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1.
Exp Parasitol ; 218: 107987, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32891601

RESUMO

The protozoan parasite Leishmania spp. causes leishmaniases, a group of diseases creating serious health problems in many parts of the world with significant resistance to existing drugs. Insect derived antimicrobial peptides are promising alternatives to conventional drugs against several human disease-causing pathogens because they do not generate resistance. Halictine-2, a novel antimicrobial peptide from the venom of eusocial honeybee, Halictus sexcinctus showed significant anti-leishmanial activity in vitro, towards two life forms of the dimorphic parasite, the free-swimming infective metacyclic promastigotes and the intracellular amastigotes responsible for the systemic infection. The anti-leishmanial activity of the native peptide (P5S) was significantly enhanced by serine to threonine substitution at position 5 (P5T). The peptide showed a propensity to form α-helices after substitution at position-5, conferring amphipathicity. Distinct pores observed on the promastigote membrane after P5T exposure suggested a mechanism of disruption of cellular integrity. Biochemical alterations in the promastigotes after P5T exposure included generation of increased oxygen radicals with mitochondrial Ca2+ release, loss of mitochondrial membrane potential, reduction in total ATP content and increased mitochondrial mass, resulting in quick bioenergetic and chemiosmotic collapse leading to cell death characterized by DNA fragmentation. P5T was able to reduce intracellular amastigote burden in an in vitro model of Leishmania infection but did not alter the proinflammatory cytokines like TNF-α and IL-6. The ability of the P5T peptide to kill the Leishmania parasite with negligible haemolytic activity towards mouse macrophages and human erythrocytes respectively, demonstrates its potential to be considered as a future antileishmanial drug candidate.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antiprotozoários/farmacologia , Apoptose , Leishmania tropica/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Antiprotozoários/química , Venenos de Abelha/química , Cálcio/análise , Cálcio/metabolismo , Linhagem Celular , Dicroísmo Circular , Fragmentação do DNA , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Fluorometria , Humanos , Leishmania tropica/ultraestrutura , Leishmaniose Visceral/parasitologia , Macrófagos Peritoneais , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia Eletrônica de Varredura , Mitocôndrias/química , Tamanho Mitocondrial , Espécies Reativas de Oxigênio/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Superóxidos/isolamento & purificação
2.
Adv Exp Med Biol ; 1267: 117-133, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32894480

RESUMO

Antibiotic resistance is a global epidemic, becoming increasingly pressing due to its rapid spread. There is thus a critical need to develop new therapeutic approaches. In addition to searching for new antibiotics, looking into existing mechanisms of natural host defense may enable researchers to improve existing defense mechanisms, and to develop effective, synthetic drugs guided by natural principles. Histones, primarily known for their role in condensing mammalian DNA, are antimicrobial and share biochemical similarities with antimicrobial peptides (AMPs); however, the mechanism by which histones kill bacteria is largely unknown. Both AMPs and histones are similar in size, cationic, contain a high proportion of hydrophobic amino acids, and possess the ability to form alpha helices. AMPs, which mostly kill bacteria through permeabilization or disruption of the biological membrane, have recently garnered significant attention for playing a key role in host defenses. This chapter outlines the structure and function of histone proteins as they compare to AMPs and provides an overview of their role in innate immune responses, especially regarding the action of specific histones against microorganisms and their potential mechanism of action against microbial pathogens.


Assuntos
Antibacterianos/química , Antibacterianos/imunologia , Bactérias/imunologia , Histonas/química , Histonas/imunologia , Animais , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Histonas/farmacologia , Imunidade Inata
3.
Nat Commun ; 11(1): 3894, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753597

RESUMO

Here, we demonstrate the self-assembly of the antimicrobial human LL-37 active core (residues 17-29) into a protein fibril of densely packed helices. The surface of the fibril encompasses alternating hydrophobic and positively charged zigzagged belts, which likely underlie interactions with and subsequent disruption of negatively charged lipid bilayers, such as bacterial membranes. LL-3717-29 correspondingly forms wide, ribbon-like, thermostable fibrils in solution, which co-localize with bacterial cells. Structure-guided mutagenesis analyses supports the role of self-assembly in antibacterial activity. LL-3717-29 resembles, in sequence and in the ability to form amphipathic helical fibrils, the bacterial cytotoxic PSMα3 peptide that assembles into cross-α amyloid fibrils. This argues helical, self-assembling, basic building blocks across kingdoms of life and points to potential structural mimicry mechanisms. The findings expose a protein fibril which performs a biological activity, and offer a scaffold for functional and durable biomaterials for a wide range of medical and technological applications.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Amiloide/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Bactérias/metabolismo , Benzotiazóis , Catelicidinas/farmacologia , Cristalografia por Raios X , Gorilla gorilla , Humanos , Testes de Sensibilidade Microbiana , Micrococcus luteus/efeitos dos fármacos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Conformação Proteica , Staphylococcus hominis/efeitos dos fármacos , Difração de Raios X
4.
Proc Natl Acad Sci U S A ; 117(32): 19446-19454, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32723829

RESUMO

Antimicrobial peptides are important candidates for developing new classes of antibiotics because of their potency against antibiotic-resistant pathogens. Current research focuses on topical applications and it is unclear how to design peptides with systemic efficacy. To address this problem, we designed two potent peptides by combining database-guided discovery with structure-based design. When bound to membranes, these two short peptides with an identical amino acid composition can adopt two distinct amphipathic structures: A classic horizontal helix (horine) and a novel vertical spiral structure (verine). Their horizontal and vertical orientations on membranes were determined by solid-state 15N NMR data. While horine was potent primarily against gram-positive pathogens, verine showed broad-spectrum antimicrobial activity. Both peptides protected greater than 80% mice from infection-caused deaths. Moreover, horine and verine also displayed significant systemic efficacy in different murine models comparable to conventional antibiotics. In addition, they could eliminate resistant pathogens and preformed biofilms. Significantly, the peptides showed no nephrotoxicity to mice after intraperitoneal or intravenous administration for 1 wk. Our study underscores the significance of horine and verine in fighting drug-resistant pathogens.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/metabolismo , Antibacterianos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Infecções Bacterianas/tratamento farmacológico , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Membrana Celular/metabolismo , Bases de Dados de Proteínas , Desenho de Fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade , Resultado do Tratamento
5.
Sci Rep ; 10(1): 9392, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32523049

RESUMO

The rising incidence of antibiotic-resistant lung infections has instigated a much-needed search for new therapeutic strategies. One proposed strategy is the use of exogenous surfactants to deliver antimicrobial peptides (AMPs), like CATH-2, to infected regions of the lung. CATH-2 can kill bacteria through a diverse range of antibacterial pathways and exogenous surfactant can improve pulmonary drug distribution. Unfortunately, mixing AMPs with commercially available exogenous surfactants has been shown to negatively impact their antimicrobial function. It was hypothesized that the phosphatidylglycerol component of surfactant was inhibiting AMP function and that an exogenous surfactant, with a reduced phosphatidylglycerol composition would increase peptide mediated killing at a distal site. To better understand how surfactant lipids interacted with CATH-2 and affected its function, isothermal titration calorimetry and solid-state nuclear magnetic resonance spectroscopy as well as bacterial killing curves against Pseudomonas aeruginosa were utilized. Additionally, the wet bridge transfer system was used to evaluate surfactant spreading and peptide transport. Phosphatidylglycerol was the only surfactant lipid to significantly inhibit CATH-2 function, showing a stronger electrostatic interaction with the peptide than other lipids. Although diluting the phosphatidylglycerol content in an existing surfactant, through the addition of other lipids, significantly improved peptide function and distal killing, it also reduced surfactant spreading. A synthetic phosphatidylglycerol-free surfactant however, was shown to further improve CATH-2 delivery and function at a remote site. Based on these in vitro experiments synthetic phosphatidylglycerol-free surfactants seem optimal for delivering AMPs to the lung.


Assuntos
Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/química , Galinhas/metabolismo , Surfactantes Pulmonares/química , Animais , Anti-Infecciosos/administração & dosagem , Anti-Infecciosos/química , Sistemas de Liberação de Medicamentos/métodos , Excipientes/química , Lipídeos/química , Pulmão/efeitos dos fármacos , Fosfatidilgliceróis/química , Pseudomonas aeruginosa/efeitos dos fármacos
6.
Nat Commun ; 11(1): 3184, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576824

RESUMO

Peptide antibiotics are an abundant and synthetically tractable source of molecular diversity, but they are often cationic and can be cytotoxic, nephrotoxic and/or ototoxic, which has limited their clinical development. Here we report structure-guided optimization of an amphipathic peptide, arenicin-3, originally isolated from the marine lugworm Arenicola marina. The peptide induces bacterial membrane permeability and ATP release, with serial passaging resulting in a mutation in mlaC, a phospholipid transport gene. Structure-based design led to AA139, an antibiotic with broad-spectrum in vitro activity against multidrug-resistant and extensively drug-resistant bacteria, including ESBL, carbapenem- and colistin-resistant clinical isolates. The antibiotic induces a 3-4 log reduction in bacterial burden in mouse models of peritonitis, pneumonia and urinary tract infection. Cytotoxicity and haemolysis of the progenitor peptide is ameliorated with AA139, and the 'no observable adverse effect level' (NOAEL) dose in mice is ~10-fold greater than the dose generally required for efficacy in the infection models.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Animais , Carbapenêmicos/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Colistina/farmacologia , Modelos Animais de Doenças , Descoberta de Drogas , Feminino , Proteínas de Helminto/química , Proteínas de Helminto/farmacologia , Humanos , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Peritonite/tratamento farmacológico , Peritonite/microbiologia , Pneumonia/tratamento farmacológico , Pneumonia/microbiologia , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia
7.
Science ; 368(6490)2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32355003

RESUMO

Antimicrobial peptides (AMPs) are essential components of immune defenses of multicellular organisms and are currently in development as anti-infective drugs. AMPs have been classically assumed to have broad-spectrum activity and simple kinetics, but recent evidence suggests an unexpected degree of specificity and a high capacity for synergies. Deeper evaluation of the molecular evolution and population genetics of AMP genes reveals more evidence for adaptive maintenance of polymorphism in AMP genes than has previously been appreciated, as well as adaptive loss of AMP activity. AMPs exhibit pharmacodynamic properties that reduce the evolution of resistance in target microbes, and AMPs may synergize with one another and with conventional antibiotics. Both of these properties make AMPs attractive for translational applications. However, if AMPs are to be used clinically, it is crucial to understand their natural biology in order to lessen the risk of collateral harm and avoid the crisis of resistance now facing conventional antibiotics.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Farmacorresistência Bacteriana , Evolução Molecular , Animais , Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/farmacologia , Sinergismo Farmacológico , Humanos , Polimorfismo Genético , Pesquisa Médica Translacional
8.
Exp Parasitol ; 215: 107930, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32464221

RESUMO

Trypanosoma cruzi, the etiological agent of Chagas disease, is responsible for the infection of millions of people worldwide and it is a public health problem, without an effective cure. Four fragments with antimicrobial potential from the hemocyanin of Penaeus monodon shrimp were identified using a computer software AMPA. The present study aimed to evaluate the antichagasic effect of these four peptides (Hmc364-382, Hmc666-678, Hmc185-197 and Hmc476-498). The peptides were tested against the epimastigote, trypomastigote and amastigote forms of Trypanosoma cruzi Y strain (benznidazole-resistant strain) and cytotoxicity in mammalian cells was evaluated against LLC-MK2 lineage cells. Two fragments (Hmc364-382, Hmc666-678) showed activity against the epimastigote and trypomastigote forms and their selectivity index (SI) was calculated. The Hmc364-382 peptide was considered the most promising (SI > 50) one and it was used for further studies, using flow cytometry analyses with specific molecular probes and scanning electron microscopy (SEM). Hmc364-382 was able to induce cell death in T. cruzi through necrosis, observed by loss of membrane integrity in flow cytometry analyses and pore formation in SEM. Overall, Hmc364-382 open perspectives to the development of new antichagasic agents.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Hemocianinas/farmacologia , Penaeidae/química , Trypanosoma cruzi/efeitos dos fármacos , Animais , Peptídeos Catiônicos Antimicrobianos/toxicidade , Linhagem Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doença de Chagas/tratamento farmacológico , Citometria de Fluxo , Hemocianinas/toxicidade , Concentração Inibidora 50 , Macaca mulatta , Microscopia Eletrônica de Varredura , Fatores de Tempo , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/ultraestrutura
9.
World J Microbiol Biotechnol ; 36(5): 77, 2020 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-32399738

RESUMO

Protease mediated proteolysis has been widely implicated in virulence of necrotrophic fungal pathogens. This is counteracted in plants by evolving new and effective antimicrobial peptides (AMP) that constitute important components of innate immune system. Peptide extraction from rhizome of Zingiber zerumbet was optimized using ammonium sulphate (50-80% w/v) and acetone (60 and 100% v/v) with maximal protein recovery of 1.2 ± 0.4 mg/g obtained using 100% acetone. Evaluation of inhibitory potential of Z. zerumbet rhizome protein extract to prominent hydrolases of necrotrophic Pythium myriotylum revealed maximal inhibition of proteases (75.8%) compared to other hydrolytic enzymes. Protein was purified by Sephacryl S200HR resin resulting in twofold purification and protease inhibition of 84.4%. Non-reducing polyacrylamide gel electrophoresis (PAGE) of the fractions yielded two bands of 75 kDa and 25 kDa molecular size. Peptide mass fingerprint of the protein bands using matrix assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectroscopy (MS) and subsequent MASCOT searches revealed peptide match to methylesterase from Arabidopsis thaliana (15%) and to hypothetical protein from Oryza sativa (98%) respectively. Further centrifugal filter purification using Amicon Ultra (10,000 MW cut-off) filter, yielded a prominent band of 25 kDa size. Concentration dependent inhibition of zoospore viability by Z. zerumbet AMP designated as ZzAMP was observed with maximal inhibition of 89.5% at 4 µg protein and an IC50 value of 0.59 µg. Studies are of particular relevance in the context of identifying the molecules involved in imparting below ground defense in Z. zerumbet as well in development of AMPs as potential candidate molecules for control of necrotrophic pathogens of agricultural relevance.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeo Hidrolases/efeitos dos fármacos , Extratos Vegetais/farmacologia , Pythium/efeitos dos fármacos , Rizoma/microbiologia , Zingiberaceae/microbiologia , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Arabidopsis , Inibidores Enzimáticos , Fungos/efeitos dos fármacos , Oryza , Peptídeos/farmacologia , Extratos Vegetais/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Zingiberaceae/crescimento & desenvolvimento
10.
Biochim Biophys Acta Biomembr ; 1862(8): 183282, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32376222

RESUMO

Antimicrobial peptides are considered promising candidates for the development of novel antimicrobial agents to combat infections by multi-drug-resistant (MDR) bacteria. Here, we describe the identification and characterization of the synthetic peptide TC19, derived from the human thrombocidin-1-derived peptide L3. Biophysical experiments into the interaction between TC19 and mimics of human and bacterial plasma membranes demonstrated that the peptide is highly selective for bacterial membranes. In agreement, TC19 combined low cytotoxicity towards human fibroblasts with efficient and rapid killing in human plasma of MDR strains of several bacterial species of the ESKAPE panel. In addition, TC19 induced minor resistance in vitro, neutralized pro-inflammatory activity of bacterial cell envelope components while displaying slight chemotactic activity for human neutrophils. Importantly, topical application of TC19-containing hypromellose gel significantly reduced numbers of viable methicillin-resistant Staphylococcus aureus (MRSA) and MDR Acinetobacter baumannii in a superficial wound infection in mice. Together, TC19 is an attractive candidate for further development as a novel agent against (MDR) bacterial skin wound infections.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Biofilmes/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Infecção dos Ferimentos/tratamento farmacológico , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Humanos , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , Pele/efeitos dos fármacos , Pele/microbiologia , Pele/patologia , Infecção dos Ferimentos/genética , Infecção dos Ferimentos/microbiologia , Infecção dos Ferimentos/patologia
11.
Soft Matter ; 16(21): 5032-5043, 2020 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-32452495

RESUMO

Amphipathic peptides that partition into lipid bilayers affect the curvature elastic properties of their host. Some of these peptides are able to shift the Gaussian modulus to positive values, thus triggering an instability with respect to the formation of saddle curvatures. To characterize the generic aspects of the underlying mechanism, we employ a molecular lipid model that accounts for the interfacial tension between the polar and apolar regions of the membrane, for interactions between the lipid headgroups, and for the energy to stretch or compress the hydrocarbon chains. Peptides are modeled as cylinders that partition into the host membrane in a parallel orientation where they diminish the space available to the lipid headgroups and chains. The penetration depth into the membrane is determined by the angular size of the peptide's hydrophilic region. We demonstrate that only peptides with a small angular size of their hydrophilic region have an intrinsic tendency to render the Gaussian modulus more positive, and we identify conditions at which the Gaussian modulus adopts a positive sign upon increasing the peptide concentration. Our model allows us to also incorporate electrostatic interactions between cationic peptides and anionic lipids on the level of the linear Debye-Hückel model. We show that electrostatic interactions tend to shift the Gaussian modulus toward more positive values. Steric and electrostatic lipid-peptide interactions jointly decrease the effective interaction strength in the headgroup region of the host membrane thus suggesting a generic mechanisms of how certain amphipathic peptides are able to induce the formation of saddle curvatures.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Peptídeos Catiônicos Antimicrobianos/metabolismo , Ligação Proteica , Domínios Proteicos
12.
Mar Drugs ; 18(4)2020 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-32290449

RESUMO

The antimicrobial peptide (AMP) piscidin was identified from Epinephelus lanceolatus and demonstrated to possess antimicrobial and immune-related functions. Supplementation of feed with recombinant Epinephelus lanceolatus piscidin (rEP)-expressing yeast pellets may minimize the excessive use of antibiotics and control pathogens in aquaculture or animal husbandry. However, before implementing rEP as a supplement, it is necessary to understand whether it harbors any toxicity. Since toxicological information on the topic is scarce, the present investigation was carried out to test whether rEP exhibits allergenic and/or toxic effects. In an oral acute toxicity test (OECD 425), Sprague Dawley (SD) rats were administered rEP dissolved in reverse osmosis water, yielding an LD50 > 5000 mg/kg (no observed animal death). The compound was therefore classified as non-toxic by oral administration. In an acute respiratory toxicity test (OECD 403), heads and noses of SD rats were exposed to liquid aerosol for 4 h (the highest concentration that could be administered without causing any animal death), and a lethal concentration (LC50) > 0.88 mg/L was obtained. The mass medium aerodynamics diameter (MMAD) of rEP aerosol particles was 8.18 µm and mass medium aerodynamics diameter (GSD) was 3.04, which meant that 25.90% could enter the airway (<4 µm) of a rat, and 58.06% (<10 µm) could be inhaled by humans. An ocular irritation test (OECD 405) with rEP powder was performed on New Zealand White (NZW) rabbits. Signs of irritation included conjunctival swelling and diffuse flushing 1 h after administration. The signs were less apparent after 24 h and disappeared after 72 h. The classification assigned to the powder was mild eye irritation. Skin sensitization was performed for a local lymphoproliferative test (OECD 442B) using BALB/c mice, with the highest soluble concentration of the rEP considered to be 100% test substance; formulations were diluted to 50% and 25%, and bromodeoxyuridine (BrdU) incorporation was used to measure the degree of lymphocyte proliferation. The stimulation indexes (SIs) were 1.06 (100%), 0.44 (50%), and 0.77 (25%), all of which were less than the cutoff value for a positive sensitization result (1.6). Negative response was also seen in the bacterial reverse mutation test (OECD 471), and no chromosomal effects on Chinese hamster ovary (CHO)-K1 cells were observed (OECD 487). Based on these six toxicity tests, rEP showed neither acute toxic effects in experimental animals nor mutagenicity. Thus, rEP can be considered safe for use in subsequent research on its application as a feed additive for poultry, cattle, or aquatic animals.


Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Peixes/química , Peixes , Animais , Antibacterianos/farmacologia , Antibacterianos/toxicidade , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/toxicidade , Feminino , Proteínas de Peixes/farmacologia , Proteínas de Peixes/toxicidade , Dose Letal Mediana , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese , Pichia/genética , Coelhos , Ratos , Ratos Sprague-Dawley , Testes de Toxicidade
13.
Biochim Biophys Acta Proteins Proteom ; 1868(8): 140429, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32298805

RESUMO

Several D-amino acid-containing peptides (DAACPs) with antimicrobial, cardio-excitatory, or neuronal activities have been found in several species. Here, we demonstrated the chiral separation of the antimicrobial peptide diastereomers, D-phenylseptin and L-phenylseptin using (S) and (R) 3,3'-phenyl-1,1'-binaphthyl-18-crown-6-ether columns (CR-I (+) and CR-I (-), respectively) and also investigated the underlying mechanism. First, using D-amino acid-containing tripeptide Phe-Phe-Phe-OH, we found that CR-I (+) could be used to recognize diastereomeric tripeptides containing an L-amino acid as the first residue. On the contrary, CR-I (-) enabled separation of a series of diastereomers with D-amino acid as the first residue. Therefore, we achieved separation of the stereoisomers using the chiral columns depending on the position of the D- amino acid in the peptide and demonstrated the orthogonality of separations of the chiral columns. Then, using CR-I (+), we separated amphibian antimicrobial peptide diastereomers, L- and D-phenylseptin, which have the sequences, L-Phe-L-Phe-L-Phe and L-Phe-D-Phe-L-Phe at their N-termini, respectively. In order to understand the host-guest interactions, we performed molecular dynamics simulations for L-Phe-L-Phe-L-Phe tripeptide-CR-I molecule complex systems. Three hydrogen bonds between the N-terminal amine group -NH3+ and the crown ether oxygens were the dominant interactions. The hydrophobic interactions between phenyl-rings in the chiral selector unit of CR-I (+) and the side chains of 2nd and 3rd residues of the peptide also contributed to the affinity. Our results show that the CR-I (+)-column can be applied for the separation of endogenous DAACPs generated by the post-translational modification.


Assuntos
Proteínas de Anfíbios/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Éteres de Coroa/química , Oligopeptídeos/isolamento & purificação , Aminoácidos/química , Proteínas de Anfíbios/química , Animais , Peptídeos Catiônicos Antimicrobianos/química , Anuros , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Oligopeptídeos/química , Estereoisomerismo
14.
Proc Natl Acad Sci U S A ; 117(15): 8437-8448, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32241895

RESUMO

Novel classes of antibiotics and new strategies to prevent and treat infections are urgently needed because the rapid rise in drug-resistant bacterial infections in recent decades has been accompanied by a parallel decline in development of new antibiotics. Membrane permeabilizing antimicrobial peptides (AMPs) have long been considered a potentially promising, novel class of antibiotic, especially for wound protection and treatment to prevent the development of serious infections. Yet, despite thousands of known examples, AMPs have only infrequently proceeded as far as clinical trials, especially the chemically simple, linear examples. In part, this is due to impediments that often limit their applications in vivo. These can include low solubility, residual toxicity, susceptibility to proteolysis, and loss of activity due to host cell, tissue, and protein binding. Here we show how synthetic molecular evolution can be used to evolve potentially advantageous antimicrobial peptides that lack these impediments from parent peptides that have at least some of them. As an example of how the antibiotic discovery pipeline can be populated with more promising candidates, we evolved and optimized one family of linear AMPs into a new generation with high solubility, low cytotoxicity, potent broad-spectrum sterilizing activity against a panel of gram-positive and gram-negative ESKAPE pathogens, and antibiofilm activity against gram-positive and gram-negative biofilms. The evolved peptides have these activities in vitro even in the presence of concentrated host cells and also in vivo in the complex, cell- and protein-rich environment of a purulent animal wound model infected with drug-resistant bacteria.


Assuntos
Antibacterianos/administração & dosagem , Antibacterianos/síntese química , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/síntese química , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Biofilmes/efeitos dos fármacos , Farmacorresistência Bacteriana , Animais , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Bactérias/genética , Infecções Bacterianas/microbiologia , Evolução Molecular Direcionada , Feminino , Humanos , Camundongos , Testes de Sensibilidade Microbiana
15.
Biochim Biophys Acta Biomembr ; 1862(8): 183291, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32234322

RESUMO

Antimicrobial peptides (AMPs) selectively kill bacteria by disrupting their cell membranes, and are promising compounds to fight drug-resistant microbes. Biophysical studies on model membranes have characterized AMP/membrane interactions and the mechanism of bilayer perturbation, showing that accumulation of cationic peptide molecules in the external leaflet leads to the formation of pores ("carpet" mechanism). However, similar quantitative studies on real cells are extremely limited. Here, we investigated the interaction of the dansylated PMAP23 peptide (DNS-PMAP23) with a Gram-positive bacterium, showing that 107 bound peptide molecules per cell are needed to kill it. This result is consistent with our previous finding for Gram-negative strains, where a similar high threshold for killing was determined, demonstrating the general relevance of the carpet model for real bacteria. However, in the case of the Gram-positive strain, this number of molecules even exceeds the total surface available on the bacterial membrane. The high affinity of DNS-PMAP23 for the anionic teichoic acids of the Gram-positive cell wall, but not for the lipopolysaccharides of Gram-negative bacteria, provides a rationale for this finding. To better define the role of anionic lipids in peptide/cell association, we studied DNS-PMAP23 interaction with E. coli mutant strains lacking phosphatidylglycerol and/or cardiolipin. Surprisingly, these strains showed a peptide affinity similar to that of the wild type. This finding was rationalized by observing that these bacteria have an increased content of other anionic lipids, thus maintaining the total membrane charge essentially constant. Finally, studies of DNS-PMAP23 association to dead bacteria showed an affinity an order of magnitude higher compared to that of live cells, suggesting strong peptide binding to intracellular components that become accessible after membrane perturbation. This effect could play a role in population resistance to AMP action, since dead bacteria could protect the surviving cells by sequestering significant amounts of peptide molecules. Overall, our data indicate that quantitative studies of peptide association to bacteria can lead to a better understanding of the mechanism of action of AMPs.


Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Parede Celular/efeitos dos fármacos , Relação Estrutura-Atividade , Sequência de Aminoácidos/genética , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Parede Celular/química , Parede Celular/ultraestrutura , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/patogenicidade , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/patogenicidade , Humanos , Lipopolissacarídeos/química , Testes de Sensibilidade Microbiana
16.
Biochim Biophys Acta Biomembr ; 1862(8): 183302, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32311341

RESUMO

All antibiotics have to engage bacterial amphiphilic barriers such as the lipopolysaccharide-rich outer membrane or the phospholipid-based inner membrane in some manner, either by disrupting them outright and/or permeating them and thereby allow the antibiotic to get into bacteria. There is a growing class of cyclic antibiotics, many of which are of bacterial origin, that exhibit activity against Gram-negative bacteria, which constitute an urgent problem in human health. We examine a diverse collection of these cyclic antibiotics, both natural and synthetic, which include bactenecin, polymyxin B, octapeptin, capreomycin, and Kirshenbaum peptoids, in order to identify what they have in common when they interact with bacterial lipid membranes. We find that they virtually all have the ability to induce negative Gaussian curvature (NGC) in bacterial membranes, the type of curvature geometrically required for permeation mechanisms such as pore formation, blebbing, and budding. This is interesting since permeation of membranes is a function usually ascribed to antimicrobial peptides (AMPs) from innate immunity. As prototypical test cases of cyclic antibiotics, we analyzed amino acid sequences of bactenecin, polymyxin B, and capreomycin using our recently developed machine-learning classifier trained on α-helical AMP sequences. Although the original classifier was not trained on cyclic antibiotics, a modified classifier approach correctly predicted that bactenecin and polymyxin B have the ability to induce NGC in membranes, while capreomycin does not. Moreover, the classifier was able to recapitulate empirical structure-activity relationships from alanine scans in polymyxin B surprisingly well. These results suggest that there exists some common ground in the sequence design of hybrid cyclic antibiotics and linear AMPs.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/química , Bactérias Gram-Negativas/patogenicidade , Humanos , Aprendizado de Máquina , Testes de Sensibilidade Microbiana , Fosfolipídeos/química , Relação Estrutura-Atividade
17.
Biochim Biophys Acta Biomembr ; 1862(7): 183305, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32298679

RESUMO

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.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/química , Bicamadas Lipídicas/química , Magaininas/química , Sequência de Aminoácidos/genética , Peptídeos Catiônicos Antimicrobianos/genética , Membrana Celular/genética , Dimerização , Humanos , Magaininas/genética , Fosfolipídeos/química , Fosfolipídeos/genética , Porosidade
18.
Chemistry ; 26(39): 8524-8531, 2020 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-32250484

RESUMO

Natural products (NPs) are an important inspirational source for developing drugs and chemical probes. In 1999, the group of Omura reported the constitutional elucidation of zelkovamycin. Although largely unrecognized so far, this NP displays structural similarities as well as differences to the argyrin NP family, a class of peptidic NPs with promising anticancer activities and diverse mode-of-action at the molecular level. By a combination of structure elucidation experiments, the first total synthesis of zelkovamycin and bioassays, the zelkovamycin configuration was determined and its previously proposed molecular structure was revised. The full structure assignment proves zelkovamycin as an additional member of the argyrins with however unique OXPHOS inhibitory properties. Zelkovamycin may therefore not only serve as a new starting point for chemical inhibitors of the OXPHOS system, but also guide customized argyrin NP isolation and biosynthesis studies.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Produtos Biológicos/farmacologia , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Produtos Biológicos/química , Estrutura Molecular
19.
Biochim Biophys Acta Biomembr ; 1862(8): 183246, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32142818

RESUMO

The filamentous fungus Penicillium chrysogenum Q176 secretes the antimicrobial proteins (AMPs) PAF and PAFB, which share a compact disulfide-bond mediated, ß-fold structure rendering them highly stable. These two AMPs effectively inhibit the growth of human pathogenic fungi in micromolar concentrations and exhibit antiviral potential without causing cytotoxic effects on mammalian cells in vitro and in vivo. The antifungal mechanism of action of both AMPs is closely linked to - but not solely dependent on - the lipid composition of the fungal cell membrane and requires a strictly regulated protein uptake into the cell, indicating that PAF and PAFB are not canonical membrane active proteins. Variations in their antifungal spectrum and their killing dynamics point towards a divergent mode of action related to their physicochemical properties and surface charge distribution. In this review, we relate characteristic features of PAF and PAFB to the current knowledge about other AMPs of different sources. In addition, we present original data that have never been published before to substantiate our assumptions and provide evidences that help to explain and understand better the mechanistic function of PAF and PAFB. Finally, we underline the promising potential of PAF and PAFB as future antifungal therapeutics.


Assuntos
Antifúngicos/química , Peptídeos Catiônicos Antimicrobianos/química , Proteínas Fúngicas/química , Micoses/tratamento farmacológico , Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Apoptose/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Cisteína/genética , Proteínas Fúngicas/genética , Humanos , Lipídeos de Membrana/química , Micoses/genética , Micoses/microbiologia , Penicillium chrysogenum/química , Penicillium chrysogenum/genética
20.
Sci Rep ; 10(1): 4710, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170095

RESUMO

The amphipathic α-helical antimicrobial peptide MSI-103 (aka KIA21) can form stable transmembrane pores when the bilayer takes on a positive spontaneous curvature, e.g. by the addition of lyso-lipids. Solid-state 31P- and 15N-NMR demonstrated an enrichment of lyso-lipids in these toroidal wormholes. Anionic lyso-lipids provided additional stabilization by electrostatic interactions with the cationic peptides. The remaining lipid matrix did not affect the nature of the pore, as peptides maintained the same orientation independent of lipid charge, and a change in membrane thickness did not considerably affect their tilt angle. Under optimized conditions (i.e. in the presence of lyso-lipids and appropriate bilayer thickness), stable and well-aligned pores could be obtained for solid-state 2H-NMR analysis. These data revealed for the first time the complete 3D alignment of this representative amphiphilic peptide in fluid membranes, which is compatible with either monomeric helices as constituents, or left-handed supercoiled dimers as building blocks from which the overall toroidal wormhole is assembled.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/química , Bicamadas Lipídicas/química , Lipídeos/química , Conformação Proteica em alfa-Hélice , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Ressonância Magnética Nuclear Biomolecular , Relação Estrutura-Atividade
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