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1.
World J Microbiol Biotechnol ; 36(1): 18, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31912374

RESUMO

In this paper, we report the antimicrobial activity of AMEP412 (a protein elicitor from Bacillus subtilis) against Streptomyces scabiei, which is the potato common scab pathogen. The purified protein samples showed an obvious inhibition zone on an S. scabiei agar plate, and the minimum inhibition concentration detected was 50 µg mL-1. The fluorescence localization assay revealed that AMEP412 could bind to aerial mycelia and spores. The stability test showed that AMEP412 was stable at 60 °C for 30 min and in pH values from 5.0 to 10.0. Its antimicrobial activity was not sensitive to metal cations. However, its activity declined by 23% when treated with Proteinase K, and was completely abrogated with Tween 80 treatment. Three antimicrobial peptides (GS21, GY20 and GY23) were identified from AMEP412, which further verified its antimicrobial activity. This research reveals the antimicrobial function of AMEP412, which not only enriches the function of the protein elicitor, but also provides a candidate for the biocontrol of potato common scab.


Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/metabolismo , Proteínas de Bactérias/farmacologia , Streptomyces/efeitos dos fármacos , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Bactérias/química , Temperatura Alta , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Micélio/efeitos dos fármacos , Doenças das Plantas/prevenção & controle , Solanum tuberosum/microbiologia , Esporos Bacterianos/efeitos dos fármacos , Streptomyces/crescimento & desenvolvimento
2.
Arch Insect Biochem Physiol ; 103(1): e21626, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31562754

RESUMO

Insects can produce various antimicrobial peptides (AMPs) upon immune stimulation. One class of AMPs are characterized by their high proline content in certain fragments. They are generally called proline-rich antimicrobial peptides (PrAMPs). We previously reported the characterization of Spodoptera litura lebocin-1 (SlLeb-1), a PrAMP proprotein. Preliminary studies with synthetic polypeptides showed that among the four deductive active fragments, the C-terminal fragment SlLeb-1 (124-158) showed strong antibacterial activities. Here, we further characterized the antibacterial and antifungal activities of 124-158 and its four subfragments: 124-155, 124-149, 127-158, and 135-158. Only 124-158 and 127-158 could agglutinate bacteria, while 124-158 and four subfragments all could agglutinate Beauveria bassiana spores. Confocal microscopy showed that fluorescent peptides were located on the microbial surface. Fragment 135-158 lost activity completely against Escherichia coli and Staphylococcus aureus, and partially against Bacillus subtilis. Only 124-149 showed low activity against Serratia marcescens. Negative staining, transmission, and scanning electron microscopy of 124-158 treated bacteria showed different morphologies. Flow cytometry analysis of S. aureus showed that 124-158 and four subfragments changed bacterial subpopulations and caused an increase of DNA content. These results indicate that active fragments of SlLeb-1 may have diverse antimicrobial effects against different microbes. This study may provide an insight into the development of novel antimicrobial agents.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Insetos/farmacologia , Spodoptera/química , Animais , Peptídeos Catiônicos Antimicrobianos/química , Bacillus subtilis/efeitos dos fármacos , Beauveria/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Proteínas de Insetos/química , Serratia marcescens/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos
3.
Biosci Biotechnol Biochem ; 84(1): 143-153, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31549575

RESUMO

Brevinin-GR23 (B-GR23) was a brevinin-2 like antimicrobial peptide, which had antimicrobial activity against Staphylococcus aureus with minimum inhibitory concentration (MIC) of 16 µM. B-GR23 increased the bacterial membrane permeation, leading to the damage of membrane integrity and the leakage of genomic DNA, then causing the cell death. The peptide nearly inhibited all plantonic bacteria to start the initial attachment of biofilm at the concentration of 1 × MIC. Whereas the disruption rates on immature and mature biofilm decreased from 60% to 20%. B-GR23 reduced the production of extracellular polysaccharides (EPS) in the planktonic growth of S. aureus, which is a crucial structure of biofilm formation. B-GR23 with the concentration of ½ × MIC inhibited 50% water-soluble EPS, and 48% water-insoluble EPS, which contributed to the antibiofilm activity. B-GR23 had no significant toxicity to human blood cells under-tested concentration (200 µM), making it a potential template for designing antimicrobial peptides.


Assuntos
Proteínas de Anfíbios/farmacologia , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Biofilmes/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Animais , Antibacterianos/síntese química , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Permeabilidade da Membrana Celular/efeitos dos fármacos , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/metabolismo , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana/métodos , Polissacarídeos Bacterianos/antagonistas & inibidores , Conformação Proteica em alfa-Hélice , Estabilidade Proteica/efeitos da radiação , Ranidae , Infecções Estafilocócicas/tratamento farmacológico
4.
Eur J Med Chem ; 185: 111814, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678742

RESUMO

The emergence of multidrug-resistant (MDR) Pseudomonas aeruginosa, coupled with shrinking antibiotic pipelines, has increased the demand for new antimicrobials with novel mechanisms of action. As the indiscriminate nature of broad-spectrum antimicrobial toxicity may have negative clinical consequences and increase the incidence of resistance, we have developed a P. aeruginosa-selective antimicrobial peptide capable of preferentially killing P. aeruginosa relative to benign microorganisms. A targeting peptide (PA2) that binds specifically to OprF porin on P. aeruginosa was identified by phage display peptide library screening, and a hybrid peptide was constructed by addition of the targeting peptide to GNU7, a potent antimicrobial peptide. The resulting hybrid peptide PA2-GNU7 exhibited potent antimicrobial activity against P. aeruginosa without causing host toxicity. Confocal laser scanning microscopy analysis and time-kill experiments demonstrated that PA2-GNU7 exhibited a high degree of specificity for P. aeruginosa, and rapidly and selectively killed P. aeruginosa cells in mixed cultures. In addition, in vivo treatment efficacy of PA2-GNU7 was significantly greater than that of conventional antibiotics in a mouse model of MDR P. aeruginosa infection. Taken together, the data suggest that PA2-GNU7 may be a promising template for further development as a novel anti-MDR P. aeruginosa therapeutic agent.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Animais , Antibacterianos/síntese química , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Relação Dose-Resposta a Droga , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/citologia , Relação Estrutura-Atividade
5.
Chemistry ; 26(7): 1511-1517, 2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-31867761

RESUMO

Solid-state 19 F NMR is a powerful method to study the interactions of biologically active peptides with membranes. So far, in labelled peptides, the 19 F-reporter group has always been installed on the side chain of an amino acid. Given the fact that monofluoroalkenes are non-hydrolyzable peptide bond mimics, we have synthesized a monofluoroalkene-based dipeptide isostere, Val-Ψ[(Z)-CF=CH]-Gly, and inserted it in the sequence of two well-studied antimicrobial peptides: PGLa and (KIGAKI)3 are representatives of an α-helix and a ß-sheet. The conformations and biological activities of these labeled peptides were studied to assess the suitability of monofluoroalkenes for 19 F NMR structure analysis.


Assuntos
Alcenos/química , Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/química , Espectroscopia de Ressonância Magnética , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/síntese química , Flúor/química , Conformação Proteica em alfa-Hélice , Coloração e Rotulagem/métodos
6.
Protein Pept Lett ; 27(1): 41-47, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31438823

RESUMO

BACKGROUND: Thanatin is the smallest member of Beta-hairpin class of cationic peptide derived from insects with vast activities against various pathogens. OBJECTIVE: In this study, the antimicrobial activity of this peptide against some species of human bacterial pathogens as well as its toxicity on NIH cells were evaluated. METHODS: Thanatin DNA sequence was cloned into pcDNA3.1+ vector and transformed into a DH5α bacterial strain. Then the recombinant plasmids were transfected into HEK-293 cells by calcium phosphate co-precipitation. After applying antibiotic treatment, the supernatant medium containing thanatin was collected. The peptide quantity was estimated by SDS-PAGE and GelQuant software. The antimicrobial activity of this peptide was performed with Minimum Inhibitory Concentration (MIC) method. In addition, its toxicity on NIH cells were evaluated by MTT assay. RESULTS: The peptide quantity was estimated approximately 164.21 µmolL-1. The antibacterial activity of thanatin was estimated between 0.99 and 31.58 µmolL-1 using MIC method. The result of cytotoxicity test on NIH cell line showed that the peptide toxicity up to the concentration of 394.10 µmolL-1 and for 48 hours, was not statistically significant from negative control cells (P>0.05). The antimicrobial assay demonstrated that thanatin had an antibacterial effect on some tested microorganisms. The results obtained in this study also showed that thanatin had no toxicity on mammalian cell lines including HEK293 and NIH. CONCLUSION: Antimicrobial peptides such as thanatin are considered to be appropriate alternatives to conventional antibiotics in treating various human pathological diseases bacteria.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas Recombinantes/farmacologia , Animais , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Sequência de Bases , Fosfatos de Cálcio/química , Sobrevivência Celular , Expressão Gênica , Células HEK293 , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Células NIH 3T3 , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transfecção
7.
Protein Pept Lett ; 27(1): 4-16, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31438824

RESUMO

Antimicrobial peptides in recent years have gained increased interest among scientists, health professionals and the pharmaceutical companies owing to their therapeutic potential. These are low molecular weight proteins with broad range antimicrobial and immuno modulatory activities against infectious bacteria (Gram positive and Gram negative), viruses and fungi. Inability of micro-organisms to develop resistance against most of the antimicrobial peptide has made them as an efficient product which can greatly impact the new era of antimicrobials. In addition to this these peptides also demonstrates increased efficacy, high specificity, decreased drug interaction, low toxicity, biological diversity and direct attacking properties. Pharmaceutical industries are therefore conducting appropriate clinical trials to develop these peptides as potential therapeutic drugs. More than 60 peptide drugs have already reached the market and several hundreds of novel therapeutic peptides are in preclinical and clinical development. Rational designing can be used further to modify the chemical and physical properties of existing peptides. This mini review will discuss the sources, mechanism and recent therapeutic applications of antimicrobial peptides in treatment of infectious diseases.


Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Interações de Medicamentos , Resistência Microbiana a Medicamentos , Fungos/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Peso Molecular , Resultado do Tratamento , Vírus/efeitos dos fármacos
8.
J Photochem Photobiol B ; 200: 111645, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31671371

RESUMO

Antimicrobial peptide W3R6 was derived from chensinin-1b and showed potential as a novel antibiotics. However, W3R6 was susceptible to protease cleavage, which limited its therapeutic application. To improve the proteolytic resistance of W3R6, D-amino acids were incorporated into its sequence by specific amino acid substitution or whole sequence substitution according to the specificity of the cleavage site. In this study, partially substituted analog D-Arg-W3R6 and completely substituted D-enantiomer D-W3R6 were synthesized. The resistance of D-Arg-W3R6 and D-W3R6 to cleavage by the tested protease increased, particularly of D-W3R6. The antimicrobial activity of D-Arg-W3R6 was almost the same as that of the parent peptide W3R6, but the antimicrobial activity of D-W3R6 was slightly decreased. The hemolytic activity of both D-Arg-W3R6 and D-W3R6 was negligible. The CD spectrum of D-W3R6 exhibited symmetry with that of W3R6 in a membrane-mimetic environment. The membrane interaction between the D-amino acid substituted analogs and a real/mimic bacterial cell membrane was examined. The outer membrane depolarization, inner membrane permeability and dye leakage in three types of liposomes treated with D-Arg-W3R6 and D-W3R6 were not obviously different from W3R6, which could be due to the similar physical and chemical properties. In addition, these three peptides showed the binding ability with LPS micelles detected by ITC, and their ability to disrupt the LPS micelles was examined by DLS experiment and even neutralize the surface negative charge of E. coli cells. These results suggest that D-Arg-W3R6 is a promising antibiotic molecule.


Assuntos
Aminoácidos/química , Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/química , Anti-Infecciosos/síntese química , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Parede Celular/química , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Lipossomos/química , Lipossomos/metabolismo , Peptídeos/química , Permeabilidade/efeitos dos fármacos , Estabilidade Proteica
9.
Analyst ; 144(24): 7242-7249, 2019 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-31687669

RESUMO

Rapid detection and identification of bacteria is important for human health, biodefense, and food safety. Small arrays of different antimicrobial peptides (AMPs) enable the identification of lipopolysaccharide (LPS) samples from a variety of bacterial species and strains. A model system for examining how peptide presentation affects LPS detection is the sheep myeloid antimicrobial peptide (SMAP-29), which contains a helix-turn-helix motif. Varying the cysteine attachment site on SMAP-29 controls the three-dimensional presentation of the peptide on the surface, altering the ability of the peptide to discriminate between LPS samples. A small array of only SMAP-29 variants-and no other peptides-is capable of discriminating among LPS samples from multiple bacterial species, as well as between different strains within the same species, with high accuracy.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Proteínas Sanguíneas/química , Catelicidinas/química , Lipopolissacarídeos/química , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/metabolismo , Sítios de Ligação , Cisteína/química , Humanos , Lipopolissacarídeos/antagonistas & inibidores , Ovinos , Especificidade da Espécie , Ressonância de Plasmônio de Superfície
10.
Chem Commun (Camb) ; 55(100): 15020-15032, 2019 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-31782426

RESUMO

With the rapid spread of resistance among parasites and bacterial pathogens, antibiotic-resistant infections have drawn much attention worldwide. Consequently, there is an urgent need to develop new strategies to treat neglected diseases and drug-resistant infections. Here, we outline several new strategies that have been developed to counter pathogenic microorganisms by designing and constructing antimicrobial peptides (AMPs). In addition to traditional discovery and design mechanisms guided by chemical biology, synthetic biology and computationally-based approaches offer useful tools for the discovery and generation of bioactive peptides. We believe that the convergence of such fields, coupled with systematic experimentation in animal models, will help translate biological peptides into the clinic. The future of anti-infective therapeutics is headed towards specifically designed molecules whose form is driven by computer-based frameworks. These molecules are selective, stable, and active at therapeutic doses.


Assuntos
Anti-Infecciosos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/química , Doenças Transmissíveis/tratamento farmacológico , Algoritmos , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Biofilmes/efeitos dos fármacos , Doenças Transmissíveis/patologia , Desenho de Drogas , Humanos , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Fatores Imunológicos/uso terapêutico , Engenharia de Proteínas , Pseudomonas/fisiologia
11.
Int J Mol Sci ; 20(19)2019 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-31581682

RESUMO

Recently, bioactive peptides have attracted attention for their therapeutic applications in the pharmaceutical industry. Among them, antimicrobial peptides are candidates for new antibiotic drugs. Since pseudin-2 (Ps), isolated from the skin of the paradoxical frog Pseudis paradoxa, shows broad-spectrum antibacterial activity with high cytotoxicity, we previously designed Ps-K18 with a Lys substitution for Leu18 in Ps, which showed high antibacterial activity and low toxicity. Here, we examined the potency of Ps-K18, aiming to develop antibiotics derived from bioactive peptides for the treatment of Gram-negative sepsis. We first investigated the antibacterial mechanism of Ps-K18 based on confocal micrographs and field emission scanning electron microscopy, confirming that Ps-K18 targets the bacterial membrane. Anti-inflammatory mechanism of Ps-K18 was investigated by secreted alkaline phosphatase reporter gene assays and RT-PCR, which revealed that Ps-K18 activates innate defense via Toll-like receptor 4-mediated nuclear factor-kappa B signaling pathways. Moreover, we investigated the antiseptic effect of Ps-K18 using a lipopolysaccharide or Escherichia coli K1-induced septic shock mouse model. Ps-K18 significantly reduced bacterial growth and inflammatory responses in the septic shock model. Ps-K18 showed low renal and liver toxicity and attenuated lung damage effectively. This study suggests that Ps-K18 is a potent peptide antibiotic that could be applied therapeutically to Gram-negative sepsis.


Assuntos
Proteínas de Anfíbios/química , Anti-Infecciosos Locais/farmacologia , Anti-Inflamatórios/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/química , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Endotoxemia/tratamento farmacológico , Endotoxemia/microbiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/ultraestrutura , Humanos , Macrófagos , Camundongos , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
12.
Zool Res ; 40(6): 488-505, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31592585

RESUMO

The discovery of antibiotics marked a golden age in the revolution of human medicine. However, decades later, bacterial infections remain a global healthcare threat, and a return to the pre-antibiotic era seems inevitable if stringent measures are not adopted to curb the rapid emergence and spread of multidrug resistance and the indiscriminate use of antibiotics. In hospital settings, multidrug resistant (MDR) pathogens, including carbapenem-resistant Pseudomonas aeruginosa, vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and extended-spectrum ß-lactamases (ESBL) bearing Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae are amongst the most problematic due to the paucity of treatment options, increased hospital stay, and exorbitant medical costs. Antimicrobial peptides (AMPs) provide an excellent potential strategy for combating these threats. Compared to empirical antibiotics, they show low tendency to select for resistance, rapid killing action, broad-spectrum activity, and extraordinary clinical efficacy against several MDR strains. Therefore, this review highlights multidrug resistance among nosocomial bacterial pathogens and its implications and reiterates the importance of AMPs as next-generation antibiotics for combating MDR superbugs.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla , Animais , Peptídeos Catiônicos Antimicrobianos/química , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/microbiologia , Humanos
13.
Fish Shellfish Immunol ; 94: 861-870, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31585246

RESUMO

The main advantage of antimicrobial peptides (AMPs) used as the effectors in the innate immunity system of invertebrates is that the high specificity is not indispensable. And they play important roles in the systemic defenses against microbial invasion. In this study, a new full-length cDNA of the crustins molecule was identified in red swamp crayfish, P. clarkii (named Pc-crustin 4). The ORF of Pc-crustin 4 contained 369 bp which encoded a protein of 122 amino acids, with a 20-amino-acid signal peptide sequence. On the base of the classification method established by Smith et al., Pc-crustin 4 belonged to Type Ⅰ crustin molecule. The Pc-crustin 4 transcripts were expressed in hemocytes at relatively high level, and relatively low level in hepatopancreas, gills, and intestine in normal crayfish. After respectively challenged with S. aureus or E. ictaluri, the expression levels of Pc-crustin 4 showed up-regulation trends at different degrees in the hemocytes, hepatopancreas, gills, and intestine tissues. Besides, the results of liquid antibacterial assay showed that rPc-crustin 4 inhibited obviously the growth of S. aureus and E. ictaluri. The results of bacteria binding assay showed that rPc-crustin 4 could bind strongly to S. aureus and E. ictaluri. Finally, RNAi assay was performed to study the immunity roles of Pc-crustin 4 in crayfish in vivo. Taken together, Pc-crustin 4 is an important immunity effector molecule, which plays crucial roles in defending against bacterial infection in crayfish.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Astacoidea/genética , Astacoidea/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Edwardsiella ictaluri/fisiologia , Perfilação da Expressão Gênica , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Alinhamento de Sequência , Staphylococcus aureus/fisiologia
14.
Biochimie ; 167: 198-206, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31639404

RESUMO

The study investigates conformational analysis and the in vitro cytokine-mediated immunomodulatory and insulin-releasing activities of rhinophrynin-27 (ELRLPEIARPVPEVLPARLPLPALPRN; RP-27), a proline-arginine-rich peptide first isolated from skin secretions of the Mexican burrowing toad Rhinophrynus dorsalis (Rhinophrynidae). In both water and 50% trifluoroethanol-water, the peptide adopts a polyproline type II helical conformation with a high degree of deviation from the canonical collagen-like folding and a pronounced bend in the molecule at the Glu13 residue. Incubation of mouse peritoneal cells with RP-27 significantly (P < 0.05) inhibited production of the pro-inflammatory cytokines TNF-α and IL-1ß and stimulated production of the anti-inflammatory cytokine IL-10. The peptide significantly (P < 0.01) stimulated release of insulin from BRIN-BD11 rat clonal ß-cells at concentrations ≥ 1 nM while maintaining the integrity of the plasma membrane and also stimulated insulin release from isolated mouse islets at a concentration of 10-6 M. Increasing the cationicity of RP-27 by substituting glutamic acid residues in the peptide by arginine and increasing hydrophobicity by substituting alanine residues by tryptophan did not result in analogues with increased activity with respect to cytokine production and insulin release. The combination of immunosuppressive and insulinotropic activities together with very low cytotoxicity suggests that RP-27 may represent a template for the development of an agent for use in anti-inflammatory and Type 2 diabetes therapies.


Assuntos
Anti-Inflamatórios , Peptídeos Catiônicos Antimicrobianos , Hipoglicemiantes , Células Secretoras de Insulina/imunologia , Proteínas de Anfíbios/química , Proteínas de Anfíbios/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Células Cultivadas , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/citologia , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Conformação Molecular , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/metabolismo
15.
Soft Matter ; 15(37): 7509-7526, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31528961

RESUMO

Antimicrobial peptides (AMPs) are naturally-occurring peptide antibiotics. AMPs are typically cationic and utilize their electrostatic interactions with the bacterial membrane to selectively attack bacteria. The way they work has inspired a vigorous search for optimized peptides for fighting resistant bacteria. Here, we present a physical model of membrane selectivity of AMPs. The challenge for theoretical modeling of membrane-peptide systems arises from the simultaneous presence of several competing effects, including lipid demixing and peptide-peptide interactions on the membrane surface. We first examine critically a number of models of peptide-membrane interactions and map out one, which incorporates adequately these competing effects as well as the geometry of various regions in membranes, occupied by bound peptides, anionic lipids within the interaction range of each peptide, and those outside this range. This effort leads to a systematically-improved model for peptide selectivity. Using the model, we relate peptide's intrinsic (Ccell-independent) selectivity to an apparent, Ccell-dependent one, and clarify the relative roles of peptide parameters and cell densities in determining their selectivity. This relationship suggests that the selectivity is more sensitive to peptide parameters at low cell densities; as a result, the optimal peptide charge, at which the selectivity is maximized, increases with the cell density in such a manner that this notion becomes less meaningful at high cell densities.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/química , Termodinâmica , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/efeitos dos fármacos , Bicamadas Lipídicas/química , Modelos Teóricos
16.
Eur J Med Chem ; 183: 111686, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31520928

RESUMO

The rapid increase of Methicillin-resistant Staphylococcus aureus (MRSA) infections and the cross-resistance of MRSA to other antibiotics create an urgent demand for new therapeutic agents. Antimicrobial peptides (AMPs) are one of the most promising options for next-generation antibiotics. In this study, novel peptides were designed based on antimicrobial peptide fragments derived from Aristicluthys nobilia interferon-I to promote anti-MRSA activity and decrease adverse effects. Design strategies included substitutions of charged or hydrophobic amino acid residues for noncharged polar residues to promote amphipathicity. Two designed peptides, P5 (YIRKIRRFFKKLKKILKK-NH2) and P9 (SYERKINRHFKTLKKNLKKK-NH2), showed potent antimicrobial activities against both sensitive Staphylococcus aureus clinical isolates and MRSA strains without significant hemolysis or cytotoxicity to human hemocytes and renal epithelial cells. Scanning Electronic Microscopy (SEM) and qRT-PCR were employed to investigate the effects of P5 and P9 on S. aureus biofilm formation, morphology, and virulence-related gene expression. P5 and P9 significantly inhibited the biofilm and destroyed the cell membrane integrity, in addition to down-regulating several virulence factor genes and biofilm formation-related genes including spa, hld, and sdrC. P5 and P9 could be promising candidate antibacterial agents for the treatment of MRSA infections.


Assuntos
Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Biofilmes/efeitos dos fármacos , Células HEK293 , Hemólise/efeitos dos fármacos , Humanos
17.
Fish Shellfish Immunol ; 94: 398-406, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31521782

RESUMO

Crustin is an antimicrobial peptide (AMP) that plays a key role in the innate immunity of crustaceans. This study cloned a new crustin from Pacific white shrimp Litopenaeus vannamei, which we designated as LvCrustinB, using rapid amplification of cDNA ends (RACE). The full-length cDNA of LvCrustinB is 751 bp with an open reading frame (ORF) of 591 bp encoding a peptide of 196 amino acids that includes a putative signal sequence. LvCrustinB is a type II crustin that has a glycine-rich region and a single whey acidic protein domain (WAP) domain. The mRNA transcript of LvCrustinB was detected in all examined tissues and was found to be most abundantly expressed in the epithelium and muscle. The expression of LvCrustinB in hemocytes was significantly upregulated after L. vannamei was challenged with LPS, Vibrio parahaemolyticus, and white spot syndrome virus (WSSV). When LvCrustinB was knocked down with RNAi, the mortality rate of L. vannamei significantly increased after V. parahaemolyticus or WSSV infection. Recombinant LvCrustinB was produced using Pichia pastoris GS115 and was shown to bind to 2 g-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and 2 g-negative bacteria (Escherichia coli and V. parahaemolyticus) via polysaccharides, which included PGN, LTA, and LPS. In vivo, the recombinant LvCrustinB remarkably protected L. vannamei from V. parahaemolyticus infection. These results suggest that LvCrustinB plays an important role in innate immunity and may be potentially utilized as antibacterial agents in shrimp.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Hemócitos/metabolismo , Lipopolissacarídeos/farmacologia , Filogenia , Interferência de RNA , Alinhamento de Sequência , Vibrio parahaemolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
18.
J Photochem Photobiol B ; 199: 111620, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31522113

RESUMO

Current scenario of bio-nanotechnology, successfully fabrication of ultrafine titanium dioxide nanoparticles (TiO2NPs) using various biological protein sources for the multipurpose targets. The present research report involves synthesis of TiO2NPs using antimicrobial peptide (AMP) crustin (Cr). Crustin previously purified from the blue crab, Portunus pelagicus haemolymph, by blue Sepharose CL-6B matrix assisted affinity column chromatography. Synthesized Cr-TiO2NPs was physico-chemically characterized by UV-Visible spectroscopy (UV-Visible), X-ray Diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), High-resolution transmission electron microscopy (HR-TEM) and zeta potential examination. X-ray diffraction analysis for crystalline nature and phase identification of titanium dioxide nanoparticles was absorbed. Functional groups were found through FTIR ranges between 1620 and 1700 cm-1. HR-TEM analysis showed that the synthesized Cr-TiO2NPs tetragonal shape and sizes ranging from 10 to 50 nm. Finally, the surface charge of the Cr-TiO2NPs was confirmed through zeta potential analysis. Furthermore, the characterized Cr-TiO2NPs exhibited good biofilm inhibition against GPB - S. mutans (Gram Positive Bacteria- Streptococcus mutans), GNB - P. vulgaris (Gram Negative Bacteria- Proteus vulgaris) and fungal Candida albicans. Moreover, photocatalysis demonstrated that the Cr-TiO2NPs was effectively explored the degradation of dyes. The results suggest that Cr-TiO2NPs is an excellent bactericidal, fungicidal and photocatalytic agent that can be supportively used for biomedical and industrial applications.


Assuntos
Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/química , Inseticidas/química , Nanocápsulas/química , Processos Fotoquímicos , Titânio/química , Animais , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Biofilmes , Braquiúros/química , Candida albicans/efeitos dos fármacos , Catálise , Sobrevivência Celular/efeitos dos fármacos , Culicidae , Liberação Controlada de Fármacos , Humanos , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Luz , Estrutura Molecular , Proteus vulgaris/efeitos dos fármacos , Streptococcus mutans/efeitos dos fármacos
19.
Int J Mol Sci ; 20(19)2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31557910

RESUMO

The interactions between chiral molecules and cell membranes have attracted more and more attention in recent decades, due to their importance in molecular science and medical applications. It is observed that some peptides composed of different chiral amino acids may have distinct interactions with a membrane. How does the membrane exhibit a selective behavior related to the chirality of the peptides? Microscopically, the interactions between the peptides and the membrane are poorly understood. In this work, we study the interactions between an amphipathic peptide (C6) and POPC membrane with simulations. The kinetics and thermodynamics of peptide enantiomers during the adsorption to the membrane are characterized with direct simulations and umbrella sampling. It is observed that there are slow kinetics for the peptide composed of D-type amino acids. Along the observed pathways, the free energy landscapes are determined with umbrella sampling techniques. A free-energy barrier for the peptide composed of D-amino acids is observed, which is consistent with the kinetic observations. The results indicate the concurrent adsorption and rotation of the peptide helix. The local interactions between the peptides and the membrane are examined in detail, including the contact interactions between the peptides and the membrane, and the distributions of the lipids around the peptide. There are observable differences of the local interactions for the cases related to different peptide enantiomers. These results further demonstrate the importance of the rotation of peptide helix during the adsorption. More interestingly, all these kinetic differences between peptide enantiomers can be explained based on the conformations of the residue Trp and interactions between Trp and lipid molecules. These results give us a molecular understanding of the mechanism of the chirality-dependent peptide-membrane interactions, and may provide clues to designing systems which are sensitive to the chirality of membranes.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Fosfatidilcolinas/química , Adsorção , Sequência de Aminoácidos , Bicamadas Lipídicas , Conformação Molecular , Simulação de Dinâmica Molecular
20.
J Microbiol Biotechnol ; 29(11): 1707-1716, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31546301

RESUMO

The development of new antimicrobial agents is essential for the effective treatment of diseases such as sepsis. We previously developed a new short peptide, Pap12-6, using the 12 N-terminal residues of papiliocin, which showed potent and effective antimicrobial activity against multidrug-resistant Gram-negative bacteria. Here, we investigated the antimicrobial mechanism of Pap12-6 and a newly designed peptide, Pap12-7, in which the 12th Trp residue of Pap12-6 was replaced with Val to develop a potent peptide with high bacterial selectivity and a different antibacterial mechanism. Both peptides showed high antimicrobial activity against Gram-negative bacteria, including multidrug-resistant Gram-negative bacteria. In addition, the two peptides showed similar anti-inflammatory activity against lipopolysaccharide-stimulated RAW 264.7 cells, but Pap12-7 showed very low toxicities against sheep red blood cells and mammalian cells compared to that showed by Pap12-6. A calcein dye leakage assay, membrane depolarization, and confocal microscopy observations revealed that the two peptides with one single amino acid change have different mechanisms of antibacterial action: Pap12-6 directly targets the bacterial cell membrane, whereas Pap12-7 appears to penetrate the bacterial cell membrane and exert its activities in the cell. The therapeutic efficacy of Pap12-7 was further examined in a mouse model of sepsis, which increased the survival rate of septic mice. For the first time, we showed that both peptides showed anti-septic activity by reducing the infiltration of neutrophils and the production of inflammatory factors. Overall, these results indicate Pap12-7 as a novel non-toxic peptide with potent antibacterial and anti-septic activities via penetrating the cell membrane.


Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Sequência de Aminoácidos , Animais , Anti-Infecciosos/uso terapêutico , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Bactérias Gram-Negativas/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Oligopeptídeos/uso terapêutico , Células RAW 264.7 , Sepse/tratamento farmacológico , Ovinos , Especificidade da Espécie , Relação Estrutura-Atividade , Resultado do Tratamento
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