RESUMO
In a previous study, we synthesized a series of peptides containing simple sequence repeats, (KW)(n)-NH(2) (n = 2,3,4 and 5) and determined their antimicrobial and hemolytic activities, as well as their mechanism of antimicrobial action. However, (KW)(5) showed undesirable cytotoxicity against RBC cells. In order to identify the mechanisms behind the hemolytic and cytotoxic activities of (KW)(5), we measured the ability of these peptides to induce aggregation of liposomes. In addition, their binding and permeation activities were assessed by Trp fluorescence, calcein leakage and circular dichrorism using artificial phospholipids that mimic eukaryotic liposomes, including phosphatidylcholine (PC), PC/sphingomyelin (SM) (2:1, w/w) and PC/cholesterol (CH) (2:1, w/w). Experiments confirmed that only (KW)(5) induced aggregation of all liposomes; it formed much larger aggregates with PC:CH (2:1, w/w) than with PC or PC:SM (2:1, w/w). Longer peptide (KW)(5), but not (KW)(3) or (KW)(4), strongly bound and partially inserted into PC:CH compared to PC or PC:SM (2:1, w/w). Calcein release experiments showed that (KW)(5) induced calcein leakage from the eukaryotic membrane. Greater calcein leakage was induced by (KW)(5) from PC:CH than from PC:SM (2:1, w/w) or PC, whereas (KW)(4) did not induce calcein leakage from any of the liposomes. Circular dichroism measurements indicated that (KW)(5) showed higher conformational transition compared to (KW)(4) due to peptide-liposome interactions. Taken together, our results suggest that (KW)(5) reasonably mediates the aggregation and permeabilization of eukaryotic membranes, which could in turn explain why (KW)(5) displays efficient hemolytic activity.
Assuntos
Permeabilidade da Membrana Celular , Membrana Celular/química , Células Eucarióticas/química , Membranas Artificiais , Modelos Químicos , Peptídeos/química , Motivos de Aminoácidos , Lisina/química , Triptofano/químicaRESUMO
Circular dichroism (CD) is a useful technique for monitoring changes in the conformation of antimicrobial peptides or gelatin. In this study, interactions between cationic peptides and gelatin were observed without affecting the triple helical content of the gelatin, which was more strongly affected by anionic surfactant. The peptides did not adopt a secondary structure in the presence of aqueous solution or Tween 80, but a peptide secondary structure formed upon the addition of sodium dodecyl sulfate (SDS). The peptides bound to the phosphate group of lipopolysaccharide (LPS) and displayed an alpha-helical conformation while (KW)(4) adopted a folded conformation. Further, the peptides did not specifically interact with the fungal cell wall components of mannan or laminarin. Tryptophan blue shift assay indicated that these peptides interacted with SDS, LPS, and gelatin but not with Tween 80, mannan, or laminarin. The peptides also displayed antibacterial activity against P. aeruginosa without cytotoxicity against HaCaT cells at MIC, except for HPA3NT3-analog peptide. In this study, we used a CD spectroscopic method to demonstrate the feasibility of peptide characterization in numerous environments. The CD method can thus be used as a screening method of gelatin-peptide interactions for use in wound healing applications.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Dicroísmo Circular/métodos , Gelatina/química , Sequência de Aminoácidos , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/toxicidade , Linhagem Celular , Parede Celular/química , Glucanos/química , Humanos , Lipopolissacarídeos/química , Mananas/química , Dados de Sequência Molecular , Polissorbatos , Conformação Proteica , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/efeitos dos fármacos , Dodecilsulfato de Sódio , Espectrometria de Fluorescência , Tensoativos , Triptofano/químicaRESUMO
In this study, a HPA3NT3-analog (FKKLKKLFKKILKLK-NH2) peptide was designed. In this analog, two Trp residues (positions 12, 14) were replaced with Leu, and Arg and Asn (positions 3, 13) were replaced with Lys to investigate the role of amino acid substitution and increased cationicity on antimicrobial and hemolytic activities. In fungal and Gram-negative bacterial cells, HPA3NT3-analog activity was unchanged or slightly enhanced when compared to the HPA3NT3 peptide. In addition, a twofold decrease in activity against Gram-positive bacteria was observed. The HPA3NT3-analog also induced less hemolysis (4.2%) than the HPA3NT3 peptide (71%) at 200 microM. Circular dichroism (CD) spectra revealed that the HPA3NT3-analog peptide had an unordered structure in buffer and egg yolk L-2-phosphatidyl choline (EYPC), but adapted an alpha-helical conformation in 50% 2,2,2-trifluoroethanol (TFE) and negatively charged egg yolk L-2-phosphatidyl glycerol (EYPG), while the parent peptide showed an ordered structure in the EYPC. Additionally, the HPA3NT3-analog peptide induced the leakage of calcein from egg yolk L-2-phosphatidyl ethanolamine (EYPE)/EYPG (7:3 w/w) large unilamellar vesicles (LUVs); however, the activity was slightly weaker than that of the HPA3NT3 peptide. The molecular dynamics (MD) structures revealed that the amino acid substitutions induced a significant variation in peptide structure. These results suggest that the substitutions of Arg and Asn with Lys and two Trp with Leu resulted in small changes in HPA3NT3-analog activity and significant decreases in hemolytic activity.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Candida albicans/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Leucina/química , Lisina/química , Trichosporon/efeitos dos fármacos , Substituição de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Dicroísmo Circular , Simulação por Computador , Fluoresceínas/química , Hemólise/efeitos dos fármacos , Humanos , Lipossomos/química , Testes de Sensibilidade Microbiana , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
HP (2-20) is a 19-aa, amphipathic, α-helical peptide with antimicrobial properties that was derived from the N-terminus of Helicobacter pylori ribosomal protein L1. We previously showed that increasing the net hydrophobicity of HP (2-20) by substituting Trp for Gln(17) and Asp(19) (Anal 3) increased the peptide's antimicrobial activity. In hydrophobic medium, Anal 3 forms an amphipathic structure consisting of an N-terminal random coil region (residues 2-5) and an extended helical region (residues 6-20). To investigate the structure-activity relationship of Anal 3, we substituted Pro for Glu(9) (Anal 3-Pro) and then examined the new peptide's three-dimensional structure, antimicrobial activity and mechanism of action. Anal 3-Pro had an α-helical structure in the presence of trifluoroethanol (TFE) and sodium dodecyl sulfate (SDS). NMR spectroscopic analysis of Anal 3-Pro's tertiary structure in SDS micelles confirmed that the kink potential introduced by Pro(10) was responsible for the helix distortion. We also found that Anal 3-Pro exhibited about 4 times greater antimicrobial activity than Anal 3. Fluorescence activated flow cytometry and confocal fluorescence microscopy showed that incorporating a Pro-hinge into Anal 3 markedly reduced its membrane permeability so that it accumulated in the cytoplasm without remaining in the cell membrane. To investigate the translocation mechanism, we assessed its ability to release of FITC-dextran. The result showed Anal 3-Pro created a pore <1.8 nm in diameter, which is similar to buforin II. Notably, scanning electron microscopic observation of Candida albicans revealed that Anal 3-Pro and buforin II exert similar effects on cell membranes, whereas magainin 2 exerts a different, more damaging, effect. In addition, Anal 3-Pro assumed a helix-hinge-helix structure in the presence of biological membranes and formed micropores in both bacterial and fungal membranes, through which it entered the cytoplasm and tightly bound to DNA. These results indicate that the bending region of Anal 3- Pro peptide is prerequisite for effective antibiotic activity and may facilitate easy penetration of the lipid bilayers of the cell membrane.