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1.
Gene ; 764: 145101, 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-32877747

RESUMO

India is the world's largest milk producing country because of massive contribution made by cattle and buffaloes. In the present investigation, comprehensive comparative profiling of transcriptomic landscape of milk somatic cells of Sahiwal cattle and Murrah buffaloes was carried out. Genes with highest transcript abundance in both species were enriched for biological processes such as lactation, immune response, cellular oxidant detoxification and response to hormones. Analysis of differential expression identified 377 significantly up-regulated and 847 significantly down-regulated genes with fold change >1.5 in Murrah buffaloes as compared to Sahiwal cattle (padj <0.05). Marked enrichment of innate and adaptive immune response related GO terms and higher expression of genes for various host defense peptides such as lysozyme, defensin ß and granzymes were evident in buffaloes. Genes related to ECM-receptor interaction, complement and coagulation cascades, cytokine-cytokine receptor interaction and keratinization pathway showed more abundant expression in cattle. Network analysis of the up-regulated genes delineated highly connected genes representing immunity and haematopoietic cell lineage (CBL, CD28, CD247, PECAM1 and ITGA4). For the down-regulated dataset, genes with highest interactions were KRT18, FGFR1, GPR183, ITGB3 and DKK3. Our results lend support to more robust immune mechanisms in buffaloes, possibly explaining lower susceptibility to mammary infections as compared to cattle.


Assuntos
Búfalos/imunologia , Bovinos/imunologia , Imunidade/genética , Transcriptoma/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Búfalos/genética , Bovinos/genética , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Regulação para Baixo/imunologia , Feminino , Hematopoese/genética , Hematopoese/imunologia , Índia , Lactação/genética , Lactação/imunologia , Leite/citologia , Leite/imunologia , RNA-Seq , Transcriptoma/genética , Regulação para Cima/imunologia
2.
DNA Cell Biol ; 39(10): 1872-1885, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32936023

RESUMO

Hyenas (family Hyaenidae) occupy a variety of different niches, of which the striped hyena (Hyaena hyaena) scavenges mainly on the carcasses of animals. We compared its genome with the genomes of nine other mammals, focusing on similarities and differences in chemoreception, detoxification, digestive, and immune systems. The results showed that the striped hyena's immune and digestive system-related gene families have significantly expanded, which was likely to be an adaptive response to its scavenging lifestyle. In addition, 88 and 26 positive selected genes (PSGs) were identified in the immune system and digestive system, respectively, which may be the molecular basis for immune defense system to effectively resist pathogen invasion. Functional enrichment analysis of PSGs revealed that most of them were involved in the immune regulation process. Among them, eight specific missense mutations were found in two PSGs (MHC class II antigen DOA and MHC class II antigen DOB), suggesting important reorganization of the immune system in the striped hyena. Moreover, we identified one cathelicidin gene and four defensin genes in the striped hyenas by genome mining, which have high-efficiency and broad-spectrum antimicrobial activity. Of particular interest, a striped hyena-specific missense mutation was found in the cathelicidin gene. PolyPhen-2 classified the missense mutation as a harmful mutation, which may have aided in immune adaptation to carrion feeding. Our genomic analyses on the striped hyena provided insights into its success in the adaptation to the scavenging lifestyle.


Assuntos
Comportamento Alimentar , Genoma , Hyaenidae/genética , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Defensinas/genética , Digestão/genética , Antígenos de Histocompatibilidade/genética , Hyaenidae/fisiologia , Imunidade Inata/genética , Mutação
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.
Mol Immunol ; 126: 1-7, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32712503

RESUMO

Rel/nuclear factor (NF)-κB family of transcription factors paly vital roles in innate immunity response to bacterial and viral infection. Here, we cloned and identified a dorsal homologue (named as MnDorsal) from Macrobrachium nipponense. The full-length cDNA of MnDorsal is 2573 bp with a 1986 bp open reading frame that encodes 661 amino acids. Predicted MnDorsal protein contained a RHD (Rel homology domain), an IPT (Iglike, plexins, and transcriptions factors) domain, and two low complexity regions. Phylogenetic analysis showed that MnDorsal has a closer genetic distance with dorsal homologues from invertebrates. MnDorsal was widely expressed in a variety of tissues, including hemocytes, heart, hepatopancreas, gills, stomach, and intestine. Expression patterns analysis showed that the transcriptional level of MnDorsal in the gills was evidently up-regulated after Staphylococcus aureus, Vibrio parahaemolyticus, white spot syndrome virus, or polyinosinic-polycytidylic acid challenge, suggesting that MnDorsal participates in the immune defenses against pathogens and stimulant challenges. Additionally, the dsRNA-mediated RNA interference analysis showed that knockdown of MnDorsal can significantly inhibit the expression of anti-lipopolysaccharide factor (ALF) and crustin. Further studies revealed that the up-regulated expression of ALFs (MnALF2, MnALF3, and MnALF4) and crustins (MnCrustin3 and MnCrustin4) caused by S. aureus infection were obviously decreased after silencing MnDorsal. These findings suggest that MnDorsal positively regulate the expression of antibacterial peptides (AMPs) during S. aureus infection. Our study will promote to better understand the role of Toll-Dorsal-AMPs pathway in innate immunity response to gram-positive bacterial infection in crustacean.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Artrópodes/metabolismo , Imunidade Inata/genética , Palaemonidae/imunologia , Fatores de Transcrição/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Aquicultura , Proteínas de Artrópodes/genética , Regulação da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Palaemonidae/genética , Palaemonidae/metabolismo , Palaemonidae/microbiologia , Poli I-C/imunologia , Staphylococcus aureus/imunologia , Fatores de Transcrição/genética , Vibrio parahaemolyticus/imunologia , Vírus da Síndrome da Mancha Branca 1/imunologia
5.
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
6.
Arch Microbiol ; 202(7): 1785-1794, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32382765

RESUMO

Bacillus spp. have a wide range of activities in the biocontrol potential against various phytopathogens. This study focuses on the biocontrol potential of two species belonging to the same genera, as Bacillus subtilis (SSR2I) and Bacillus flexus (AIKDL) have contrasting activity under in vivo and in vitro conditions. In this study, two medicinal plants-associated bacteria showing antagonistic activity against wilt-causing pathogens were selected and identified as B. subtilis (SSR2I) and B. flexus (AIKDL) based on 16S rRNA gene sequencing. Crude extracts of these bacteria showed that chloroform extracts of AIKDL, and ethyl acetate extraction of SSR2I showed effective potential inhibition of both the wilt-causing pathogens in the well-diffusion method. PCR-based detection of antimicrobial peptide genes revealed the presence of five genes in B. subtilis and none in B. flexus. On the basis of in vivo analysis, the isolate SSR2I showed reduced disease incidence and enhanced biocontrol efficiency against Ralstonia solanacearum and Fusarium oxysporum compared with AIKDL and control plants. Further, the isolates SSR2I also enhanced the induced systemic resistance (ISR) against both the pathogens compared to the control. However, the isolate AIKDL showed enhanced ISR against F. oxysporum-treated plants, but not against R. solanacearum-treated plants. The results indicated that even though the isolates had strong antagonistic potential under in vitro conditions, their biocontrol efficiency differed in in vivo condition. On the basis of the overall performance, the isolate SSR2I could be formulated as biocontrol agents against both the wilt-causing pathogens tested in this study.


Assuntos
Bacillus/genética , Bacillus/metabolismo , Fenômenos Fisiológicos Bacterianos , Fungos/fisiologia , Interações Microbianas , Controle Biológico de Vetores , Plantas/microbiologia , Peptídeos Catiônicos Antimicrobianos/genética , Bacillus/classificação , Desenvolvimento Vegetal/fisiologia , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , RNA Ribossômico 16S/genética
7.
BMC Biotechnol ; 20(1): 19, 2020 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-32228563

RESUMO

BACKGROUND: Foodborne pathogens and their biofilms are considered as one of the most serious problems in human health and food industry. Moreover, safety of foods is a main global concern because of the increasing use of chemical food additives. Ensuring food safety enhances interest in discovery of new alternative compounds such as antimicrobial peptides (AMPs), which can be used as bio-preservatives in the food industry. In this study, the most important antimicrobial peptides of camel milk lactoferrin (lactoferrampin and lactoferricin) were recombinantly expressed in the form of chimeric peptide (cLFchimera) in a food-grade L. lactis strain. P170 expression system was used to express secreted cLFchimera using pAMJ1653 expression vector which harbors a safe (non-antibiotic) selectable marker. RESULTS: Peptide purification was carried out using Ni-NTA agarose column from culture medium with concentration of 0.13 mg/mL. The results of disk diffusion test revealed that cLFchimera had considerable antimicrobial activity against a number of major foodborne bacteria. Furthermore, this chimeric peptide showed strong and weak inhibitory effect on biofilm formation against P. aeruginosa, S. aureus E. faecalis, and E. coli, respectively. Antioxidant activity and thermal stability of the chimeric peptide was determined. The results showed that cLFchimera had antioxidant activity (IC50: 310 µ/mL) and its activity was not affected after 40 min of boiling. Finally, we evaluated the interaction of the peptide with LPS and DNA in bacteria using molecular dynamic simulation as two main intra and extra cellular targets for AMPs, respectively. Our in silico analysis showed that cLFchimera had strong affinity to both of these targets by positive charged residues after 50 ns molecular dynamic simulation. CONCLUSIONS: Overall, the engineered food-grade L. lactis generated in the present study successfully expressed a secreted chimeric peptide with antimicrobial properties and could be considered as a promising bio-preservative in the food industry.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Lactococcus lactis/crescimento & desenvolvimento , Lactoferrina/química , Fragmentos de Peptídeos/química , Engenharia de Proteínas/métodos , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Biofilmes/efeitos dos fármacos , Camelus , Simulação por Computador , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Enterococcus faecalis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Microbiologia de Alimentos , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos
8.
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
9.
PLoS One ; 15(3): e0230021, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32160226

RESUMO

Supplementing chicken feed with antibiotics can improve survival and prevent disease outbreaks. However, overuse of antibiotics may promote the development of antibiotic-resistant bacteria. Recently, antimicrobial peptides have been proposed as alternatives to antibiotics in animal husbandry. Here, we evaluate the effects of antimicrobial peptide, Epinephelus lanceolatus piscidin (EP), in Gallus gallus domesticus. The gene encoding EP was isolated, sequenced, codon-optimized and cloned into a Pichia pastoris recombinant protein expression system. The expressed recombinant EP (rEP) was then used as a dietary supplement for G. g. domesticus; overall health, growth performance and immunity were assessed. Supernatant from rEP-expressing yeast showed in vitro antimicrobial activity against Gram-positive and Gram-negative bacteria, according to an inhibition-zone diameter (mm) assay. Moreover, the antimicrobial peptide function of rEP was temperature independent. The fermentation broth yielded a spray-dried powder formulation containing 262.9 µg EP/g powder, and LC-MS/MS (tandem MS) analysis confirmed that rEP had a molecular weight of 4279 Da, as expected for the 34-amino acid peptide; the DNA sequence of the expression vector was also validated. We then evaluated rEP as a feed additive for G. g. domesticus. Treatment groups included control, basal diet and rEP at different doses (0.75, 1.5, 3.0, 6.0 and 12%). Compared to control, rEP supplementation increased G. g. domesticus weight gain, feed efficiency, IL-10 and IFN-γ production. Our results suggest that crude rEP could provide an alternative to traditional antibiotic feed additives for G. g. domesticus, serving to enhance growth and health of the animals.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Galinhas/imunologia , Sistema Imunitário/metabolismo , Perciformes/metabolismo , Sequência de Aminoácidos , Animais , Anti-Infecciosos/análise , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/análise , Peptídeos Catiônicos Antimicrobianos/classificação , Peptídeos Catiônicos Antimicrobianos/genética , Galinhas/crescimento & desenvolvimento , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Suplementos Nutricionais , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Sistema Imunitário/efeitos dos fármacos , Interferon gama/metabolismo , Interleucina-10/metabolismo , Filogenia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência , Espectrometria de Massas em Tandem , Temperatura
10.
Res Microbiol ; 171(3-4): 115-121, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32119904

RESUMO

Listeria ivanovii is one of the two pathogenic species within the genus Listeria, the other being Listeria monocytogenes. In this study, we generated a stable pediocin resistant mutant Liv-r1 of a L. ivanovii strain, compared phenotypic differences between the wild-type and the mutant, localised the pediocin-induced mutations in the chromosome, and analysed the mechanisms behind the bacteriocin resistance. In addition to pediocin resistance, Liv-r1 was also less sensitive to nisin. The growth of Liv-r1 was significantly reduced with glucose and mannose, but less with cellobiose. The cells of Liv-r1 adsorbed less pediocin than the wild-type cells. Consequently, with less pediocin on the cell surface, the mutant was also less leaky, as shown as the release of intracellular lactate dehydrogenase to the supernatant. The surface of the mutant cells was more hydrophobic than that of the wild-type. Whole genome sequencing revealed numerous changes in the Liv-r1 chromosome. The mutations were found e.g., in genes encoding sigma-54-dependent transcription regulator and internalin B, as well as in genes involved in metabolism of carbohydrates such as glucose and cellobiose. Genetic differences observed in the mutant may be responsible for resistance to pediocin but no direct evidence is provided.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Farmacorresistência Bacteriana , Listeria/efeitos dos fármacos , Listeria/genética , Listeriose/microbiologia , Pediocinas/genética , Pediocinas/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Metabolismo dos Carboidratos , Genoma Bacteriano , Genômica/métodos , Listeria/metabolismo , Listeriose/tratamento farmacológico , Testes de Sensibilidade Microbiana , Pediocinas/química , Sequenciamento Completo do Genoma
11.
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
12.
Biochim Biophys Acta Biomembr ; 1862(4): 183242, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32135146

RESUMO

Antimicrobial Peptides (AMPs) are host defense molecules that initiate microbial death by binding to the membrane. On membrane binding, AMPs undergo changes in conformation and aggregation state to enable killing action. Depending on the AMP and cell membrane characteristics, the nature of binding can be aggregating or non-aggregating, with high/low cooperativity, at single or multiple sites with high/low affinity leading to a unique killing action that needs to be studied individually. In the present study, a steady-state model that simulates AMP-membrane interaction was developed and was used to predict the mechanism of AMP binding. The predictions obtained from the model were validated with experimentally deciphered values available in literature. The model was further used to predict the mechanism for a set of designed AMPs with high sequence similarity to Myeloid Antimicrobial Peptide (MAP) family. Depending on the predicted mechanism, a unique half saturation constant and steepness of response (Hill coefficient) was obtained which was further validated with available data from literature. The model could reliably predict the mechanism, the half saturation constant and the Hill coefficient values. Further based on the analysis, it was observed that aggregation and oligomerization result in drastic killing action in a short range of peptide concentration owing to high Hill coefficient values. Mechanisms such as monomers binding at multiple sites with/without cooperativity result in antimicrobial activity at low half saturation constant though the killing action may not be steep. Thus, the methodology developed here can be used to develop hypothesis for studying AMP-membrane interaction mechanisms.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/química , Proteínas de Membrana/química , Modelos Moleculares , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Membrana Celular/genética , Membrana Celular/ultraestrutura , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Testes de Sensibilidade Microbiana , Ligação Proteica/genética , Biologia de Sistemas
13.
Biochim Biophys Acta Biomembr ; 1862(8): 183228, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32126228

RESUMO

Antimicrobial peptides (AMPs) constitute a diverse family of peptides with the ability to protect their host against microbial infections. In addition to their ability to kill microorganisms, several AMPs also exhibit selective cytotoxicity towards cancer cells and are collectively referred to as anticancer peptides (ACPs). Here a large library of AMPs, mainly derived from the porcine cathelicidin peptide, tritrpticin (VRRFPWWWPFLRR), were assessed for their anticancer activity against the Jurkat T cell leukemia line. These anticancer potencies were compared to the cytotoxicity of the peptides towards normal cells isolated from healthy donors, namely peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs; where hemolytic activity was assessed). Among the active tritrpticin derivatives, substitution of Arg by Lys enhanced the selectivity of the peptides towards Jurkat cells when compared to PBMCs. Additionally, the side chain length of the Lys residues was also optimized to further enhance the tritrpticin ACP selectivity at low concentrations. The mechanism of action of the peptides with high selectivity involved the permeabilization of the cytoplasmic membrane of Jurkat cells, without formation of apoptotic bodies. The incorporation of non-natural Lys-based cationic amino acids could provide a new strategy to improve the selectivity of other synthetic ACPs to enhance their potential for therapeutic use against leukemia cells.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Antineoplásicos/farmacologia , Oligopeptídeos/genética , Peptídeos/genética , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/química , Dicroísmo Circular , Eritrócitos/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Oligopeptídeos/química , Peptídeos/química , Peptídeos/farmacologia , Suínos
14.
Biochim Biophys Acta Biomembr ; 1862(7): 183236, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32126226

RESUMO

Piscidins are host-defense peptides (HDPs) from fish that exhibit antimicrobial, antiviral, anti-cancer, anti-inflammatory, and wound-healing properties. They are distinctively rich in histidine and contain an amino terminal copper and nickel (ATCUN) binding motif due to the presence of a conserved histidine at position 3. Metallation lowers their total charge and provides a redox center for the formation of radicals that can convert unsaturated fatty acids (UFAs) into membrane-destabilizing oxidized phospholipids (OxPLs). Here, we focus on P1, a particularly membrane-active isoform, and investigate how metallating it and making OxPL available influence its membrane activity. First, we quantify through dye leakage experiments the permeabilization of the apo- and holo-forms of P1 on model membranes containing a fixed ratio of anionic phosphatidylglycerol (PG) and zwitterionic phosphatidylcholine (PC) but varying amounts of Aldo-PC, an OxPL derived from the degradation of several UFAs. Remarkably, metallating P1 increases membranolysis by a factor of five in each lipid system. Conversely, making Aldo-PC available improves permeabilization by a factor of two for each peptide form. Second, we demonstrate through CD-monitored titrations that the strength of the peptide-membrane interactions is similar in PC/PG and PC/PG/Aldo-PC. Thus, peptide-induced membrane activity is boosted by properties intrinsic to the peptide (e.g., charge and structural changes associated with metallation) and bilayer (e.g., reversal of sn-2 chain due to oxidation). Third, we show using oriented-sample 15N solid-state NMR that the helical portion of P1 lies parallel to the bilayer surface in both lipid systems. 31P NMR experiments show that both the apo- and holo-states interact more readily with PC in PC/PG. However, the presence of Aldo-PC renders the holo-, but not the apo-state, more specific to PG. Hence, the membrane disruptive effects of P1 and its specificity for the anionic lipids found on pathogenic cell membrane surfaces are simultaneously optimized when it is metallated and the OxPL is present. Overall, this study deepens our insights into how OxPLs affect peptide-lipid interactions and how host defense metallopeptides could help integrate the effects of antimicrobial agents.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Ácidos Graxos Insaturados/química , Proteínas de Peixes/genética , Metais/química , Animais , Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/genética , Sítios de Ligação , Membrana Celular , Cobre/química , Ácidos Graxos Insaturados/genética , Proteínas de Peixes/química , Histidina/química , Histidina/genética , Humanos , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Lipídeos de Membrana/genética , Níquel/química , Fosfolipídeos/química , Fosfolipídeos/genética
15.
Biochim Biophys Acta Biomembr ; 1862(7): 183280, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32220553

RESUMO

Short linear antimicrobial peptides are attractive templates for developing new antibiotics. Here, it is described a study of the interaction between two short Trp-rich peptides, horine and verine-L, and model membranes. Isothermal titration calorimetry studies showed that the affinity of these peptides towards large unilamellar vesicles (LUV) having a lipid composition mimicking the lipid composition of S. aureus membranes is ca. 30-fold higher than that towards E. coli mimetics. The former interaction is driven by enthalpy and entropy, while the latter case is driven by entropy, suggesting differences in the forces that play a role in the binding to the two types of model membranes. Upon membrane binding the peptides acquired different conformations according to circular dichroism (CD) studies; however, in both cases CD studies indicated stacked W-residues. Peptide-induced membrane permeabilization, lipid flip-flop, molecular packing at the membrane-water interface, and lateral lipid segregation were observed in all cases. However, the extent of these peptide-induced changes on membrane properties was always higher in S. aureus than E. coli mimetics. Both peptides seem to act via a similar mechanism of membrane permeabilization of S. aureus membrane mimetics, while their mechanisms seem to differ in the case of E. coli. This may be the result of differences in both the peptides´ structure and the membrane lipid composition between both types of bacteria.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Conformação Molecular , Sequência de Aminoácidos/genética , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Biomimética , Calorimetria , Dicroísmo Circular , Escherichia coli/química , Escherichia coli/patogenicidade , Humanos , Staphylococcus aureus/química , Staphylococcus aureus/patogenicidade , Termodinâmica , Triptofano/química , Triptofano/genética , Lipossomas Unilamelares/química
16.
Int J Med Sci ; 17(2): 191-206, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32038103

RESUMO

Epigenetic alteration of host DNA is a common occurrence in both low- and high-risk human papillomavirus (HPV) infection. Although changes in promoter methylation have been widely studied in HPV-associated cancers, they have not been the subject of much investigation in HPV-induced warts, which are a temporary manifestation of HPV infection. The present study sought to examine the differences in promoter methylation between warts and normal skin. To achieve this, DNA was extracted from 24 paired wart and normal skin samples and inputted into the Infinium MethylationEPIC BeadChip microarray. Differential methylation analysis revealed a clear pattern of hyper- and hypomethylation in warts compared to normal skin, and the most differentially methylated promoters were found within the EIF3EP2, CYSLTR1, C10orf99, KRT6B, LAMA4, and H3F3B genes as well as the C9orf30 pseudogene. Moreover, pathway analysis showed that the H3F3A, CDKN1A, and MAPK13 genes were the most common regulators among the most differentially methylated promoters. Since the tissue samples were excised from active warts, however, this differential methylation could either be a cellular response to HPV infection or an HPV-driven process to establish the wart and/or promote disease progression. Conclusively, it is apparent that HPV infection alters the methylation status of certain genes to possibly initiate the formation of a wart and maintain its presence.


Assuntos
Epigênese Genética/genética , Epigenoma/genética , Regiões Promotoras Genéticas/genética , Verrugas/genética , Peptídeos Catiônicos Antimicrobianos/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Histonas/genética , Humanos , Queratina-6/genética , Laminina/genética , Masculino , Proteína Quinase 13 Ativada por Mitógeno/genética , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/genética , Receptores de Leucotrienos/genética , Sequenciamento Completo do Genoma
17.
Sci Rep ; 10(1): 2696, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32060388

RESUMO

Cutaneous secretions of amphibians have bioactive compounds, such as peptides, with potential for biotechnological applications. Therefore, this study aimed to determine the primary structure and investigate peptides obtained from the cutaneous secretions of the amphibian, Leptodactylus vastus, as a source of bioactive molecules. The peptides obtained possessed the amino acid sequences, GVVDILKGAAKDLAGH and GVVDILKGAAKDLAGHLASKV, with monoisotopic masses of [M + H]± = 1563.8 Da and [M + H]± = 2062.4 Da, respectively. The molecules were characterized as peptides of the class of ocellatins and were named as Ocellatin-K1(1-16) and Ocellatin-K1(1-21). Functional analysis revealed that Ocellatin-K1(1-16) and Ocellatin-K1(1-21) showed weak antibacterial activity. However, treatment of mice with these ocellatins reduced the nitrite and malondialdehyde content. Moreover, superoxide dismutase enzymatic activity and glutathione concentration were increased in the hippocampus of mice. In addition, Ocellatin-K1(1-16) and Ocellatin-K1(1-21) were effective in impairing lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) formation and NF-kB activation in living microglia. We incubated hippocampal neurons with microglial conditioned media treated with LPS and LPS in the presence of Ocellatin-K1(1-16) and Ocellatin-K1(1-21) and observed that both peptides reduced the oxidative stress in hippocampal neurons. Furthermore, these ocellatins demonstrated low cytotoxicity towards erythrocytes. These functional properties suggest possible to neuromodulatory therapeutic applications.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Anuros/metabolismo , Hipocampo/efeitos dos fármacos , Infecções/tratamento farmacológico , Neurônios/efeitos dos fármacos , Sequência de Aminoácidos/genética , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Hipocampo/metabolismo , Infecções/induzido quimicamente , Infecções/genética , Infecções/microbiologia , Lipopolissacarídeos/toxicidade , Camundongos , Microglia/efeitos dos fármacos , NF-kappa B/genética , Neurônios/metabolismo , Nitritos/antagonistas & inibidores , Nitritos/metabolismo , Espécies Reativas de Oxigênio/metabolismo
18.
Biochim Biophys Acta Biomembr ; 1862(8): 183212, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32057757

RESUMO

The LAH4 family of amphipathic peptides exhibits pronounced antimicrobial, cell penetrating and nucleic acid transfection activities. Furthermore, variants were designed with potent lentiviral transduction enhancement. When viewed along a helical wheel the four histidines are arranged to form an amphipathic structure. In order to optimize some of these biological activities the number of leucine and alanine residues exposed to the hydrophilic surface was systematically varied which resulted in the design of vectofusin a peptide with strong lentiviral transduction enhancement activities. Here the series of peptides with varying numbers of alanine or leucine residues, respectively, framed by the histidines was tested for their calcein release activity. Interestingly, the membrane pore formation and DNA transfection activities show a clear correlation with the hydrophilic angle. In contrast the membrane partitioning and the propensity to adopt helical conformations was hardly affected as long as the hydrophilic angle did not exceed a limiting value of 150°.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , DNA/genética , Histidina/genética , Membranas/efeitos dos fármacos , Alanina/genética , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/farmacologia , DNA/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Lentivirus/genética , Leucina/genética , Membranas/metabolismo , Porosidade , Transfecção
19.
Fish Shellfish Immunol ; 98: 508-514, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32004613

RESUMO

At present, several reports have indicated that the C-terminal peptides of tissue factor pathway inhibitor 1 (TFPI-1) were active antibacterial peptides. However, the functions of TFPI-1 C-terminal peptides in teleost are still very limited. In this study, a C-terminal peptide, TC26 (with 26 amino acids), derived from common carp (Cyprinus carpio) TFPI-1, was synthesized and investigated for its antibacterial spectrum, action mechanism, as well as the in vivo effects on bacterial invasion. Our results showed that TC26 was active against Gram-positive bacteria Micrococcus luteus and Staphylococcus aureus, as well as Gram-negative bacterium Vibrio vulnificus. TC26 treatment facilitated the bactericidal process of erythromycin by enhancing the out-membrane permeability of V. vulnificus. During the bactericidal process, TC26 killed the target bacterial cells Vibrio vulnificus, by destroying cell membrane integrity, penetrating into the cytoplasm and inducing degradation of genomic DNA and total RNA. In vivo study showed that administration of turbot with TC26 before bacterial infection significantly reduced pathogen dissemination and replication in tissues. These results indicated that TC26 is a novel and active antibacterial peptide and may play a vital role in fighting pathogenic infection in aquaculture.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Carpas/metabolismo , Proteínas de Peixes/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/veterinária , DNA Bacteriano , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Linguados
20.
Parasit Vectors ; 13(1): 20, 2020 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-31931867

RESUMO

BACKGROUND: MF6p/host defense molecules (HDMs) are a broad family of small proteins secreted by helminth parasites. Although the physiological role of MF6p/HDMs in trematode parasites is not fully understood, their potential biological function in maintaining heme homeostasis and modulating host immune response has been proposed. METHODS: A gene encoding the MF6p/HDM of Clonorchis sinensis (CsMF6p/HDM) was cloned. Recombinant CsMF6p/HDM (rCsMF6p/HDM) was expressed in Escherichia coli. The biochemical and immunological properties of rCsMF6/HDM were analyzed. CsMF6p/HDM induced pro-inflammatory response in RAW 264.7 cells was analyzed by cytokine array assay, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. The structural feature of CsMF6p/HDM was analyzed by three-dimensional modeling and molecular docking simulations. RESULTS: The CsMF6p/HDM shares a high level of amino acid sequence similarity with orthologs from other trematodes and is expressed in diverse developmental stages of the parasite. The rCsMF6p/HDM bound to bacteria-derived lipopolysaccharide (LPS), without effectively neutralizing LPS-induced inflammatory response in RAW 264.7 macrophage cells. Rather, the rCsMF6p/HDM induced pro-inflammatory immune response, which is characterized by the expression of TNF-α and IL-6, in RAW 264.7 cells. The rCsMF6p/HDM-induced pro-inflammatory immune response was regulated by JNK and p38 MAPKs, and was effectively down-regulated via inhibition of NF-κB. The structural analysis of CsMF6p/HDM and the docking simulation with LPS suggested insufficient capture of LPS by CsMF6p/HDM, which suggested that rCsMF6p/HDM could not effectively neutralize LPS-induced inflammatory response in RAW 264.7 cells. CONCLUSIONS: Although rCsMF6p/HDM binds to LPS, the binding affinity may not be sufficient to maintain a stable complex of rCsMF6p/HDM and LPS. Moreover, the rCsMF6p/HDM-induced pro-inflammatory response is characterized by the release of IL-6 and TNF-α in RAW 264.7 macrophage cells. The pro-inflammatory response induced by rCsMF6p/HDM is mediated via NF-κB-dependent MAPK signaling pathway. These results collectively suggest that CsMF6p/HDM mediates C. sinensis-induced inflammation cascades that eventually lead to hepatobiliary diseases.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Clonorchis sinensis/metabolismo , Macrófagos/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Clonagem Molecular , Clonorquíase/etiologia , Citocinas/metabolismo , Doenças do Sistema Digestório/etiologia , Doenças do Sistema Digestório/parasitologia , Escherichia coli , Imunidade Celular , Inflamação/metabolismo , Sistema de Sinalização das MAP Quinases , Macrófagos/parasitologia , Camundongos , Simulação de Acoplamento Molecular/métodos , NF-kappa B/metabolismo , Células RAW 264.7 , Trematódeos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
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