RESUMEN
Mixtures of Magainin 2 and PGLa are simulated with 94 nm-sized bilayers composed of phospholipids and lyso-phospholipids for 3 µs using coarse-grained force fields. Calculation of the bilayer bending modulus shows that bilayers become more flexible in the presence of lyso-lipids or peptides, in agreement with experiments. Starting with the initial configuration of peptides randomly distributed on the bilayer surface, peptides aggregate, insert to the bilayer, and form pores. Aggregated peptides do not retain side-by-side heterodimeric structure but instead show the anchoring between C-terminal groups of magainin 2 and PGLa, which allows the deeper insertion of PGLa into the bilayer. In particular, due to the anchoring of magainin 2 and PGLa, the deeply inserted PGLa pull magainin 2 into contact with the edge of the opposite leaflet of the bilayer, which stabilizes the pore. In addition to these biophysical insights, anionic unsaturated-phospholipid bilayers are also simulated to mimic bacterial cell membranes, showing less extent of PGLa insertion and no pore formation. These simulation findings indicate that these synergistic heterodimers have the anchoring structure rather than the side-by-side structure, which supports the experimental observations suggesting the deeper insertion of PGLa and pore formation via the anchoring between anionic C-terminus of magainin 2 and cationic C-terminus of PGLa.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Membrana Celular/química , Membrana Dobles de Lípidos/química , Magaininas/química , Secuencia de Aminoácidos/genética , Péptidos Catiónicos Antimicrobianos/genética , Membrana Celular/genética , Dimerización , Humanos , Magaininas/genética , Fosfolípidos/química , Fosfolípidos/genética , PorosidadRESUMEN
Antimicrobial peptides are small molecule polypeptides with biological activity, which can avoid the drug resistance. Magainin and thanatin are antimicrobial peptides with a broad spectrum of inhibitory microbes, and the core sequence of magainin is linked to a core sequence of thanatin. Here, the hybrid magainin-thanatin (MT) antimicrobial peptide was designed through bioinformatics analysis. The recombinant MT antimicrobial peptide was successfully expressed and purified in Escherichia coli BL21 (DE3). The molecular weight of the hybrid MT antimicrobial peptide was about 3.35 kDa. Moreover, the target protein indeed has an inhibitory effect on Staphylococcus aureus, E. coli DH5α, and Bacillus subtilis, with the minimum inhibitory concentrations 16.5, 20, and 9 µM, respectively. The rational designed hybrid MT antimicrobial peptide will hopefully provide large-scale fermentable antimicrobial peptides in the industrial production in the future.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Magaininas/farmacología , Proteínas Recombinantes de Fusión/farmacología , Péptidos Catiónicos Antimicrobianos/genética , Bacillus subtilis/efectos de los fármacos , Secuencia de Bases , Biología Computacional , Diseño de Fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Magaininas/genética , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/genética , Staphylococcus aureus/efectos de los fármacosRESUMEN
Due to emergence of multidrug resistance in pathogens, the attention of the scientific community is now directed towards strengthening the reservoir of antimicrobial compounds. Prior to in vivo studies, the interaction and penetration of a hybrid peptide K11 in bacterial cells using confocal microscopy was assessed which was observed as early as 10 min after incubation with the peptide. Cell lysis along with leakage of cytoplasmic content was confirmed by electron microscopy. To evaluate the in vivo performance of the peptide, it was contained in carbopol hydrogel. Efficacy of the hydrogel formulation was then evaluated against Acinetobacter baumannii-infected wounds using a murine excision model. Treatment resulted in restoration of body weight, complete clearance of infection from the wound by day 7 and 99% wound enclosure by day 21, in contrast to the persistence of infection and 70% wound enclosure in the infected group. Further, this treatment resulted in a 2.6-fold decrease in the levels of malondialdehyde along with a 4.5-fold increase in the levels of catalase on day 3. Appearance of normal histo-architecture was observed in the treatment group. Based on these results, the peptide hydrogel can be exploited in future as one of the strategies for developing a topical anti-infective therapeutic agent.
Asunto(s)
Infecciones por Acinetobacter/tratamiento farmacológico , Antiinfecciosos/administración & dosificación , Péptidos Catiónicos Antimicrobianos/administración & dosificación , Magaininas/administración & dosificación , Meliteno/administración & dosificación , Proteínas Recombinantes de Fusión/administración & dosificación , Infección de Heridas/tratamiento farmacológico , Proteínas de Xenopus/administración & dosificación , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/ultraestructura , Animales , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/farmacología , Modelos Animales de Enfermedad , Hidrogel de Polietilenoglicol-Dimetacrilato/administración & dosificación , Magaininas/genética , Magaininas/farmacología , Meliteno/genética , Meliteno/farmacología , Ratones , Microscopía Confocal , Microscopía Electrónica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Factores de Tiempo , Resultado del Tratamiento , Cicatrización de Heridas , Infección de Heridas/microbiología , Proteínas de Xenopus/genética , Proteínas de Xenopus/farmacologíaRESUMEN
BACKGROUND: Antibiotic residues can cause antibiotic resistance in livestock and their food safety-related issues have increased the consumer demand for products lacking these residues. Hence, developing safe and effective antibiotic alternatives is important to the animal feed industry. With their strong antibacterial actions, antimicrobial peptides have potential as antibiotic alternatives. RESULTS: We investigated the antibacterial and immunomodulatory activities and the mechanisms of action of an antimicrobial peptide. The hybrid antimicrobial peptide magainin II-cecropin B (Mag II-CB) gene was transformed into the medicinal Cordyceps militaris fungus. Recombinant Mag II-CB exhibited broad-spectrum antibacterial activity in vitro and its antibacterial and immunomodulatory functions were evaluated in BALB/c mice infected with Escherichia coli (ATCC 25922). Histologically, Mag II-CB ameliorated E. coli-related intestinal damage and maintained the integrity of the intestinal mucosal barrier by up-regulating tight junction proteins (zonula occludens-1, claudin-1 and occludin). The intestinal microbial flora was positively modulated in the Mag II-CB-treated mice infected with E. coli. Mag II-CB treatment also supported immune functioning in the mice by regulating their plasma immunoglobulin and ileum secreted immunoglobulin A levels, by attenuating their pro-inflammatory cytokine levels, and by elevating their anti-inflammatory cytokines levels. Moreover, directly feeding the infected mice with the C. militaris mycelium producing Mag II-CB further proofed the antibacterial and immunomodulatory functions of recombinant hybrid antimicrobial peptide. CONCLUSION: Our findings suggest that both purified recombinant AMPs and C. militaris mycelium producing AMPs display antibacterial and immunomodulatory activities in mice. And C. militaris producing AMPs has the potential to become a substitute to antibiotics as a feed additive for livestock in future.
Asunto(s)
Antiinfecciosos/farmacología , Cecropinas/genética , Cordyceps/genética , Escherichia coli/efectos de los fármacos , Magaininas/genética , Micelio/genética , Alimentación Animal , Animales , Antibacterianos/efectos adversos , Antiinfecciosos/metabolismo , Cecropinas/metabolismo , Cecropinas/farmacología , Cordyceps/química , Inmunomodulación , Intestinos/efectos de los fármacos , Intestinos/microbiología , Magaininas/metabolismo , Magaininas/farmacología , Ratones , Ratones Endogámicos BALB C , Micelio/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Magainin 2 (MAG2) and PGLa are two related antimicrobial peptides found in the skin of the African frog Xenopus laevis with a pronounced synergistic activity, which act by permeabilizing bacterial membranes. To probe the influence of hydrophobic peptide-lipid and peptide-peptide interactions on the antimicrobial activity and on synergy, the sequence of MAG2 was modified by replacing single amino acids either with a small alanine or with the stiff and bulky hydrophobic 3-(trifluoromethyl)-L-bicyclopent-[1.1.1]-1-ylglycine side chain. The minimum inhibitory concentration of 14 MAG2 analogs was strongly influenced by these single substitutions: the antimicrobial activity was consistently improved when the hydrophobicity was increased on the hydrophobic face of the amphiphilic helix, while the activity decreased when the hydrophobicity was reduced. The synergy with PGLa, on the other hand, was rather insensitive to mutations of hydrophobic residues. It thus seems that the antimicrobial effect of MAG2 on its own depends strongly on the hydrophobicity of the peptide, while the synergy with PGLa does not depend on the overall hydrophobicity of MAG2.
Asunto(s)
Sustitución de Aminoácidos , Bacterias/efectos de los fármacos , Magaininas/química , Magaininas/farmacología , Proteínas de Xenopus/química , Proteínas de Xenopus/farmacología , Antiinfecciosos/química , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Dicroismo Circular , Sinergismo Farmacológico , Interacciones Hidrofóbicas e Hidrofílicas , Magaininas/genética , Pruebas de Sensibilidad Microbiana , Estructura Secundaria de Proteína , Proteínas de Xenopus/genéticaRESUMEN
Twelve novel peptides (Pxt-1 to Pxt-12) were isolated from the skin of Xenopus tropicalis, diploid frogs, using topological MS analysis. Among them, Pxt-8, Pxt-9, and Pxt-10 were the N terminus of Pxt-1, N terminus of Pxt-3 and C terminus of Pxt-11, respectively. The Pxt-3 and Pxt-11 peptides shared significant sequence homologies with magainins 1, -2 and levitide, respectively, which all isolated from X. laevis. Pxt-12 was identical to the X. tropicalis XT-6-like precursor previously isolated by ESI-MS/MS. None of the Pxt peptides contained any Cys, Asp, Tyr or Trp, although Leu and Lys were frequently found as typical frog-skin peptides. RT-PCR analysis confirmed the gene expressions of Pxt-2, Pxt-3, Pxt-4, Pxt-5, Pxt-7 and Pxt-11 in X. tropicalis skin. Several ion peaks corresponding to all identified Pxt peptides were observed with MALDI-MS analysis of X. tropicalis secretory fluids, collected after in vivo stimulation, which suggested that Pxt peptides were definitely secretory molecules. CD studies and Schiffer-Edmundson helical wheel projections suggested that Pxt-5, as well as mastoparan, at least, could form a typical amphiphilic α helix without a phospholipid or a membrane-mimetic solvent (trifluoroethanol). Moreover, Pxt-2 and Pxt-5 showed growth inhibitory effects on both Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive). Measurements of dynamic light scattering and the surface tensions of Pxt peptides solutions suggested that both Pxt-2 and Pxt-5 could form associations as micelles and behave like a general surfactant. Moreover, the remarkable foaming properties of Pxt-2 and Pxt-5 were observed, as well as those of the secretory fluids of X. tropicalis.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Piel/química , Proteínas de Xenopus/aislamiento & purificación , Xenopus/metabolismo , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Magaininas/genética , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Xenopus/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMEN
The antimicrobial peptide magainin II is expressed in the skin of the African clawed frog, Xenopus laevis, and exhibits a broad spectrum of antimicrobial activity as well as tumoricidal properties at low concentrations. In addition, magaininII plays a synergistic role during antimicrobial and tumoricidal processes with another antimicrobial peptide PGLa that is also expressed in Xenopus laevis. The optimized cDNA sequence of magainin II and magainin II-PGLa hybrid peptide according to E. coli or Pichia pastoris codon usage frequency were synthesized and sub-cloned into prokaryotic expression vector pGEX and Pichia pastoris secreted expression vector pPIC9k. The resulting recombinant plasmids were named as pGEX-magainin II and pPIC9k-magainin II-PGLa. The GST-magainin II fusion protein was highly expressed in E. coli. Furthermore, magainin II was successfully purified by digestion with PreScission Protease to cleave the GST tag. Additionally, our data obtained from the ELISA revealed that magainin II -PGLa hybrid peptide was successfully expressed in Pichia pastoris. These experiments establish a useful system for further studies of these antimicrobial peptides.
Asunto(s)
Magaininas/biosíntesis , Péptidos/metabolismo , Proteínas de Xenopus/biosíntesis , Animales , Escherichia coli/metabolismo , Vectores Genéticos , Magaininas/genética , Péptidos/genética , Pichia/metabolismo , Plásmidos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
Magainin-2 (MAG2) is a polycationic antimicrobial peptide isolated from the skin of the African clawed frog Xenopus laevis. It has a wide spectrum of antimicrobial activities against gram-positive and gram-negative bacteria, fungi, and induces osmotic lysis of protozoa. MAG2 also possesses antiviral and antitumoral properties. These activities make this peptide a promising candidate for therapeutic applications. Recombinant expression systems are necessary for the affordable production of large amounts of the biologically active peptide. In this work, MAG2 has been cloned to the N-terminal of a family III carbohydrate-binding module fused to the linker sequence (LK-CBM3) from Clostridium thermocellum; a formic acid recognition site was introduced between the two modules for chemical cleavage of the peptide. The recombinant protein MAG2-LK-CBM3 was expressed in Escherichia coli BL21 (DE3) and MAG2 was successfully cleaved and purified from the fusion partner LK-CBM3. Its functionality was confirmed by testing its activity against gram-negative bacteria.
Asunto(s)
Magaininas/genética , Magaininas/aislamiento & purificación , Proteínas de Xenopus/genética , Proteínas de Xenopus/aislamiento & purificación , Animales , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
The hybrid peptide CA(1-7)-M(2-12) gene was designed according to the N-terminal 1-7 amino acid sequence of the antimicrobial peptide cecropin A (CA) and the N-terminal 2-12 amino acid sequence of maganin (M) and synthesized using Pichia pastoris preferred codons. The gene was cloned into pPICZαA and transformed into the P. pastoris recipient bacterium SMD1168, regulated by the alcohol oxidase (AOX). Expression of the cecA-mag hybrid antimicrobial peptide (MW, 1.9 kDa) revealed broad-spectrum antibiotic activity and to the ability to inhibit growth of most G(-) and G(+) bacteria. Three mutants of cecA-mag were designed and synthesized by recombination polymerase chain reaction site-directed mutagenesis to investigate the relationship between the structure and function of this antimicrobial peptide. The inhibition titers of these mutants against Staphylococcus aureus were evaluated using the agar diffusion method. Under the conditions of the same concentration and volume, the bacteriostatic diameters of three cecA-mag mutants were 1.2, 1.2 and 1.5 times, respectively, compared with the diameters of wild-type cecA-mag.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/biosíntesis , Magaininas/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Secuencia de Aminoácidos , Antiinfecciosos/metabolismo , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/metabolismo , Péptidos Catiónicos Antimicrobianos/farmacología , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Magaininas/genética , Magaininas/farmacología , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mutación , Pichia , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
We have designed and synthesized a gene encoding a fusion protein comprising Magainin2 and a carrier protein with the aim of screening for a suitable carrier protein expressing antibacterial peptides in the mammalian expression system. The antibacterial peptide Magainin2 was used as a model. Our results on mammalian cell expression showed that there was no exceptional splicing in the transfected CHO-s cells. Analysis of the transgenic mouse model revealed that the expression level of this fusion protein in one transgenic positive mouse was up to 10 g/l, which is close to the level of beta-casein in goat milk. The bioactivity analysis showed that the digested fusion protein had antibacterial activity. These results demonstrate that the synthetic gene of the carrier protein is suitable for expressing an antibacterial peptide in a mammalian cell system at high productivity and efficiency. Moreover, they demonstrate the potential for producing antibacterial peptides in a transgenic animal bioreactor on a large scale and inexpensively.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Vectores Genéticos , Magaininas/genética , Magaininas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Secuencia de Bases , Northern Blotting , Southern Blotting , Células CHO , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Caseínas/genética , Cricetinae , Cricetulus , Dosificación de Gen , Fusión Génica , Genoma , Cabras , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Transgénicos , Proteínas de la Leche/metabolismo , Leche Humana/química , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Homología de Secuencia de Ácido NucleicoRESUMEN
To enhance the antibacterial ability of Magaininl-12, its N side was joined with an alkaline peptide named Hexapeptide( RRWQWR), which would make Magaininl-12 cling to the membrane of bacterial cells even tighter. According to the partiality codon of Pichia pastoris, a new hybrid antibacterial peptide Hex-Mag was designed based on the sequence of Hexapeptide and Magainin( 1-12). Synthesized through gene splicing by overlap extension, the hybrid gene was cloned into pPIC9 to construct the expression vector pPIC9-HM. After restriction enzyme analysis and purification, the pPIC9-HM was transformed into Pichia pastoris GS115. And the positive clones screened by the phenotype were induced by methanol. After optimized the requirements for the flask-shaking culture fermentation, the hybrid antibacterial peptide was expressed on high level. The new peptide, which has a weight of 2.3kDa, could remain its inhibition activity after treating for more than 3 hours in boiled water. Detected by agrose diffusion assay, Hex-Mag showed its broad-spectrum antibacterial abilities not only to Gram-negative bacteria but also to Gram-positive bacteria. The function of additive positive charges were testified by the antibacterial experiments, and the results showed the activity of Hex-Mag was stronger than that of Magainin1-12 obviously.
Asunto(s)
Magaininas/genética , Fragmentos de Péptidos/genética , Proteínas Recombinantes de Fusión/biosíntesis , Clonación Molecular , Magaininas/química , Magaininas/farmacología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Pichia/genética , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
According to the partiality codon of Pichia pastoris, hybrid antimicrobial peptide CecA-mag gene was synthesized and cloned into pPICZa-A to construct the recombinant expression vector pPICZa-A-CA. The SacI-linearized plasmid pPICZalpha-A-CA was transformed into P. pastoris SMD1168 by electroporation. Under the control of the promoter AOX'(alcoholoxidase') , an approximately 1.9kDa cecA-mag protein was expressed. Antibacterial assays demonstrated that cecA-mag had broad spectrum of antimicrobial property against Gram-positive as well as Gram-negative bacteria especially showed potent antibacterial activity against ampicillin resistant bacteria, such as pathogenic E. coli. In addition, the hybrid antibacterial peptide showed an extreme heat stable and acid stable characteristic. These results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional cecA-mag for both research and industrial purpose. Based on these characteristics, the recombinant antibacterial peptide cecA-mag displays application foreground in the field of prevention of disease, and can be used as additives of animal feedstuff and so on.