PEDES (peptide descriptors from sequence) that allowed us to predict (Spearman's ρ = 0.74; P < 0.001) the permeabilizing activity of a new peptide generation. To study if peptide-mediated permeabilization could neutralize antibiotic resistance mechanisms, the most potent peptides were combined with antibiotics, and the antimicrobial activities of the combinations were determined on P. aeruginosa strains whose mechanisms of resistance to those antibiotics had been previously characterized. A subinhibitory concentration of compound P2-15 or P2-27 sensitized P. aeruginosa to most classes of antibiotics tested and counteracted several mechanisms of antibiotic resistance, including loss of the OprD porin and overexpression of several multidrug efflux pump systems. Using a mouse model of lethal infection, we demonstrated that whereas P2-15 and erythromycin were unable to protect mice when administered separately, concomitant administration of the compounds afforded long-lasting protection to one-third of the animals