RESUMO
For cationic antimicrobial peptides to become useful therapeutic agents, it is important to understand their mechanism of action. To obtain high resolution data, this involves studying the structure and membrane interaction of these peptides in tractable model bacterial membranes rather than directly utilizing more complex bacterial surfaces. A number of lipid mixtures have been used as bacterial mimetics, including a range of lipid headgroups, and different ratios of neutral to negatively charged headgroups. Here we examine how the structure and membrane interaction of aurein 2.2 and some of its variants depend on the choice of lipids, and how these models correlate with activity data in intact bacteria (MICs, membrane depolarization). Specifically, we investigated the structure and membrane interaction of aurein 2.2 and aurein 2.3 in 1:1 cardiolipin/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (CL/POPG) (mol/mol), as an alternative to 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)/POPG and a potential model for Gram positive bacteria such as S. aureus. The structure and membrane interaction of aurein 2.2, aurein 2.3, and five variants of aurein 2.2 were also investigated in 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)/POPG (mol/mol) lipids as a possible model for other Gram positive bacteria, such as Bacillus cereus. Solution circular dichroism (CD) results demonstrated that the aurein peptides adopted α-helical structure in all lipid membranes examined, but demonstrated a greater helical content in the presence of POPE/POPG membranes. Oriented CD and ³¹P NMR results showed that the aurein peptides had similar membrane insertion profiles and headgroup disordering effects on POPC/POPG and CL/POPG bilayers, but demonstrated reduced membrane insertion and decreased headgroup disordering on mixing with POPE/POPG bilayers at low peptide concentrations. Since the aurein peptides behaved very differently in POPE/POPG membrane, minimal inhibitory concentrations (MICs) of the aurein peptides in B. cereus strain C737 were determined. The MIC results indicated that all aurein peptides are significantly less active against B. cereus than against S. aureus and S. epidermidis. Overall, the data suggest that it is important to use a relevant model for bacterial membranes to gain insight into the mode of action of a given antimicrobial peptide in specific bacteria.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/química , Bicamadas Lipídicas/química , Membrana Celular/efeitos dos fármacos , Dicroísmo Circular , Bactérias Gram-Positivas/efeitos dos fármacos , Bicamadas Lipídicas/metabolismo , Espectroscopia de Ressonância Magnética , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismoRESUMO
Bacterial adhesion and the succeeding biofilm formation onto surfaces are responsible for implant- and device-associated infections. Bifunctional coatings integrating both nonfouling components and antimicrobial peptides (AMPs) are a promising approach to develop potent antibiofilm coatings. However, the current approaches and chemistry for such coatings are time-consuming and dependent on substrates and involve a multistep process. Also, the information is limited on the influence of the coating structure or its components on the antibiofilm activity of such AMP-based coatings. Here, we report a new strategy to rapidly assemble a stable, potent, and substrate-independent AMP-based antibiofilm coating in a nonfouling background. The coating structure allowed for the screening of AMPs in a relevant nonfouling background to identify optimal peptide combinations that work in cooperation to generate potent antibiofilm activity. The structure of the coating was changed by altering the organization of the hydrophilic polymer chains within the coatings. The coatings were thoroughly characterized using various surface analytical techniques and correlated with the efficiency to prevent biofilm formation against diverse bacteria. The coating method that allowed the conjugation of AMPs without altering the steric protection ability of hydrophilic polymer structure results in a bifunctional surface coating with excellent antibiofilm activity. In contrast, the conjugation of AMPs directly to the hydrophilic polymer chains resulted in a surface with poor antibiofilm activity and increased adhesion of bacteria. Using this coating approach, we further established a new screening method and identified a set of potent surface-tethered AMPs with high activity. The success of this new peptide screening and coating method is demonstrated using a clinically relevant mouse infection model to prevent catheter-associated urinary tract infection (CAUTI).
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Incrustação Biológica/prevenção & controle , Materiais Revestidos Biocompatíveis/farmacologia , Proteínas Imobilizadas/farmacologia , Acrilamidas/química , Animais , Antibacterianos/síntese química , Peptídeos Catiônicos Antimicrobianos/síntese química , Catéteres/microbiologia , Materiais Revestidos Biocompatíveis/síntese química , Humanos , Proteínas Imobilizadas/síntese química , Indóis/química , Masculino , Camundongos Endogâmicos BALB C , Polímeros/química , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Staphylococcus saprophyticus/efeitos dos fármacos , Staphylococcus saprophyticus/fisiologia , Infecções Urinárias/prevenção & controleRESUMO
Catheter-associated urinary tract infections (CAUTIs) are one of the most commonly occurring hospital-acquired infections. Current coating strategies to prevent catheter-associated biofilm formation are limited by their poor long-term efficiency and limited applicability to diverse materials. Here, the authors report a highly effective non-fouling coating with long-term biofilm prevention activity and is applicable to diverse catheters. The thin coating is lubricous, stable, highly uniform, and shows broad spectrum prevention of biofilm formation of nine different bacterial strains and prevents the migration of bacteria on catheter surface. The coating method is adapted to human-sized catheters (both intraluminal and extraluminal) and demonstrates long-term biofilm prevention activity over 30 days in challenging conditions. The coated catheters are tested in a mouse CAUTI model and demonstrate high efficiency in preventing bacterial colonization of both Gram-positive and Gram-negative bacteria. Furthermore, the coated human-sized Foley catheters are evaluated in a porcine CAUTI model and show consistent efficiency in reducing biofilm formation by Escherichia coli (E. coli) over 95%. The simplicity of the coating method, the ability to apply this coating on diverse materials, and the high efficiency in preventing bacterial adhesion increase the potential of this method for the development of next generation infection resistant medical devices.
Assuntos
Infecções Relacionadas a Cateter , Animais , Antibacterianos , Biofilmes , Infecções Relacionadas a Cateter/prevenção & controle , Escherichia coli , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Camundongos , Suínos , Cateteres UrináriosRESUMO
Previous studies on aurein 2.2 and 2.3 in DMPC/DMPG and POPC/POPG membranes have shown that bilayer thickness and phosphatidylglycerol content have a significant impact on the interaction of these peptides with membrane bilayers. Further examination with the DiSC(3)5 assay has indicated that aurein 2.2 induces greater membrane leakage than aurein 2.3 in Staphylococcus aureus C622. The only difference between these peptides is a Leu to Ile mutation at residue 13. To better understand the importance of this residue, the structure and activity of the L13A, L13F, and L13V mutants were investigated. In addition, we investigated a number of peptides with truncations at the C-terminus to determine whether the C-terminus, which contains residue 13, is crucial for antimicrobial activity. Solution circular dichroism results demonstrated that the L13F mutation and the truncation of the C-terminus by six residues resulted in decreased helical content, whereas the L13A or L13V mutation and the truncation of the C-terminus by three residues showed little to no effect on the structure. Oriented circular dichroism results demonstrated that only an extensive C-terminal truncation reduced the ability of the peptide to insert into lipid bilayers. (31)P NMR spectroscopy showed that all peptides disorder the headgroups. The implications of these results in terms of antimicrobial activity and the ability of these peptides to induce leakage in S. aureus are discussed. The results suggest that the presence of the 13th residue in aurein 2.2 is important for structure and activity, but the exact nature of residue 13 is less important as long as it is a hydrophobic residue.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Fenômenos Biofísicos , Dicroísmo Circular , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
The naturally occurring host defense peptide (HDP), aurein 2.2, secreted by the amphibian Litoria aurea, acts as a moderate antibacterial, affecting Gram positive bacteria such as Staphylococcus aureus by forming selective ion pores. In a quest to find more active analogues of aurein 2.2, peptides 73 and 77 were discovered. These peptides were rich in arginine and tryptophan and found to have MICs of 4 µg/mL. Here we examined what impact the increased charge from +2 to +3 and a slight increase in hydrophobic moment relative to aurein 2.2 had on the mechanism of action of these two analogues. Using a time-kill assay, both peptides 73 and 77 were found to kill bacteria more effectively than the parent peptide. Using solution CD and NMR, the peptides were found to not adopt a continuous α-helical structure, i.e. the analogues were not helical from residue 1-13 like the parent peptide. Results obtained from oriented CD (OCD), DiSC35 and pyranine assays and a gel retardation experiment showed that the peptides did not function by membrane perturbation and further showed that peptide 73 and 77 did not interact with DNA. Overall, the data were consistent with these peptides acting as cell penetrating peptides with intracellular targets, which did not appear to be DNA.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Anfíbios/química , Anfíbios , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Penetradores de Células/química , Descoberta de Drogas , Bactérias Gram-Positivas/efeitos dos fármacos , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
The effects of hydrophobic thickness and the molar phosphatidylglycerol (PG) content of lipid bilayers on the structure and membrane interaction of three cationic antimicrobial peptides were examined: aurein 2.2, aurein 2.3 (almost identical to aurein 2.2, except for a point mutation at residue 13), and a carboxy C-terminal analog of aurein 2.3. Circular dichroism results indicated that all three peptides adopt an alpha-helical structure in the presence of a 3:1 molar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPC/DMPG), and 1:1 and 3:1 molar mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG). Oriented circular dichroism data for three different lipid compositions showed that all three peptides were surface-adsorbed at low peptide concentrations, but were inserted into the membrane at higher peptide concentrations. The (31)P solid-state NMR data of the three peptides in the DMPC/DMPG and POPC/POPG bilayers showed that all three peptides significantly perturbed lipid headgroups, in a peptide or lipid composition-dependent manner. Differential scanning calorimetry results demonstrated that both amidated aurein peptides perturbed the overall phase structure of DMPC/DMPG bilayers, but perturbed the POPC/POPG chains less. The nature of the perturbation of DMPC/DMPG bilayers was most likely micellization, and for the POPC/POPG bilayers, distorted toroidal pores or localized membrane aggregate formation. Calcein release assay results showed that aurein peptide-induced membrane leakage was more severe in DMPC/DMPG liposomes than in POPC/POPG liposomes, and that aurein 2.2 induced higher calcein release than aurein 2.3 and aurein 2.3-COOH from 1:1 and 3:1 POPC/POPG liposomes. Finally, DiSC(3)5 assay data further delineated aurein 2.2 from the others by showing that it perturbed the lipid membranes of intact S. aureus C622 most efficiently, whereas aurein 2.3 had the same efficiency as gramicidin S, and aurein 2.3-COOH was the least efficient. Taken together, these data show that the membrane interactions of aurein peptides are affected by the hydrophobic thickness of the lipid bilayers and the PG content.
Assuntos
Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/química , Animais , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Anuros , Benzotiazóis , Carbocianinas , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Dimiristoilfosfatidilcolina/química , Fluoresceínas , Gramicidina/farmacologia , Bicamadas Lipídicas , Potenciais da Membrana , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Estrutura Secundária de Proteína , Staphylococcus aureusRESUMO
Antimicrobial peptides have been the focus of considerable research; however, issues associated with toxicity and aggregation have the potential to limit clinical applications. Here, a derivative of a truncated version of aurein 2.2 (aurein 2.2Δ3), namely peptide 73, was investigated, along with its d-amino acid counterpart (D-73) and a retro-inverso version (RI-73). A version that incorporated a cysteine residue to the C-terminus (73c) was also generated, as this form is required to covalently attach antimicrobial peptides to polymers (e.g., polyethylene glycol (PEG) or hyperbranched polyglycerol (HPG)). The antimicrobial activity of the 73-derived peptides was enhanced 2- to 8-fold, and all the derivatives eradicated preformed Staphylococcus aureus biofilms. Formulation of the peptides with compatible polyethylene glycol (PEG)-modified phospholipid micelles alleviated toxicity toward human cells and reduced aggregation. When evaluated in vivo, the unformulated d-enantiomers aggregated when injected under the skin of mice, but micelle encapsulated peptides were well absorbed. Pegylated micelle formulated peptides were investigated for their potential as therapeutic agents for treating high-density infections in a murine cutaneous abscess model. Formulated peptide 73 reduced abscess size by 36% and bacterial loads by 2.2-fold compared to the parent peptide aurein 2.2Δ3. Micelle encapsulated peptides 73c and D-73 exhibited superior activity, further reducing abscess sizes by 85% and 63% and lowering bacterial loads by 510- and 9-fold compared to peptide 73.
Assuntos
Antibacterianos/administração & dosagem , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Infecções Cutâneas Estafilocócicas/tratamento farmacológico , Animais , Composição de Medicamentos , Feminino , Humanos , Staphylococcus aureus Resistente à Meticilina/fisiologia , Camundongos , Micelas , Testes de Sensibilidade Microbiana , Fosfolipídeos/química , Polietilenoglicóis/química , Infecções Cutâneas Estafilocócicas/microbiologiaRESUMO
Dissecting the mechanism of action of surface-tethered antimicrobial and immunomodulatory peptides is critical to the design of optimized anti-infection coatings on biomedical devices. To address this, we compared the biomembrane interactions of host defense peptide IDR-1010cys (1) in free form, (2) as a soluble polymer conjugate, and (3) with one end tethered to a solid support with model bacterial and mammalian lipid membranes. Our results show that IDR-1010cys in all three distinct forms interacted with bacterial and mammalian lipid vesicles, but the extent of the interactions as monitored by the induction of secondary structure varied. The enhanced interaction of surface-tethered peptides is well correlated with their very good antimicrobial activities. Our results demonstrate that there may be a difference in the mechanism of action of surface-tethered versus free IDR-1010cys.
Assuntos
Bicamadas Lipídicas/metabolismo , Peptídeos/química , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Imobilizadas/síntese química , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Bicamadas Lipídicas/química , Modelos Biológicos , Peptídeos/síntese química , Peptídeos/metabolismo , Polímeros/química , Estrutura Secundária de Proteína , Quartzo/química , Propriedades de SuperfícieRESUMO
Bacterial colonization on implant surfaces and subsequent infections are one of the most common reasons for the failure of many indwelling devices. Several approaches including antimicrobial and antibiotic-eluting coatings on implants have been attempted; however, none of these approaches succeed in vivo. Here we report a polymer brush based implant coating that is non-toxic, antimicrobial and biofilm resistant. These coating consists of covalently grafted hydrophilic polymer chains conjugated with an optimized series of antimicrobial peptides (AMPs). These tethered AMPs maintained excellent broad spectrum antimicrobial activity in vitro and in vivo. We found that this specially structured robust coating was extremely effective in resisting biofilm formation, and that the biofilm resistance depended on the nature of conjugated peptides. The coating had no toxicity to osteoblast-like cells and showed insignificant platelet activation and adhesion, and complement activation in human blood. Since such coatings can be applied to most currently used implant surfaces, our approach has significant potential for the development of infection-resistant implants.
Assuntos
Antibacterianos/química , Biofilmes/crescimento & desenvolvimento , Materiais Revestidos Biocompatíveis/efeitos adversos , Próteses e Implantes/efeitos adversos , Próteses e Implantes/microbiologia , Animais , Antibacterianos/farmacologia , Linhagem Celular Tumoral , Dicroísmo Circular , Feminino , Humanos , Microscopia de Força Atômica , Peptídeos/efeitos adversos , Peptídeos/química , Peptídeos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Polímeros/efeitos adversos , Polímeros/química , Polímeros/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
The structure and function of the synthetic innate defense regulator peptide 1018 was investigated. This 12 residue synthetic peptide derived by substantial modification of the bovine cathelicidin bactenecin has enhanced innate immune regulatory and moderate direct antibacterial activities. The solution state NMR structure of 1018 in zwitterionic dodecyl phosphocholine (DPC) micelles indicated an α-helical conformation, while secondary structures, based on circular dichroism measurements, in anionic sodium dodecyl sulfate (SDS) and phospholipid vesicles (POPC/PG in a 1:1 molar ratio) and simulations revealed that 1018 can adopt a variety of folds, tailored to its different functions. The structural data are discussed in light of the ability of 1018 to potently induce chemokine responses, suppress the LPS-induced TNF-α response, and directly kill both Gram-positive and Gram-negative bacteria.
Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Fatores Imunológicos/química , Peptídeos/química , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bovinos , Dicroísmo Circular , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Micelas , Testes de Sensibilidade Microbiana , Peptídeos Cíclicos/química , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Estrutura Secundária de Proteína , Relação Quantitativa Estrutura-Atividade , Dodecilsulfato de Sódio/química , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The phase behavior and lipid mixing properties of an equimolar mixture of nonhydroxylated palmitoyl ceramide (Cer16), palmitic acid (PA), and cholesterol have been investigated using 2H NMR and vibrational spectroscopy. This mixture is formed by the three main classes of lipids found in the stratum corneum (SC), the top layer of the epidermis, and provides an optimized hydrophobic matching. Therefore, its behavior highlights the role played by hydrophobic matching on the phase behavior of SC lipids. We found that, below 45 degrees C, the mixture is essentially formed of coexisting crystalline domains with a small fraction of lipids (less than 20%) that forms a gel or fluid phase, likely ensuring cohesion between the solid domains. Upon heating, there is the formation of a liquid ordered phase mainly composed of PA and cholesterol, including a small fraction of Cer16. This finding is particularly highlighted by correlation vibrational microspectroscopy that indicates that domains enriched in cholesterol and PA include more disordered Cer16 than those found in the Cer16-rich domains. Solubilization of Cer16 in the fluid phase occurs progressively upon further heating, and this leads to the formation of a nonlamellar self-assembly where the motions are isotropic on the NMR time scale. It is found that the miscibility of Cer16 with cholesterol and PA is more limited than the one previously observed for ceramide III extracted from bovine brain, which is heterogeneous in chain composition and includes, in addition to Cer16, analogous ceramide with longer alkyl chains that are not hydrophobically matched with cholesterol and PA. Therefore, it is inferred that, in SC, the chain heterogeneity is a stronger criteria for lipid miscibility than chain hydrophobic matching.
Assuntos
Colesterol/química , Ácido Palmítico/química , Esfingosina/análogos & derivados , Animais , Bovinos , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Transição de Fase , Espectrofotometria Infravermelho , Esfingosina/química , Água/químicaRESUMO
The structure and membrane interaction of the antimicrobial peptide aurein 2.2 (GLFDIVKKVVGALGSL-CONH(2)), aurein 2.3 (GLFDIVKKVVGAIGSL-CONH(2)), both from Litoria aurea, and a carboxy C-terminal analog of aurein 2.3 (GLFDIVKKVVGAIGSL-COOH) were studied to determine which features of this class of peptides are key to activity. Circular dichroism and solution-state NMR data indicate that all three peptides adopt an alpha-helical structure in the presence of trifluoroethanol or lipids such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a 1:1 mixture of DMPC and 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG). Oriented circular dichroism was used to determine the orientation of the peptides in lipid bilayers over a range of concentrations (peptide/lipid molar ratios (P/L) = 1:15-1:120) in DMPC and 1:1 DMPC/DMPG, in the liquid crystalline state. The results demonstrate that in DMPC all three peptides are surface adsorbed over a range of low peptide concentrations but insert into the bilayers at high peptide concentrations. This finding is corroborated by (31)P-solid-state NMR data of the three peptides in DMPC, which shows that at high peptide concentrations the peptides perturb the membrane. Oriented circular dichroism data of the aurein peptides in 1:1 DMPC/DMPG, on the other hand, show that the peptides with amidated C-termini readily insert into the membrane bilayers over the concentration range studied (P/L = 1:15-1:120), whereas the aurein 2.3 peptide with a carboxy C-terminus inserts at a threshold concentration of P/L* between 1:80 and 1:120. Overall, the data presented here suggest that all three peptides studied interact with phosphatidylcholine membranes in a manner which is similar to aurein 1.2 and citropin 1.1, as reported in the literature, with no correlation to the reported activity. On the other hand, both aurein 2.2 and aurein 2.3 behave similarly in phosphatidylcholine/phosphatidylglycerol (PC/PG) membranes, whereas aurein 2.3-COOH inserts less readily. As this does not correlate with reported activities, minimal inhibitory concentrations of the three peptides against Staphylococcus aureus (strain C622, ATCC 25923) and Staphylococcus epidermidis (strain C621--clinical isolate) were determined. The correlation between structure, membrane interaction, and activity are discussed in light of these results.