RESUMEN
ß-defensin host defense peptides are important components of the innate immune system of vertebrates. Although evidence of their broad antimicrobial, antibiofilm and immunomodulatory activities in mammals have been presented, ß-defensins from other vertebrate species, like crocodylians, remain largely unexplored. In this study, five new crocodylian ß-defensin variants from Alligator mississippiensis and Crocodylus porosus were selected for synthesis and characterization based on their charge and hydrophobicity values. Linear peptides were synthesized, folded, purified and then evaluated for their antimicrobial and antibiofilm activities against the bacterial pathogens, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Enterobacter cloacae and Acinetobacter baumannii. The Am23SK variant (SCRFSGGYCIWNWERCRSGHFLVALCPFRKRCCK) from A. mississippiensis displayed promising activity against both planktonic cells and bacterial biofilms, outperforming the human ß-defensin 3 under the experimental conditions. Moreover, Am23SK exhibited no cytotoxicity towards mammalian cells and exerted immunomodulatory effects in vitro, moderately suppressing the production of proinflammatory mediators from stimulated human bronchial epithelial cells. Overall, our results have expanded the activity landscape of crocodylian and reptilian ß-defensin in general.
Asunto(s)
Caimanes y Cocodrilos , Antibacterianos/farmacología , beta-Defensinas/química , beta-Defensinas/farmacología , Animales , Antibacterianos/química , Biopelículas/efectos de los fármacos , Línea Celular , Células Epiteliales , Humanos , Agentes Inmunomoduladores/química , Agentes Inmunomoduladores/farmacología , Pruebas de Sensibilidad Microbiana , Pliegue de Proteína , beta-Defensinas/síntesis químicaRESUMEN
Increasing antibiotic resistance is a matter of grave concern for consumers, public health authorities, farmers, and researchers. Antimicrobial peptides (AMPs) are emerging as novel and effective non-antibiotic tools to combat infectious diseases in poultry. In this study, we evaluated six avian AMPs including 2 truncated cathelicidins, [CATH-1(6-26) and CATH-2(1-15)], and 4 avian ß-defensins (ABD1, 2, 6 and 9) for their bactericidal and immunomodulatory activities. Our findings have shown CATH-1(6-26) and ABD1 being the two most potent avian AMPs effective against Gram-positive and Gram-negative bacteria investigated in these studies. Moreover, CATH-1(6-26) inhibited LPS-induced NO production and exhibited dose-dependent cytotoxicity to HD11 cells. While, ABD1 blocked LPS-induced IL-1ß gene induction and was non-toxic to HD11 cells. Importantly, in ovo administration of these AMPs demonstrated that ABD1 can offer significant protection from early chick mortality (44% less mortality in ABD1 treated group versus the control group) due to the experimental yolk sac infection caused by avian pathogenic Escherichia coli. Our data suggest that in ovo administration of ABD1 has immunomodulatory and anti-infection activity comparable with CpG ODN. Thus, ABD1 can be a significant addition to potential alternatives to antibiotics for the control of bacterial infections in young chicks.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Enfermedades de las Aves de Corral/prevención & control , Saco Vitelino/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/química , Catelicidinas/síntesis química , Catelicidinas/química , Catelicidinas/farmacología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Pollos , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Expresión Génica/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Enfermedades de las Aves de Corral/microbiología , Conformación Proteica , Salmonella/efectos de los fármacos , Salmonella/crecimiento & desarrollo , Saco Vitelino/microbiología , beta-Defensinas/síntesis química , beta-Defensinas/química , beta-Defensinas/farmacologíaRESUMEN
Human ß defensin-3-C15, an epithelium-derived cationic peptide that has antibacterial/antifungal and immuno-regulatory properties, is getting attention as potential therapeutic agent in endodontics. This study aimed to investigate if synthetic human ß defensin-3-C15 (HBD3-C15) peptides could inhibit inflammatory responses in human dental pulp cells (hDPCs), which had been induced by gram-positive endodontic pathogen. hDPC explant cultures were stimulated with Streptococcus gordonii lipoprotein extracts for 24 h to induce expression of pro-inflammatory mediators. The cells were then treated with either HBD3-C15 (50 µg/mL) or calcium hydroxide (CH, 100 µg/mL) as control for seven days, to assess their anti-inflammatory effects. Quantitative RT-PCR analyses and multiplex assays showed that S. gordonii lipoprotein induced the inflammatory reaction in hDPCs. There was a significant reduction of IL-8 and MCP-1 within 24 h of treatment with either CH or HBD3-C15 (p < 0.05), which was sustained over 1 week of treatment. Alleviation of inflammation in both medications was related to COX-2 expression and PGE2 secretion (p < 0.05), rather than TLR2 changes (p > 0.05). These findings demonstrate comparable effects of CH and HDB3-C15 as therapeutic agents for inflamed hDPCs.
Asunto(s)
Antiinflamatorios/farmacología , Lipoproteínas/inmunología , Infecciones Estreptocócicas/inmunología , Streptococcus gordonii/inmunología , beta-Defensinas/farmacología , Antiinflamatorios/síntesis química , Células Cultivadas , Pulpa Dental/citología , Pulpa Dental/efectos de los fármacos , Pulpa Dental/inmunología , Humanos , Inflamación/tratamiento farmacológico , Inflamación/etiología , Inflamación/inmunología , Modelos Moleculares , Infecciones Estreptocócicas/complicaciones , Infecciones Estreptocócicas/tratamiento farmacológico , beta-Defensinas/síntesis químicaRESUMEN
Streptococcus mutans is a major etiologic agent of human dental caries that forms biofilms on hard tissues in the human oral cavity, such as tooth and dentinal surfaces. Human ß-defensin-3 (HBD3) is a 45-amino-acid natural antimicrobial peptide that has broad spectrum antimicrobial activity against bacteria and fungi. A synthetic peptide consisting of the C-terminal 15 amino acids of HBD3 (HBD3-C15) was recently shown to be sufficient for its antimicrobial activity. Thus, clinical applications of this peptide have garnered attention. In this study, we investigated whether HBD3-C15 inhibits the growth of the representative cariogenic pathogen Streptococcus mutans and its biofilm formation. HBD3-C15 inhibited bacterial growth, exhibited bactericidal activity, and attenuated bacterial biofilm formation in a dose-dependent manner. HBD3-C15 potentiated the bactericidal and anti-biofilm activity of calcium hydroxide (CH) and chlorhexidine digluconate (CHX), which are representative disinfectants used in dental clinics, against S. mutans. Moreover, HBD3-C15 showed antimicrobial activity by inhibiting biofilm formation by S. mutans and other dentinophilic bacteria such as Enterococcus faecalis and Streptococcus gordonii, which are associated with dental caries and endodontic infection, on human dentin slices. These effects were observed for HBD3-C15 alone and for HBD3-C15 in combination with CH or CHX. Therefore, we suggest that HBD3-C15 is a potential alternative or additive disinfectant that can be used for the treatment of oral infectious diseases, including dental caries and endodontic infections.
Asunto(s)
Antibacterianos/farmacología , Desinfectantes Dentales/farmacología , Streptococcus mutans/efectos de los fármacos , beta-Defensinas/farmacología , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Hidróxido de Calcio/farmacología , Clorhexidina/análogos & derivados , Clorhexidina/farmacología , Recuento de Colonia Microbiana , Caries Dental/tratamiento farmacológico , Caries Dental/microbiología , Dentina/microbiología , Combinación de Medicamentos , Endodoncia , Enterococcus faecalis/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica , Enfermedades de la Boca/tratamiento farmacológico , Streptococcus gordonii/efectos de los fármacos , Streptococcus mutans/citología , Streptococcus mutans/crecimiento & desarrollo , beta-Defensinas/síntesis químicaRESUMEN
BACKGROUND: Avian ß-defensins (AvBD) possess broad-spectrum antimicrobial, LPS neutralizing and chemotactic properties. AvBD-12 is a chemoattractant for avian immune cells and mammalian dendritic cells (JAWSII) - a unique feature that is relevant to the applications of AvBDs as chemotherapeutic agents in mammalian hosts. To identify the structural components essential to various biological functions, we have designed and evaluated seven AvBD analogues. RESULTS: In the first group of analogues, the three conserved disulfide bridges were eliminated by replacing cysteines with alanine and serine residues, peptide hydrophobicity and charge were increased by changing negatively charged amino acid residues to hydrophobic (AvBD-12A1) or positively charged residues (AvBD-12A2 and AvBD-12A3). All three analogues in this group showed improved antimicrobial activity, though AvBD-12A3, with a net positive charge of +9, hydrophobicity of 40% and a predicted CCR2 binding domain, was the most potent antimicrobial peptide. AvBD-12A3 also retained more than 50% of wild type chemotactic activity. In the second group of analogues (AvBD-12A4 to AvBD-12A6), one to three disulfide bridges were removed via substitution of cysteines with isosteric amino acids. Their antimicrobial activity was compromised and chemotactic activity abolished. The third type of analogue was a hybrid that had the backbone of AvBD-12 and positively charged amino acid residues AvBD-6. The antimicrobial and chemotactic activities of the hybrid resembled that of AvBD-6 and AvBD-12, respectively. CONCLUSIONS: While the net positive charge and charge distribution have a dominating effect on the antimicrobial potency of AvBDs, the three conserved disulfide bridges are essential to the chemotactic property and the maximum antimicrobial activity. Analogue AvBD-12A3 with a high net positive charge, a moderate degree of hydrophobicity and a CCR2-binding domain can serve as a template for the design of novel antimicrobial peptides with chemotactic property and salt resistance.
Asunto(s)
Aves/inmunología , Disulfuros/química , Interacciones Hidrofóbicas e Hidrofílicas , beta-Defensinas/síntesis química , beta-Defensinas/farmacología , Alanina/química , Secuencia de Aminoácidos , Animales , Antiinfecciosos/síntesis química , Antiinfecciosos/farmacología , Bacterias/efectos de los fármacos , Bacterias/crecimiento & desarrollo , Carga Bacteriana , Línea Celular/efectos de los fármacos , Quimiotaxis , Pollos , Recuento de Colonia Microbiana/métodos , Cisteína/química , Células Dendríticas/inmunología , Combinación de Medicamentos , Macrófagos/efectos de los fármacos , Pruebas de Sensibilidad Microbiana/métodos , Microscopía Electrónica de Rastreo , Péptidos/síntesis química , Conformación Proteica , Serina/química , Cloruro de Sodio/administración & dosificación , Cloruro de Sodio/farmacología , beta-Defensinas/administración & dosificaciónRESUMEN
Human defensins are innate immune defense peptides with a remarkably broad repertoire of anti-pathogen activities. In addition to modulating immune response, inflammation, and angiogenesis, disintegrating bacterial membranes, and inactivating bacterial toxins, defensins are known to intercept various viruses at different stages of their life cycles, while remaining relatively benign towards human cells and proteins. Recently we have found that human defensins inactivate proteinaceous bacterial toxins by taking advantage of their low thermodynamic stability and acting as natural "anti-chaperones", i.e. destabilizing the native conformation of the toxins. In the present study we tested various proteins produced by several viruses (HIV-1, PFV, and TEV) and found them to be susceptible to destabilizing effects of human α-defensins HNP-1 and HD-5 and the synthetic θ-defensin RC-101, but not ß-defensins hBD-1 and hBD-2 or structurally related plant-derived peptides. Defensin-induced unfolding promoted exposure of hydrophobic groups otherwise confined to the core of the viral proteins. This resulted in precipitation, an enhanced susceptibility to proteolytic cleavage, and a loss of viral protein activities. We propose, that defensins recognize and target a common and essential physico-chemical property shared by many bacterial toxins and viral proteins - the intrinsically low thermodynamic protein stability.
Asunto(s)
Péptidos/química , Proteínas Virales/química , alfa-Defensinas/química , Precipitación Química , VIH-1/química , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos/síntesis química , Potyvirus/química , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estabilidad Proteica , Desplegamiento Proteico , Proteolisis , Virus Espumoso de los Simios/química , Termodinámica , alfa-Defensinas/síntesis química , beta-Defensinas/síntesis química , beta-Defensinas/químicaRESUMEN
Human beta defensin-3 (HßD-3) is a host-defense protein exhibiting antibacterial activity towards both Gram-negative and Gram-positive bacteria. There is considerable interest in the function of this protein due to its increased salt tolerance and activity against Gram-positive Staphylococcus aureus. In this study, analogs of HßD-3 devoid of N and C terminal regions are investigated to determine the influence of specific structural motif on antimicrobial activity and selectivity between Gram-positive and Gram-negative bacteria. Circular dichroism, fluorescence and solid-state NMR experiments have been used to investigate the conformation and mode of action of HßD3 analogs with various model membranes to mimic bacterial inner and outer membranes and also mammalian membranes. Our studies specifically focused on determining four major characteristics: (i) interaction of HßD3 analogs with phospholipid vesicles composed of zwitterionic PC or anionic PE:PG vesicles and LPS; (ii) conformation of HßD3-peptide analogs in the presence of PC or PE:PG vesicles; (iii) ability of HßD3 analogs to permeate phospholipid vesicles composed of PC or PE:PG; and (iv) activities on bacteria cells and erythrocytes. Our results infer that the linear peptide L25P and its cyclic form C25P are more active than L21P and C21P analogs. However, they are less active than the parent peptide, thus pointing towards the importance of the N terminal domain in its biological activity. The variation in the activities of L21P/C21P and L25P/C25P also suggest the importance of the positively charged residues at the C terminus in providing selectivity particularly to Gram-negative bacteria.
Asunto(s)
Antibacterianos/farmacología , Membrana Celular/efectos de los fármacos , Péptidos/farmacología , Salmonella enterica/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , beta-Defensinas/farmacología , Secuencia de Aminoácidos , Animales , Antibacterianos/síntesis química , Transporte Biológico/efectos de los fármacos , Membrana Celular/química , Permeabilidad de la Membrana Celular , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Humanos , Membranas Artificiales , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/síntesis química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Fosfatidilgliceroles/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Salmonella enterica/crecimiento & desarrollo , Salmonella enterica/metabolismo , Ovinos , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/metabolismo , Electricidad Estática , Relación Estructura-Actividad , beta-Defensinas/síntesis químicaRESUMEN
Gallin is a 41-residue protein, first identified as a minor component of hen egg white and found to be antimicrobial against Escherichia coli. Gallin may participate in the protection of the embryo during its development in the egg. Its sequence is related to antimicrobial ß-defensin peptides. In the present study, gallin was chemically synthesized 1) to further investigate its antimicrobial spectrum and 2) to solve its three-dimensional NMR structure and thus gain insight into structure-function relationships, a prerequisite to understanding its mode(s) of action. Antibacterial assays confirmed that gallin was active against Escherichia coli, but no additional antibacterial activity was observed against the other Gram-positive or Gram-negative bacteria tested. The three-dimensional structure of gallin, which is the first ovodefensin structure to have been solved to date, displays a new five-stranded arrangement. The gallin three-dimensional fold contains the three-stranded antiparallel ß-sheet and the disulfide bridge array typical of vertebrate ß-defensins. Gallin can therefore be unambiguously classified as a ß-defensin. However, an additional short two-stranded ß-sheet reveals that gallin and presumably the other ovodefensins form a new structural subfamily of ß-defensins. Moreover, gallin and the other ovodefensins calculated by homology modeling exhibit atypical hydrophobic surface properties, compared with the already known vertebrate ß-defensins. These specific structural features of gallin might be related to its restricted activity against E. coli and/or to other yet unknown functions. This work provides initial understanding of a critical sequence-structure-function relationship for the ovodefensin family.
Asunto(s)
Pollos/metabolismo , beta-Defensinas/química , Secuencia de Aminoácidos , Animales , Imagenología Tridimensional , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Pliegue de Proteína , beta-Defensinas/síntesis químicaRESUMEN
In this study, the influence of isoleucine and arginine on the biological activity and peptide-membrane interactions of linear avian ß-defensin-4 (RL38) analogs was investigated. Results of biological activities showed that the antimicrobial activities of AvBD-4 analogs were closely related to hydrophobicity and amphipathicity. The peptide GLI19 with high hydrophobicity value and amphipathicity displayed broad spectrum antimicrobial activity against both gram-negative and gram-positive, whereas GLR19 with increasing multiple charges only exhibited activity against gram-negative. The interaction between peptides and the liposome membrane demonstrated that the peptides preferentially bound to negatively charged phospholipids over zwitterionic phospholipids, which supported the antimicrobial activity data. The outer membranes assay further demonstrated that GLI19 had a greater capacity than the other tested peptides to penetrate the cell membrane at a low concentration. Collectively, the peptides derived from the bactericidal domain of linear ß- defensins by truncation and hydrophobic amino acid substitution may be effective high-potential antibacterial agents.
Asunto(s)
Arginina/química , Isoleucina/química , Péptidos/síntesis química , beta-Defensinas/química , Sustitución de Aminoácidos , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Antibacterianos/farmacología , Aves , Dicroismo Circular , Escherichia coli/efectos de los fármacos , Interacciones Hidrofóbicas e Hidrofílicas , Liposomas/química , Membranas/química , Péptidos/química , Péptidos/farmacología , Salmonella enterica/efectos de los fármacos , Relación Estructura-Actividad , beta-Defensinas/síntesis química , beta-Defensinas/farmacologíaRESUMEN
Human ß-defensins (HBDs) are cationic antimicrobial peptides that are components of the innate immune system. They are characterized by three disulfide bridges. However, the number of cationic residues as well as the presence of lysine and arginine residues vary. In HBD4, the cationic residues occur predominantly in the N-terminal segment, unlike in HBD1-3. We have examined the antimicrobial activity of peptides spanning the N- and C-terminal segments of HBD4. We have introduced one, two and three disulfide bridges in the peptides corresponding to the N-terminal segments. Peptides corresponding to the N-terminal segment had identical sequences and variation was only in the number and spacing of cysteines and disulfide bridges. Antimicrobial activity to varying extents was observed for all the peptides. When two disulfide bridges were present, decrease in antimicrobial potency as well as sensitivity of activity to salt was observed. Enhanced antimicrobial activity was observed when three disulfide bridges were present. The antimicrobial potency was similar to HBD4 except against Escherichia coli and was attenuated in the presence of salt. While the presence of three disulfide bridges did not constrain the peptide to a rigid ß-sheet, the activity was considerably more as compared to the peptides with one or two disulfide bridges. The peptides enter bacterial and fungal cells rapidly without membrane permeabilization and appear to exert their activity inside the cells rather than at the membrane.
Asunto(s)
Antibacterianos/farmacología , Antifúngicos/farmacología , Bacterias/efectos de los fármacos , Candida albicans/efectos de los fármacos , Disulfuros/química , beta-Defensinas/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Antifúngicos/síntesis química , Antifúngicos/química , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Relación Estructura-Actividad , beta-Defensinas/síntesis química , beta-Defensinas/químicaRESUMEN
Defensins are important components in host defense systems. The therapeutic use of ß-defensins is limited by their innate toxicity and high cost due to the size and complex disulfide pairing. In this study, we used linear avian ß- defensin-4 (RL38) without disulfide bonds as model peptide to derive two peptides by the truncation. GL23 is the C-terminal truncated sequence of RL38, and GLI23 is the derivative of GL23 by the replacement of cysteines with isoleucines. Results showed that these peptides exhibited strong antibacterial activity against gram-negative and gram-positive bacteria. An exception was that GL23 showed weak antimicrobial activity against gallinaceous pathogenic bacteria Salmonella Pullorum C79-13. Two truncated peptides GL23 and GLI23 displayed no or weak hemolysis, which was in accordance with little blue shifts of the peptides in the presence of synthetic eukaryotic membranes. CD spectroscopy demonstrated that these peptides appeared to be unfolded in aqueous solution but acquire structure in the presence of membrane- mimicking phospholipids. GLI23 kept the antibacterial activity at high concentrations of NaCl or low concentration of divalent cations (Mg2+ and Ca2+). The peptides preferentially bound to negatively charged phospholipids over zwitterionic phospholipids, which led to greater cell selectivity. The outer and inner membranes assay displayed that GLI23 killed bacteria by targeting the cell membrane. These results suggest the peptides derived by truncation of linear ß-defensins may be a promising candidate for future antibacterial agent.
Asunto(s)
Antibacterianos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Péptidos/farmacología , beta-Defensinas/farmacología , Secuencia de Aminoácidos , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Permeabilidad de la Membrana Celular/efectos de los fármacos , Pollos , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Hemólisis/efectos de los fármacos , Humanos , Liposomas/química , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , beta-Defensinas/síntesis química , beta-Defensinas/químicaRESUMEN
The yeast-like algae of the genus Prototheca are ubiquitous saprophytes causing infections in immunocompromised patients and granulomatous mastitis in cattle. Few available therapies and the rapid spread of resistant strains worldwide support the need for novel drugs against protothecosis. Host defence antimicrobial peptides inactivate a wide array of pathogens and are a rich source of leads, with the advantage of being largely unaffected by microbial resistance mechanisms. Three structurally diverse bovine peptides [BMAP-28, Bac5 and lingual antimicrobial peptide (LAP)] have thus been tested for their capacity to inactivate Prototheca spp. In minimum inhibitory concentration (MIC) assays, they were all effective in the micromolar range against clinical mastitis isolates as well as a Prototheca wickerhamii reference strain. BMAP-28 sterilized Prototheca cultures within 30-60 min at its MIC, induced cell permeabilization with near 100% release of cellular adenosine triphosphate and resulted in extensive surface blebbing and release of intracellular material as observed by scanning electron microscopy. Bac5 and LAP inactivated Prototheca following 3-6 h incubation at fourfold their MIC and did not result in detectable surface damage despite 70-90% killing, suggesting they act via non-lytic mechanisms. In circular dichroism studies, the conformation of BMAP-28, but not that of Bac5 or LAP, was affected by interaction with liposomes mimicking algal membranes. Our results indicate that BMAP-28, Bac5 and LAP kill Prototheca with distinct potencies, killing kinetics, and modes of action and may be appropriate for protothecal mastitis treatment. In addition, the ability of Bac5 and LAP to act via non-lytic mechanisms may be exploited for the development of target-selective drugs.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas en los Gránulos del Eosinófilo/farmacología , Proteínas/farmacología , Prototheca/efectos de los fármacos , beta-Defensinas/farmacología , Secuencia de Aminoácidos , Animales , Antibacterianos/síntesis química , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/química , Bovinos , Membrana Celular/efectos de los fármacos , Enterobacteriaceae/efectos de los fármacos , Proteínas en los Gránulos del Eosinófilo/síntesis química , Proteínas en los Gránulos del Eosinófilo/química , Femenino , Mastitis Bovina/microbiología , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Permeabilidad , Estructura Secundaria de Proteína , Proteínas/síntesis química , Proteínas/química , Prototheca/aislamiento & purificación , Prototheca/ultraestructura , Staphylococcus/efectos de los fármacos , Streptococcus/efectos de los fármacos , beta-Defensinas/síntesis química , beta-Defensinas/químicaRESUMEN
Human and murine immature DCs (iDCs) are highly efficient in antigen capture and processing, while as mature cells they present antigen and are potent initiators of cell-mediated immune responses. Consequently, iDCs are logical targets for vaccine antigens. Originally discovered for their antimicrobial activity, and thought of as strictly part of the innate immune system, studies with defensins such as human ß (beta)-defensin 2 (hBD2) and murine ß-defensin 2 (mBD2) have shown that they can function as chemo-attractant for iDCs and, in vaccination strategies, can enhance antigen-specific adaptive immune responses. Most studies to date have been conducted in mice. In contrast, little is known about defensins in cattle. To expand our understanding of the role of defensins in modulating immune responses in cattle, DCs were generated from bovine monocytes and the immature state of these bovine DCs was characterized phenotypically and through functional assays. By day 3 (DC3), bovine monocyte-derived DCs stained positively for DC-specific receptors CD1, CD80/86, CD205, DC-Lamp and MMR. When compared to conventional 6-day DC cultures or DCs cultured for 10 days with and without maturation factors, these DC3 were functionally at their most immature stage. Fourteen of the 16 known bovine ß-defensins were synthesized and the synthetic peptides were screened for their ability to attract bovine iDCs. Bovine DC3 were consistently attracted to BNBD3, an analog of BNBD3 (aBNBD3), BNBD9 and bovine EBD in vitro and to aBNBD3 in vivo. These results are the first to describe chemotactic ability of synthetic bovine ß-defensins for immature bovine monocyte-derived DCs.
Asunto(s)
Bovinos/inmunología , Quimiotaxis/inmunología , Células Dendríticas/inmunología , beta-Defensinas/inmunología , Secuencia de Aminoácidos , Animales , Presentación de Antígeno , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/inmunología , Bovinos/genética , Diferenciación Celular , Células Dendríticas/citología , Disulfuros/química , Humanos , Inmunofenotipificación , Linfocitos/citología , Linfocitos/inmunología , Ratones , Datos de Secuencia Molecular , Monocitos/citología , Monocitos/inmunología , Homología de Secuencia de Aminoácido , Piel/inmunología , beta-Defensinas/síntesis química , beta-Defensinas/química , beta-Defensinas/genéticaRESUMEN
Understanding the molecular mechanisms of antimicrobial peptide-membrane interactions is crucial in predicting the design of useful synthetic antimicrobial peptide analogues. Defensins are small (3-5 kDa) cysteine-rich cationic proteins which constitute the front line of host innate immunity. In this study, a series of eight 10 AA C-terminal analogues of hBD3 [sequence: RGRKXXRRKK, X = W, F, Y, V, L, I, H, C(Acm); net charge = +7, coded as W2, F2, Y2, V2, L2, I2, H2, and C2] and covalent V2-dimer [(RGRKVVRR)(2)KK] (18 AA, net charge = +11) were synthesized using solid phase peptide synthesis (SPPS) in Fmoc chemistry. Wild-type hBD3 was used as a control in all analyses. W2, V2, and especially Y2 showed high activity selectively against Gram-negative bacteria Pseudomonas aeruginosa in the concentration range of 4.3-9.7 microM. The covalent dimeric form of V2-monomer, V2-dimer, showed increased antibacterial killing compared to the monomeric form, V2-monomer. Cytotoxicity assays on a human conjunctival epithelial cell line (IOBA-NHC cells) showed that no change in viable cell number 24 h after constant exposure to all the eight peptide analogues even at concentrations up to 200 microg/ml. Fluorescence correlation spectroscopy (FCS) was used to study the interaction of these peptides against POPC vesicles (neutral; mammalian cell membrane mimic) and POPG vesicles (negatively charged; bacterial cell membrane mimic). Using FCS, significant aggregation and some leakage of Rhodamine dye were observed with POPG with Y2, W2 and V2 at the concentration of 5-10 mmicroM and no significant aggregation or disruption of vesicles was observed for all peptide analogues tested against POPC. V2-dimer induced more leakage and aggregation than the monomeric form. Overall, V2-dimer is the most effective antimicrobial peptide, with aggregation of POPG vesicles observed at concentrations as low as 1 microM. The concentration of 5-10 microM for Y2 from FCS correlated with the concentration of 5 microM (6.25 microg/ml), at which Y2 showed a cooperative increase in the activity. This suggests a structural transition of Y2 in the 2.5-5 microM concentration range resulting in the correlated increased antimicrobial activity. These results and the FCS together with previous NMR and molecular dynamics (MD) suggested that the charge density-based binding affinity, stable covalent dimerization, the ability to dimerize or even oligomerize and adopt a well-defined structure are important physicochemical properties distinguishing more effective cationic antimicrobial peptides.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Citotoxinas/química , Citotoxinas/farmacología , Células Epiteliales/efectos de los fármacos , beta-Defensinas/química , beta-Defensinas/farmacología , Secuencia de Aminoácidos , Línea Celular , Citotoxinas/síntesis química , Dimerización , Bacterias Gramnegativas/efectos de los fármacos , Humanos , Estructura Molecular , beta-Defensinas/síntesis químicaRESUMEN
To enhance the potential therapeutic efficacy of an antimicrobial peptide human beta-defensin 3, two fusion peptides, a bactericidal-immunomodulatory fusion peptide human beta-defensin 3-mannose-binding lectin and a bactericidal-bactericidal fusion peptide human beta-defensin 3-lysozyme were synthesized and the bactericidal activities in vitro and in vivo against methicillin-resistant Staphylococcus aureus N315 were demonstrated in this study. Peptide human beta-defensin 3-lysozyme showed the best bactericidal activity in vitro, but human beta-defensin 3-mannose-binding lectin showed a significant improvement in angiogenesis and tissue reconstruction. Our results illustrated that outstanding bactericidal activity in vitro is not essential in the development of antimicrobial peptides. Fusion strategy and immunomodulatory factors should be utilized in novel antimicrobial peptide development.
Asunto(s)
Factores Inmunológicos/uso terapéutico , Lectina de Unión a Manosa/uso terapéutico , Péptidos/uso terapéutico , Proteínas Recombinantes de Fusión/uso terapéutico , Infecciones Cutáneas Estafilocócicas/tratamiento farmacológico , beta-Defensinas/uso terapéutico , Secuencia de Aminoácidos , Animales , Humanos , Factores Inmunológicos/síntesis química , Factores Inmunológicos/química , Factores Inmunológicos/farmacología , Masculino , Lectina de Unión a Manosa/síntesis química , Lectina de Unión a Manosa/química , Lectina de Unión a Manosa/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Péptidos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Infecciones Cutáneas Estafilocócicas/inmunología , Infecciones Cutáneas Estafilocócicas/microbiología , Resultado del Tratamiento , Infección de Heridas/tratamiento farmacológico , Infección de Heridas/microbiología , beta-Defensinas/síntesis química , beta-Defensinas/química , beta-Defensinas/farmacologíaRESUMEN
Peptide thioesters readily prepared through N-->S acyl transfer of a specific C-terminal motif provide access to biologically active mini-proteins using native chemical ligation.
Asunto(s)
Ésteres/síntesis química , Compuestos de Sulfhidrilo/síntesis química , beta-Defensinas/química , beta-Defensinas/síntesis química , Ésteres/química , Estructura Molecular , Compuestos de Sulfhidrilo/química , Factores de TiempoRESUMEN
We have designed and chemically synthesized an artificial beta-defensin based on a minimal template derived from the comparative analysis of over 80 naturally occurring sequences. This molecule has the disulfide-bridged beta-sheet core structure of natural beta-defensins and shows a robust salt-sensitive antimicrobial activity against bacteria and yeast, as well as a chemotactic activity against immature dendritic cells. An SAR (structure-activity relationship) study using two truncated fragments or a Cys-->Ser point-mutated analogue, from which one or two of the three disulfide bridges were absent, indicated that altering the structure resulted in a different type of membrane interaction and a switch to different modes of action towards both microbial and host cells, and that covalent dimerization could favour antimicrobial activity. Comparison of the structural, aggregational and biological activities of the artificial defensin with those of three human beta-defensins and their primate orthologues provided useful information on how their mode of action may relate to specific structural features.
Asunto(s)
beta-Defensinas/química , beta-Defensinas/inmunología , Secuencia de Aminoácidos , Animales , Bacterias/efectos de los fármacos , Dimerización , Humanos , Inmunidad Innata , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Primates/inmunología , Alineación de Secuencia , Relación Estructura-Actividad , beta-Defensinas/síntesis química , beta-Defensinas/farmacologíaRESUMEN
Synthetic peptides Phd1-3 spanning the cationic carboxy-terminal region of human beta-defensins HBD-1-3 have been shown to have antibacterial activity. Gross morphological changes were seen in E. coli cells treated with these peptides. In this paper, we have studied the surface-active properties of peptides Phd1-3 and their interactions with different phospholipids using Langmuir-Blodgett monolayers. Compression isotherms and increase in pressure on insertion of peptides into lipid monolayers at different initial pressures indicate the affinity of these peptides for negatively charged lipids. Phd3 inserted less effectively into monolayers as compared to Phd1 and Phd2. The peptides differed in their ability to permeabilize the inner membrane of E. coli, with Phd3 being least effective. It is likely that the peptides kill Gram-negative bacteria by more than one mechanism. When hydrophobicity and net charge favor insertion into lipid membranes, then membrane permeabilization could be the primary event in the killing of bacteria. In cases where membrane insertion does not occur, interaction with phospholipid interface induces highly selective stress that leads to stasis and cell death, as proposed for polymyxin B and bactenecin.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Escherichia coli/química , Fosfolípidos/química , beta-Defensinas/química , Aire , Antibacterianos/síntesis química , Antibacterianos/química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Membranas Intracelulares/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Presión , Factores de Tiempo , Agua/química , beta-Defensinas/síntesis química , beta-Defensinas/farmacologíaRESUMEN
Human defensins are a family of small antimicrobial proteins found predominantly in leukocytes and epithelial cells that play important roles in the innate and adaptive immune defense against microbial infection. The most distinct molecular feature of defensins is cationicity, manifested by abundant Arg and/or Lys residues in their sequences. Sequence analysis indicates that Arg is strongly selected over Lys in alpha-defensins but not in beta-defensins. To understand this Arg/Lys disparity in defensins, we chemically synthesized human alpha-defensin 1 (HNP1) and several HNP1 analogs where three Arg residues were replaced by each of the following six alpha-amino acids: Lys, ornithine (Orn), diaminobutyric acid (Dab), diaminopropionic acid (Dap), N,N-dimethyl-Lys ((diMe)Lys), and homo-Arg ((homo)Arg). In addition, we prepared human beta-defensin 1 (hBD1) and (Lys-->Arg)hBD1 in which all four Lys residues were substituted for Arg. Bactericidal activity assays revealed the following. 1) Arg-containing HNP1 and (Lys-->Arg)hBD1 are functionally better than Lys-HNP1 and hBD1, respectively; the difference between Arg and Lys is more evident in the alpha-defensin than in the beta-defensin and is more evident at low salt concentrations than at high salt concentrations. 2) For HNP1, the Arg/Lys disparity is much more pronounced with Staphylococcus aureus than with Escherichia coli, and the Arg-rich HNP1 kills bacteria faster than its Lys-rich analog. 3) Arg and Lys appear to have optimal chain lengths for bacterial killing as shortening Lys or lengthening Arg in HNP1 invariably becomes functionally deleterious. Our findings provide insights into the Arg/Lys disparity in defensins, and shed light on the cationicity of defensins with respect to their antimicrobial activity and specificity.
Asunto(s)
Antiinfecciosos/farmacología , Arginina/química , Escherichia coli/crecimiento & desarrollo , Lisina/química , Staphylococcus aureus/crecimiento & desarrollo , beta-Defensinas/farmacología , Antiinfecciosos/síntesis química , Antiinfecciosos/química , Antiinfecciosos/inmunología , Arginina/inmunología , Cationes , Células Epiteliales/química , Células Epiteliales/inmunología , Humanos , Inmunidad Innata , Leucocitos/química , Leucocitos/inmunología , Lisina/inmunología , Modelos Moleculares , Estructura Terciaria de Proteína , Análisis de Secuencia de Proteína , Relación Estructura-Actividad , beta-Defensinas/síntesis química , beta-Defensinas/química , beta-Defensinas/inmunologíaRESUMEN
The antibacterial activities of synthetic human beta-defensin analogs, constrained by a single disulfide bridge and in the reduced form, have been investigated. The peptides span the carboxy-terminal region of human beta-defensins (HBD-1-3), which have a majority of cationic residues present in the native defensins. The disulfide constrained peptides exhibited activity against Escherichia coli and Staphylococcus aureus whereas the reduced forms were active only against E. coli. The antibacterial activities were attenuated in the presence of increasing concentrations of NaCl and divalent cations such as Ca(2+) and Mg(2+). The site of action was the bacterial membrane. Peptides spanning the carboxy-terminal region of human beta-defensins could be of help in understanding facets of antimicrobial activity of beta-defensins such as salt sensitivity and mechanisms of bacterial membrane damage.