RESUMO
Aurein 2.1, aurein 2.6 and aurein 3.1 are amphibian host defence peptides that kill bacteria via the use of lytic amphiphilic α-helical structures. The C-terminal PEGylation of these peptides led to decreased antibacterial activity (Minimum Lethal Concentration (MLCs) ↓ circa one and a half to threefold), reduced levels of amphiphilic α-helical structure in solvents (α-helicity ↓ circa 15.0%) and lower surface activity (Δπ ↓ > 1.5 mN m-1). This PEGylation of aureins also led to decreased levels of amphiphilic α-helical structure in the presence of anionic membranes and zwitterionic membranes (α-helicity↓ > 10.0%) as well as reduced levels of penetration (Δπ ↓ > 3.0 mN m-1) and lysis (lysis ↓ > 10.0%) of these membranes. Based on these data, it was proposed that the antibacterial action of PEGylated aureins involved the adoption of α-helical structures that promote the lysis of bacterial membranes, but with lower efficacy than their native counterparts. However, PEGylation also reduced the haemolytic activity of native aureins to negligible levels (haemolysis ↓ from circa 10% to 3% or less) and improved their relative therapeutic indices (RTIs ↑ circa three to sixfold). Based on these data, it is proposed that PEGylated aureins possess the potential for therapeutic development; for example, to combat infections due to multi-drug resistant strains of S. aureus, designated as high priority by the World Health Organization.
Assuntos
Proteínas de Anfíbios/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Anfíbios/farmacologia , Anfíbios/genética , Animais , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Hemólise/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Polietilenoglicóis/química , Staphylococcus aureus/efeitos dos fármacosRESUMO
Here the hypothesis that linearized esculentin 2EM (E2EM-lin) from Glandirana emeljanovi possesses pH dependent activity is investigated. The peptide showed weak activity against Gram-negative bacteria (MLCs ≥ 75.0 µM) but potent efficacy towards Gram-positive bacteria (MLCs ≤ 6.25 µM). E2EM-lin adopted an α-helical structure in the presence of bacterial membranes that increased as pH was increased from 6 to 8 (↑ 15.5-26.9%), whilst similar increases in pH enhanced the ability of the peptide to penetrate (↑ 2.3-5.1 mN m-1) and lyse (↑ 15.1-32.5%) these membranes. Theoretical analysis predicted that this membranolytic mechanism involved a tilted segment, that increased along the α-helical long axis of E2EM-lin (1-23) in the N â C direction, with - < µH > increasing overall from circa - 0.8 to - 0.3. In combination, these data showed that E2EM-lin killed bacteria via novel mechanisms that were enhanced by alkaline conditions and involved the formation of tilted and membranolytic, α-helical structure. The preference of E2EM-lin for Gram-positive bacteria over Gram-negative organisms was primarily driven by the superior ability of phosphatidylglycerol to induce α-helical structure in the peptide as compared to phosphatidylethanolamine. These data were used to generate a novel pore-forming model for the membranolytic activity of E2EM-lin, which would appear to be the first, major reported instance of pH dependent AMPs with alkaline optima using tilted structure to drive a pore-forming process. It is proposed that E2EM-lin has the potential for development to serve purposes ranging from therapeutic usage, such as chronic wound disinfection, to food preservation by killing food spoilage organisms.
Assuntos
Proteínas de Anfíbios , Antibacterianos , Peptídeos Catiônicos Antimicrobianos , Bactérias Gram-Positivas/crescimento & desenvolvimento , Proteínas de Anfíbios/química , Proteínas de Anfíbios/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Concentração de Íons de Hidrogênio , Conformação Proteica em alfa-HéliceRESUMO
Maximin H5 is an anionic antimicrobial peptide from amphibians, which carries a C-terminal amide moiety, and was found to be moderately haemolytic (20%). The α-helicity of the peptide was 42% in the presence of lipid mimics of erythrocyte membranes and was found able to penetrate (10.8 mN m(-1)) and lyse these model membranes (64 %). In contrast, the deaminated peptide exhibited lower levels of haemolysis (12%) and α-helicity (16%) along with a reduced ability to penetrate (7.8 m Nm(-1)) and lyse (55%) lipid mimics of erythrocyte membranes. Taken with molecular dynamic simulations and theoretical analysis, these data suggest that native maximin H5 primarily exerts its haemolytic action via the formation of an oblique orientated α-helical structure and tilted membrane insertion. However, the C-terminal deamination of maximin H5 induces a loss of tilted α-helical structure, which abolishes the ability of the peptide's N-terminal and C-terminal regions to H-bond and leads to a loss in haemolytic ability. Taken in combination, these observations strongly suggest that the C-terminal amide moiety carried by maximin H5 is required to stabilise the adoption of membrane interactive tilted structure by the peptide. Consistent with previous reports, these data show that the efficacy of interaction and specificity of maximin H5 for membranes can be attenuated by sequence modification and may assist in the development of variants of the peptide with the potential to serve as anti-infectives.
Assuntos
Amidas/farmacologia , Proteínas de Anfíbios/farmacologia , Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Eritrocítica/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Amidas/química , Amidas/toxicidade , Proteínas de Anfíbios/química , Proteínas de Anfíbios/toxicidade , Antibacterianos/química , Antibacterianos/toxicidade , Permeabilidade da Membrana Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Escherichia coli/crescimento & desenvolvimento , Hemólise/efeitos dos fármacos , Humanos , Lipídeos de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/toxicidade , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Staphylococcus aureus/crescimento & desenvolvimento , Relação Estrutura-Atividade , Fatores de TempoRESUMO
Aurein 2.5 (GLFDIVKKVVGAFGSL-NH2) is an antimicrobial peptide, which was seen to have activity against Stachybotris chartarum, Penicillium roseopurpureum and Aspergillus flavus with minimum fungicidal concentrations in the range 250-500 µM. S. chartarum showed enhanced susceptibility to lysis as compared to P. roseopurpureum and A. flavus, (44, 26 and 28 % respectively). Monolayers formed from lipid membrane extracts derived from S. chartarum, P. roseopurpureum and A. flavus showed maximal surface pressure changes of 13.5, 10.3 and 10.2 mN m(-1) respectively. However, aurein 2.5 adopted similar levels of α-helical structure (circa 45 %) in the presence of vesicles formed from membrane lipid extracts derived from all three fungi. These data imply that differential activity is not due to targeting and membrane association but linked to the ability of the bound peptide to lyse the cells. At sterol levels mimetic of eukaryotic systems, high levels of α-helical structure (circa 50 %) were also observed and hence similar binding. However, enhanced sterol levels (>0.6) led to a reduction in monolayer membrane interaction, suggesting that the sterols influence efficacy. Consistent with this suggestion, thermodynamic analysis showed that the peptide was able to destabilise model fungal monolayers, as indicated by negative values of ∆Gmix.
Assuntos
Antifúngicos/metabolismo , Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Fungos/citologia , Antifúngicos/química , Peptídeos Catiônicos Antimicrobianos/química , Fungos/efeitos dos fármacos , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Host defense peptides show great potential for development as new antimicrobial agents with novel mechanisms of action. However, a small number of resistance mechanisms to their action are known, and here, we report a novel bacterial resistance mechanism mediated by a lipid receptor. Maximin H5 from Bombina maxima bound anionic and zwitterionic membranes with low affinity (Kd > 225 µM) while showing a strong ability to lyse (>55%) and penetrate (π > 6.0 mN m(-1)) these membranes. However, the peptide bound Escherichia coli and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) membranes with higher affinity (Kd < 65 µM) and showed a very low ability for bilayer lysis (<8%) and partitioning (π > 1.0 mN m(-1)). Increasing levels of membrane DMPE correlated with enhanced binding by the peptide (R(2) = 0.96) but inversely correlated with its lytic ability (R(2) = 0.98). Taken with molecular dynamic simulations, these results suggest that maximin H5 possesses membranolytic activity, primarily involving bilayer insertion of its strongly hydrophobic N-terminal region. However, this region was predicted to form multiple hydrogen bonds with phosphate and ammonium groups within PE head-groups, which in concert with charge-charge interactions anchor the peptide to the surface of E. coli membranes, inhibiting its membranolytic action.
Assuntos
Resistência Microbiana a Medicamentos , Lipídeos/química , Peptídeos/química , Receptores de Superfície Celular/química , Antibacterianos/farmacologia , Dicroísmo Circular , Simulação de Dinâmica Molecular , Peptídeos/farmacologiaRESUMO
Fungal infections with multiple resistance to conventional antifungals are increasingly becoming a medical problem, and there is an urgent need for new antifungal compounds with novel mechanisms of action. Here, we show that aurein 2.5, a naturally occurring peptide antibiotic, displays activity against the fungal strains: Rhodotorula rubra and Schizosaccharomyces pombe (MICs < 130 µM). The peptide adopted high levels of membrane-interactive α-helical structure (> 65%) in the presence of lipid membranes derived from these organisms and showed strong propensities to penetrate (π ≥ 13 mN m(-1) ) and lyse them (> 70%). Based on these data, we suggest that aurein 2.5 kills yeasts via membranolytic mechanisms and may act as a template for the development of therapeutically useful antifungal agents.
Assuntos
Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Leveduras/efeitos dos fármacos , Antifúngicos/química , Antifúngicos/metabolismo , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Lipídeos/química , Lipídeos/isolamento & purificação , Estrutura Secundária de Proteína , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismoRESUMO
In order to gain an insight into the mechanism of antimicrobial peptide action, aurein 2.5 and modelin-5 were studied. When tested against Staphylococcus aureus, aurein 2.5 showed approximately 5-fold greater efficacy even though the higher net positive charge and higher helix stability shown by modelin-5 would have predicated modelin-5 to be the more effective antimicrobial. However, in the presence of S. aureus membrane mimics, aurein 2.5 showed greater helical content (75% helical) relative to modelin-5 (51% helical) indicative of increase in membrane association. This was supported by monolayer data showing that aurein 2.5 (6.6mNm(-1)) generated greater pressure changes than modelin-5 (5.3mNm(-1)). Peptide monolayers indicted that modelin-5 formed a helix horizontal to the plane of an asymmetric interface which would be supported by the even distribution of charge and hydrophobicity along the helical long axis and facilitate lysis by non-specific membrane binding. In contrast, a groove structure observed on the surface of aurein 2.5 was predicted to be the cause of enhanced lipid binding (K(d)=75µM) relative to modelin-5 (K(d)=118µM) and the balance of hydrophobicity along the aurein 2.5 long axis supported deep penetration into the membrane in a tilt formation. This oblique orientation generates greater lytic efficacy in high anionic lipid (71%) compared to modelin-5 (32%).
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Bicamadas Lipídicas/química , Staphylococcus aureus/metabolismo , Biofísica/métodos , Membrana Celular/metabolismo , Dicroísmo Circular , Escherichia coli/metabolismo , Cinética , Lipídeos/química , Peptídeos/química , Fosfatidilgliceróis/análise , Fosfolipídeos/química , Ligação Proteica , Estrutura Secundária de Proteína , Propriedades de Superfície , Termodinâmica , Fatores de TempoRESUMO
Aurein 2.5 is a naturally C-terminally amidated amphibian antimicrobial peptide. C-terminal amidation can increase efficacy and hence a comparison was made between aurein 2.5-CONH2 and its nonamidated analogue. Amidation of the C-terminal carboxyl of aurein 2.5 enhanced antimicrobial activity 2.5- fold against Klebsiella pneumonia. Our results demonstrate that both peptide analogues had high surface activities (23 mN m-1for aurein 2.5-COOH and 26 mN m-1 aurein 2.5-CONH2). Circular dichroism measurements suggest that the helical content of the amidated form, in the presence of trifluoroethanol, was significantly enhanced (33.66 % for aurein 2.5-COOH and 60.89 % aurein 2.5-CONH2). The interaction of aurein 2.5 with bacterial cell membrane mimics was investigated using Langmuir monolayers. Aurein 2.5-CONH2 induced stable surface pressure changes in monolayers formed from K. pneumonia (circa 4.7 mN m-1), however, lower surface pressure changes were observed for aurein 2.5- COOH (circa 3.8 mN m-1). The data shows that in the case of aurein 2.5, amidation is able to enhance antibacterial activity and it is proposed that the increase in effectiveness is due to stabilization of the α-helical structure at the membrane interface.
Assuntos
Antibacterianos/metabolismo , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Amidas/química , Amidas/metabolismo , Amidas/farmacologia , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Fenômenos Bioquímicos , Dicroísmo Circular , Klebsiella pneumoniae/efeitos dos fármacos , Lipídeos de Membrana/metabolismo , Membranas Artificiais , Modelos Biológicos , Conformação ProteicaRESUMO
Aurein 2.5 (GLFDIVKKVVGAFGSL-NH(2)) is an uncharacterised antimicrobial peptide. At an air/water interface, it exhibited strong surface activity (maximal surface pressure 25mNm(-1)) and molecular areas consistent with the adoption of alpha-helical structure orientated either perpendicular (1.72nm(2)molecule(-1)) or parallel (3.6nm(2)molecule(-1)) to the interface. Aurein 2.5 was strongly antibacterial, exhibiting a minimum inhibitory concentration (MIC) of 30microM against Bacillus subtilis and Escherichia coli. The peptide induced maximal surface pressure changes of 9mNm(-1) and 5mNm(-1), respectively, in monolayers mimicking membranes of these organisms whilst compression isotherm analysis of these monolayers showed DeltaG(Mix)>0, indicating destabilisation by Aurein 2.5. These combined data suggested that toxicity of the peptide to these organisms may involve membrane invasion via the use of oblique orientated alpha-helical structure. The peptide induced strong, comparable maximal surface changes in monolayers of DOPG (7.5mNm(-1)) and DOPE monolayers (6mNm(-1)) suggesting that the membrane interactions of Aurein 2.5 were driven by amphiphilicity rather than electrostatic interaction. Based on these data, it was suggested that the differing ability of Aurein 2.5 to insert into membranes of B. subtilis and E. coli was probably related to membrane-based factors such as differences in lipid packing characteristics. The peptide was active against both sessile E. coli and Staphylococcus aureus with an MIC of 125microM. The broad-spectrum antibacterial activity and non-specific modes of membrane action used by Aurein 2.5 suggested use as an anti-biofilm agent such as in the decontamination of medical devices.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Bacillus subtilis/citologia , Bacillus subtilis/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Lipídeos/química , Plâncton/citologia , Plâncton/efeitos dos fármacos , Pressão , Propriedades de Superfície/efeitos dos fármacos , Temperatura , TermodinâmicaRESUMO
VP1, a putative alpha-helical antimicrobial peptide (alpha-AMP) inhibited growth of Bacillus subtilis and Escherichia coli at 500microM. The peptide induced stable surface pressure changes in monolayers formed from B. subtilis native lipid extract (circa 4.5mNm(-1)) but transient pressure changes in corresponding E. coli monolayers (circa 1.0mNm(-1)), which led to monolayer disintegration. Synthetic lipid monolayers mimetic of the extracts were used to generate compression isotherms. Thermodynamic analysis of B. subtilis isotherms indicated membrane stabilisation by VP1 (DeltaG(Mix)<0), via a mechanism dependent upon the phosphatidylglycerol to cardiolipin ratio. Corresponding analysis of E. coli isotherms indicated membrane destabilisation by the peptide (DeltaG(Mix)>0). Destabilisation correlated with PE levels present and appeared to involve a mechanism resembling those used by tilted peptides. These data emphasise that structure/function analysis of alpha-AMPs must consider not only their structural characteristics but also the lipid make-up of the target microbial membrane.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Lipídeos de Membrana/química , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Sequência de Aminoácidos , Bacillus subtilis/química , Bacillus subtilis/efeitos dos fármacos , Escherichia coli/química , Escherichia coli/efeitos dos fármacos , Dados de Sequência Molecular , Pressão , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície , TermodinâmicaRESUMO
Theoretical analysis indicates that peptide VP1 forms a membrane interactive amphiphilic alpha-helix with antibacterial properties. Fourier transform infra-red based analyses showed VP1 to be alpha-helical (45%) in the presence of vesicle mimics of membranes from Staphylococcus aureus and to induce increases in the fluidity of these vesicles, as indicated by a rise in wavenumber of circa 0.5 to 1.0 cm(-1). The peptide induced surface pressure increases of 5 mN m(-1) in monolayer mimics of S. aureus membranes confirm the formation of a membrane interactive alpha-helix. These interactions appeared to involve significant hydrophobic and electrostatic contributions as VP1 induced comparable surface pressure changes in anionic (5.5 mN m(-1)) and zwitterionic (4 mN m(-1)) lipid monolayers. It is suggested that whilst efficacy requires further sequence specific information, the peptides generic structure provides the basis for its broad antimicrobial activity.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/efeitos dos fármacos , Oligopeptídeos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/química , Lipídeos de Membrana/química , Oligopeptídeos/química , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Staphylococcus aureus/ultraestruturaRESUMO
AP1 (GEQGALAQFGEWL) was shown by theoretical analysis to be an anionic oblique-orientated alpha-helix former. The peptide exhibited a monolayer surface area of 1.42 nm(2), implying possession of alpha-helical structure at an air/water interface, and Fourier transform infrared spectroscopy (FTIR) showed the peptide to be alpha-helical (100%) in the presence of vesicle mimics of Escherichia coli membranes. FTIR lipid-phase transition analysis showed the peptide to induce large decreases in the fluidity of these E. coli membrane mimics, and Langmuir-Blodgett trough analysis found the peptide to induce large surface pressure changes in monolayer mimics of E. coli membranes (4.6 mN.m(-1)). Analysis of compression isotherms based on mixing enthalpy (DeltaH) and the Gibbs free energy of mixing (DeltaG(Mix)) predicted that these monolayers were thermodynamically stable (DeltaH and DeltaG(Mix) each negative) but were destabilized by the presence of the peptide (DeltaH and DeltaG(Mix) each positive). The peptide was found to have a minimum lethal concentration of 3 mm against E. coli and was seen to cause lysis of erythrocytes at 5 mm. In combination, these data clearly show that AP1 functions as an anionic alpha-helical antimicrobial peptide and suggest that both its tilted peptide characteristics and the composition of its target membrane are important determinants of its efficacy of action.
Assuntos
Anti-Infecciosos/química , Desenho de Fármacos , Oligopeptídeos/química , Sequência de Aminoácidos , Ânions/química , Ânions/farmacologia , Anti-Infecciosos/farmacologia , Escherichia coli/efeitos dos fármacos , Bicamadas Lipídicas/química , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Oligopeptídeos/genética , Oligopeptídeos/farmacologia , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Termodinâmica , Fatores de TempoRESUMO
The antimicrobial activity of the anionic peptide, AP1 (GEQGALAQFGEWL), was investigated. AP1 was found to kill Staphylococcus aureus with an MLC of 3mM and to induce maximal surface pressure changes of 3.8 mN m(-1) over 1200s in monolayers formed from lipid extract of S. aureus membranes. FTIR spectroscopy showed the peptide to be alpha-helical (100%) in the presence of vesicles formed from this lipid extract and to induce increases in their fluidity (Deltanu circa 0.5 cm(-1)). These combined data show that AP1 is able to function as an alpha-helical antimicrobial peptide against Gram-positive bacteria and suggest that the killing mechanism used by the peptide involves interactions with the membrane lipid headgroup region. Moreover, this killing mechanism differs strongly from that previously reported for AP1 against Gram-negative bacteria, indicating the importance of considering the effects of membrane lipid composition when investigating the structure/function relationships of antimicrobial peptides.