RESUMO
The constant demand for new antibiotic drugs has driven efforts by the scientific community to prospect for peptides with a broad spectrum of action. In this context, antimicrobial peptides (AMPs) have acquired great scientific importance in recent years due to their ability to possess antimicrobial and immunomodulatory activity. In the last two decades, plants have attracted the interest of the scientific community and industry as regards their potential as biofactories of heterologous proteins. One of the most promising approaches is the use of viral vectors to maximize the transient expression of drugs in the leaves of the plant Nicotiana benthamiana. Recently, the MagnifectionTM expression system was launched. This sophisticated commercial platform allows the assembly of the viral particle in leaf cells and the systemic spread of heterologous protein biosynthesis in green tissues caused by Agrobacterium tumefaciens "gene delivery method". The system also presents increased gene expression levels mediated by potent viral expression machinery. These characteristics allow the mass recovery of heterologous proteins in the leaves of N. benthamiana in 8 to 10 days. This system was highly efficient for the synthesis of different classes of pharmacological proteins and contains enormous potential for the rapid and abundant biosynthesis of AMPs.
Assuntos
Agrobacterium tumefaciens/metabolismo , Peptídeos Catiônicos Antimicrobianos/biossíntese , Agricultura Molecular/métodos , Nicotiana/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Biossíntese de Proteínas , Peptídeos Catiônicos Antimicrobianos/farmacologia , Biotecnologia/métodos , Vetores Genéticos/genética , Vetores Genéticos/metabolismoRESUMO
Antimicrobial peptides (AMPs) have attracted considerable attention because of their multiple and complex mechanisms of action toward resistant bacteria. However, reports have increasingly highlighted how bacteria can escape AMP administration. Here, the molecular mechanisms involved in Escherichia coli resistance to magainin I were investigated through comparative transcriptomics. Sub-inhibitory concentrations of magainin I were used to generate four experimental groups, including magainin I-susceptible E. coli, in the absence (C) and presence of magainin I (CM); and magainin I-resistant E. coli in the absence (R) and presence of magainin I (RM). The total RNA from each sample was extracted; cDNA libraries were constructed and further submitted for Illumina MiSeq sequencing. After RNA-seq data pre-processing and functional annotation, a total of 103 differentially expressed genes (DEGs) were identified, mainly related to bacterial metabolism. Moreover, down-regulation of cell motility and chaperone-related genes was observed in CM and RM, whereas cell communication, acid tolerance and multidrug efflux pump genes (ABC transporter, major facilitator and resistance-nodulation cell division superfamilies) were up-regulated in these same groups. DEGs from the C and R groups are related to basal levels of expression of homeostasis-related genes compared to CM and RM, suggesting that the presence of magainin I is required to change the transcriptomics panel in both C and R E. coli strains. These findings show the complexity of E. coli resistance to magainin I through the rearrangement of several metabolic pathways involved in bacterial physiology and drug response, also providing information on the development of novel antimicrobial strategies targeting resistance-related transcripts and proteins herein described.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Fenômenos Fisiológicos Bacterianos/genética , Farmacorresistência Bacteriana/genética , Metabolismo Energético/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Transcriptoma/genética , Transportadores de Cassetes de Ligação de ATP/biossíntese , Proteínas de Bactérias/biossíntese , DNA Complementar/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Escherichia coli/metabolismo , Perfilação da Expressão Gênica , RNA/análiseRESUMO
Fungal Candida species are commensals present in the mammalian skin and mucous membranes. Candida spp. are capable of breaching the epithelial barrier of immunocompromised patients with neutrophil and cell-mediated immune dysfunctions and can also disseminate to multiple organs through the bloodstream. Here we examined the action of innate defense regulator 1018 (IDR-1018), a 12-amino-acid-residue peptide derived from bovine bactenecin (Bac2A): IDR-1018 showed weak antifungal and antibiofilm activity against a Candida albicans laboratory strain (ATCC 10231) and a clinical isolate (CI) (MICs of 32 and 64 µg · ml-1, respectively), while 8-fold lower concentrations led to dissolution of the fungal cells from preformed biofilms. IDR-1018 at 128 µg · ml-1 was not hemolytic when tested against murine red blood cells and also has not shown a cytotoxic effect on murine monocyte RAW 264.7 and primary murine macrophage cells at the tested concentrations. IDR-1018 modulated the cytokine profile during challenge of murine bone marrow-derived macrophages with heat-killed C. albicans (HKCA) antigens by increasing monocyte chemoattractant protein 1 (MCP-1) and interleukin-10 (IL-10) levels, while suppressing tumor necrosis factor alpha (TNF-α), IL-1ß, IL-6, and IL-12 levels. Mice treated with IDR-1018 at 10 mg · kg-1 of body weight had an increased survival rate in the candidemia model compared with phosphate-buffered saline (PBS)-treated mice, together with a diminished kidney fungal burden. Thus, IDR-1018 was able to protect against murine experimental candidemia and has the potential as an adjunctive therapy.
Assuntos
Antifúngicos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Candidemia/tratamento farmacológico , Candidemia/prevenção & controle , Fatores Imunológicos/uso terapêutico , Animais , Candida albicans/imunologia , Candida albicans/isolamento & purificação , Linhagem Celular , Quimiocina CCL2/imunologia , Modelos Animais de Doenças , Interleucina-10/imunologia , Subunidade p35 da Interleucina-12/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Células RAW 264.7 , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Antimicrobial peptides form part of the first line of defense against pathogens for many organisms. Current treatments for fungal infections are limited by drug toxicity and pathogen resistance. Cm-p5 (SRSELIVHQRLF), a peptide derived from the marine mollusk Cenchritis muricatus peptide Cm-p1, has a significantly increased fungistatic activity against pathogenic Candida albicans (minimal inhibitory concentration, 10 µg/ml; EC50, 1.146 µg/ml) while exhibiting low toxic effects against a cultured mammalian cell line. Cm-p5 as characterized by circular dichroism and nuclear magnetic resonance revealed an α-helical structure in membrane-mimetic conditions and a tendency to random coil folding in aqueous solutions. Additional studies modeling Cm-p5 binding to a phosphatidylserine bilayer in silico and isothermal titration calorimetry using lipid monophases demonstrated that Cm-p5 has a high affinity for the phospholipids of fungal membranes (phosphatidylserine and phosphatidylethanolamine), only moderate interactions with a mammalian membrane phospholipid, low interaction with ergosterol, and no interaction with chitin. Adhesion of Cm-p5 to living C. albicans cells was confirmed by fluorescence microscopy with FITC-labeled peptide. In a systemic candidiasis model in mice, intraperitoneal administration of Cm-p5 was unable to control the fungal kidney burden, although its low amphiphaticity could be modified to generate new derivatives with improved fungicidal activity and stability.
Assuntos
Antifúngicos/farmacologia , Gastrópodes/metabolismo , Moluscos/metabolismo , Peptídeos/farmacologia , Animais , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Dicroísmo Circular/métodos , Feminino , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana/métodos , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Fosfolipídeos/metabolismo , Estrutura Secundária de ProteínaRESUMO
The extraction and purification of parigidin-br3, a cyclotide analogue belonging to the "bracelet" subfamily, from Palicourea rigida leaves is discussed. Unlike conventional cyclotides, parigidin-br3 has free N- and C-termini, as identified by MALDI-TOF/TOF analysis and confirmed by gene structure elucidation, and is one of a small number of acyclotides discovered during recent years. Parigidin-br3 showed cytotoxic activity against MCF-7 (breast cancer) and CACO2 (colorectal adenocarcinoma) cells, with IC50 values of â¼2.5 µM and less than 10% hemolytic activity. Overall, parigidin-br3 is a promising new molecule with cytotoxic properties against tumor cell lines and, unlike many synthetic acyclic analogues, demonstrates that cytotoxic activity is not limited to conventional (i.e., cyclic) cyclotides.
Assuntos
Antineoplásicos Fitogênicos/isolamento & purificação , Rubiaceae/química , Sequência de Aminoácidos , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Células CACO-2 , Neoplasias Colorretais/tratamento farmacológico , Ciclotídeos/química , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Concentração Inibidora 50 , Dados de Sequência Molecular , Estrutura Molecular , Folhas de Planta/química , Proteínas de Plantas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Cyclotides are a family of plant-derived cyclic peptides comprising six conserved cysteine residues connected by three intermolecular disulfide bonds that form a knotted structure known as a cyclic cystine knot (CCK). This structural motif is responsible for the pronounced stability of cyclotides against chemical, thermal, or proteolytic degradation and has sparked growing interest in this family of peptides. Here, we isolated and characterized a novel cyclotide from Palicourea rigida (Rubiaceae), which was named parigidin-br1. The sequence indicated that this peptide is a member of the bracelet subfamily of cyclotides. Parigidin-br1 showed potent insecticidal activity against neonate larvae of Lepidoptera (Diatraea saccharalis), causing 60% mortality at a concentration of 1 µm but had no detectable antibacterial effects. A decrease in the in vitro viability of the insect cell line from Spodoptera frugiperda (SF-9) was observed in the presence of parigidin-br1, consistent with in vivo insecticidal activity. Transmission electron microscopy and fluorescence microscopy of SF-9 cells after incubation with parigidin-br1 or parigidin-br1-fluorescein isothiocyanate, respectively, revealed extensive cell lysis and swelling of cells, consistent with an insecticidal mechanism involving membrane disruption. This hypothesis was supported by in silico analyses, which suggested that parigidin-br1 is able to complex with cell lipids. Overall, the results suggest promise for the development of parigidin-br1 as a novel biopesticide.
Assuntos
Ciclotídeos/química , Ciclotídeos/isolamento & purificação , Inseticidas/química , Inseticidas/isolamento & purificação , Lepidópteros , Rubiaceae/química , Saccharum , Sequência de Aminoácidos , Animais , Linhagem Celular , Ciclotídeos/genética , Ciclotídeos/metabolismo , Feminino , Fluoresceína-5-Isotiocianato/metabolismo , Regulação da Expressão Gênica de Plantas , Inseticidas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Especificidade de Órgãos , Fosforilcolina/análogos & derivados , Fosforilcolina/metabolismo , Filogenia , Conformação Proteica , Rubiaceae/genética , Estações do Ano , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Marine organisms represent approximately half of the world's biodiversity by virtue of the sea being an immense reservoir of bioactive molecules. Here, antimicrobial crude extract activities of different marine invertebrates from the Caribbean Sea were evaluated. One of the most active, crude extracts was that marine snail Cenchritis muricatus, it was capable of totally inhibiting the development of Staphylococcus aureus and also showed a growth inhibition of 95.9% in Escherichia coli. Aiming to isolate molecules that confirm antimicrobial activity, the crude extract was purified by reversed-phase HPLC C-18 chromatography. Thereafter, one of the obtained fractions preserved this antibacterial activity. Furthermore, SDS-PAGE analysis (15%) showed the presence of two proteins of molecular masses with approximately 10 and 15 kDa, respectively. The first 19 amino acids of both proteins were sequenced by using Edman degradation, yielding unidentified primary structures compared against sequences deposited at NCBI databank. This is the first report of antibacterial proteins isolated from the mollusk Cenchritis muricatus and these proteins could be used as antibiotic alternatives in the aquacultural industry, as well as in agricultural or biomedical research.
Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Moluscos/química , Proteínas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Região do Caribe , Avaliação Pré-Clínica de Medicamentos , Escherichia coli/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Moluscos/classificação , Moluscos/metabolismo , Proteínas/química , Proteínas/metabolismo , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Klebsiella pneumoniae has been implicated in wide-ranging nosocomial outbreaks, causing severe infections without effective treatments due to antibiotic resistance. Here, we performed genome sequencing of 70 extensively drug resistant clinical isolates, collected from Brasília's hospitals (Brazil) between 2010 and 2014. The majority of strains (60 out of 70) belonged to a single clonal complex (CC), CC258, which has become distributed worldwide in the last two decades. Of these CC258 strains, 44 strains were classified as sequence type 11 (ST11) and fell into two distinct clades, but no ST258 strains were found. These 70 strains had a pan-genome size of 10â366 genes, with a core-genome size of ~4476 genes found in 95â% of isolates. Analysis of sequences revealed diverse mechanisms of resistance, including production of multidrug efflux pumps, enzymes with the same target function but with reduced or no affinity to the drug, and proteins that protected the drug target or inactivated the drug. ß-Lactamase production provided the most notable mechanism associated with K. pneumoniae. Each strain presented two or three different ß-lactamase enzymes, including class A (SHV, CTX-M and KPC), class B and class C AmpC enzymes, although no class D ß-lactamase was identified. Strains carrying the NDM enzyme involved three different ST types, suggesting that there was no common genetic origin.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Genômica , Klebsiella pneumoniae/genética , Fatores de Virulência/genética , Brasil , DNA Bacteriano/genética , Humanos , Infecções por Klebsiella/epidemiologia , Klebsiella pneumoniae/isolamento & purificação , Filogenia , Virulência/genética , beta-Lactamases/genéticaRESUMO
Biotech crops expressing Bacillus thuringiensis Cry toxins present a valuable approach for insect control. Cry8Ka5, which is highly toxic to the cotton boll weevil (Anthonomus grandis), was used as a model to study toxin-ligand interactions. Three Cry-binding proteins were detected after toxin overlay assays. Following de novo sequencing, a heat-shock cognate protein and a V-ATPase were identified, whilst a approximately 120 kDa protein remained unknown. Additional Cry8Ka5-binding proteins were visualized by two-dimensional gel electrophoresis ligand blots.
Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Sistema Digestório/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Inseticidas/metabolismo , Controle Biológico de Vetores/métodos , Gorgulhos/metabolismo , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/química , Endotoxinas/química , Proteínas de Choque Térmico HSC70/análise , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas Hemolisinas/química , Inseticidas/química , Larva/metabolismo , Ligação Proteica , ATPases Vacuolares Próton-Translocadoras/análise , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Antimicrobial peptides are sequences of amino acids, which present activity against microorganisms. These peptides were discovered over 70 years ago, and are abundant in nature from soil bacteria, insects, amphibians to mammals and plants. They vary in amino acids number, the distance between amino acids within individual peptide structure, net charge, solubility and other physical chemical properties as well as differ in mechanism of action. These peptides may provide an alternative treatment to conventional antibiotics, which encounter resistance such as the peptide nisin applied in treating methicillin resistant Staphylococcus aureus (MRSA) or may behave synergistically with known antibiotics against parasites for instance, nisin Z when used in synergy with ampicillin reported better activity against Pseudomonas fluorescens than when the antibiotic was alone. AMPs are known to be active against viruses, bacteria, fungi and protozoans. Nanotechnology is an arena which explores the synthesis, characterization and application of an array of delivery systems at a one billionth of meter scale. Such systems are implemented to deliver drugs, proteins, vaccines, and peptides. The role of nanotechnology in delivering AMPs is still at its early development stage. There are challenges of incorporating AMPs into drug delivery system. This review intends to explore in depth, the role of nanotechnology in delivering AMPs as well as presenting the current advances and accompanying challenges of the technology.
RESUMO
The use of central venous catheters (CVC) is highly associated with nosocomial blood infections and its use largely requires a systematic assessment of benefits and risks. Bacterial contamination of these tubes is frequent and may result in development of microbial consortia also known as biofilm. The woven nature of biofilm provides a practical defense against antimicrobial agents, facilitating bacterial dissemination through the patient's body and development of antimicrobial resistance. In this work, the authors describe the modification of CVC tubing by immobilizing Fe3O4-aminosilane core-shell nanoparticles functionalized with antimicrobial peptide clavanin A (clavA) as an antimicrobial prophylactic towards Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. Its anti-biofilm-attachment characteristic relies in clavA natural activity to disrupt the bacterial lipidic membrane. The aminosilane shell prevents iron leaching, which is an important nutrient for bacterial growth. Fe3O4-clavA-modified CVCs showed to decrease Gram-negative bacteria attachment up to 90% when compared to control clean CVC. Additionally, when hyperthermal treatment is triggered for 5â¯min at 80⯰C in a tubing that already presents bacterial biofilm (CVC-BF), the viability of attached bacteria reduces up to 88%, providing an efficient solution to avoid changing catheter.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Proteínas Sanguíneas/farmacologia , Óxido Ferroso-Férrico/farmacologia , Nanopartículas/química , Silanos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Proteínas Sanguíneas/química , Escherichia coli/efeitos dos fármacos , Óxido Ferroso-Férrico/química , Klebsiella pneumoniae/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Tamanho da Partícula , Pseudomonas aeruginosa/efeitos dos fármacos , Silanos/química , Staphylococcus aureus/efeitos dos fármacos , Propriedades de SuperfícieRESUMO
In recent years the antimicrobial peptides (AMPs) have been prospected and designed as new alternatives to conventional antibiotics. Indeed, AMPs have presented great potential toward pathogenic bacterial strains by means of complex mechanisms of action. However, reports have increasingly emerged regarding the mechanisms by which bacteria resist AMP administration. In this context, we performed a comparative proteomic study by using the total bacterial lysate of magainin I-susceptible and -resistant E. coli strains. After nanoUPLC-MSE analyses we identified 742 proteins distributed among the experimental groups, and 25 proteins were differentially expressed in the resistant strains. Among them 10 proteins involved in bacterial resistance, homeostasis, nutrition and protein transport were upregulated, while 15 proteins related to bacterial surface modifications, genetic information and ß-lactams binding-protein were downregulated. Moreover, 60 exclusive proteins were identified in the resistant strains, among which biofilm and cell wall formation and multidrug efflux pump proteins could be observed. Thus, differentially from previous studies that could only associate single proteins to AMP bacterial resistance, data here reported show that several metabolic pathways may be related to E. coli resistance to AMPs, revealing the crucial role of multiple "omics" studies in order to elucidate the global molecular mechanisms involved in this resistance.
Assuntos
Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Magaininas/farmacologia , Espectrometria de Massas , Nanotecnologia , Cromatografia Líquida de Alta Pressão , Proteínas de Escherichia coli/metabolismoRESUMO
Streptococcus pyogenes, also known as group A Streptococcus (GAS), is a human pathogen that causes diverse human diseases including streptococcal toxic shock syndrome (STSS). A GAS outbreak occurred in Brasilia, Brazil, during the second half of the year 2011, causing 26 deaths. Whole genome sequencing was performed using Illumina platform. The sequences were assembled and genes were predicted for comparative analysis with emm type 1 strains: MGAS5005 and M1 GAS. Genomics comparison revealed one of the invasive strains that differ from others isolates and from emm 1 reference genomes. Also, the new invasive strain showed differences in the content of virulence factors compared to other isolated in the same outbreak. The evolution of contemporary GAS strains is strongly associated with horizontal gene transfer. This is the first genomic study of a Streptococcal emm 1 outbreak in Brazil, and revealed the rapid bacterial evolution leading to new clones. The emergence of new invasive strains can be a consequence of the injudicious use of antibiotics in Brazil during the past decades.
RESUMO
Anti-infective drugs have had a key role in the contemporary world, contributing to dramatically decrease mortality rates caused by infectious diseases worldwide. Antimicrobial peptides (AMPs) are multifunctional effectors of the innate immune system of mucosal surfaces and present antimicrobial activity against a range of pathogenic viruses, bacteria, and fungi. However, the discovery and development of new antibacterial drugs is a crucial step to overcome the great challenge posed by the emergence of antibiotic resistance. In this review, we outline recent advances in the development of novel AMPs with improved antimicrobial activities that were achieved through characteristic structural design. In addition, we describe recent progress made to overcome some of the major limitations that have hindered peptide biosynthesis.
Assuntos
Antibacterianos/biossíntese , Peptídeos , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Sistemas CRISPR-Cas , Desenho de Fármacos , Farmacorresistência Bacteriana , Resistência a Múltiplos Medicamentos , Tratamento Farmacológico , Economia , Edição de Genes , Humanos , Biossíntese Peptídica , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Mudança Social , Nicotiana/metabolismoRESUMO
Diseases caused by bacterial and fungal pathogens are among the major health problems in the world. Newer antimicrobial therapies based on novel molecules urgently need to be developed, and this includes the antimicrobial peptides. In spite of the potential of antimicrobial peptides, very few of them were able to be successfully developed into therapeutics. The major problems they present are molecule stability, toxicity in host cells, and production costs. A novel strategy to overcome these obstacles is conjugation to nanomaterial preparations. The antimicrobial activity of different types of nanoparticles has been previously demonstrated. Specifically, magnetic nanoparticles have been widely studied in biomedicine due to their physicochemical properties. The citric acid-modified manganese ferrite nanoparticles used in this study were characterized by high-resolution transmission electron microscopy, which confirmed the formation of nanocrystals of approximately 5 nm diameter. These nanoparticles were able to inhibit Candida albicans growth in vitro. The minimal inhibitory concentration was 250 µg/mL. However, the nanoparticles were not capable of inhibiting Gram-negative bacteria (Escherichia coli) or Gram-positive bacteria (Staphylococcus aureus). Finally, an antifungal peptide (Cm-p5) from the sea animal Cenchritis muricatus (Gastropoda: Littorinidae) was conjugated to the modified manganese ferrite nanoparticles. The antifungal activity of the conjugated nanoparticles was higher than their bulk counterparts, showing a minimal inhibitory concentration of 100 µg/mL. This conjugate proved to be nontoxic to a macrophage cell line at concentrations that showed antimicrobial activity.
Assuntos
Anti-Infecciosos/farmacologia , Antifúngicos/farmacologia , Ácido Cítrico/farmacologia , Materiais Revestidos Biocompatíveis/farmacologia , Compostos Férricos/farmacologia , Compostos de Manganês/farmacologia , Nanopartículas/química , Peptídeos/farmacologia , Animais , Antibacterianos/farmacologia , Antifúngicos/química , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Proliferação de Células/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Nanopartículas/ultraestrutura , Células RAW 264.7 , Staphylococcus aureus/efeitos dos fármacosRESUMO
Cotton (Gossypium hirsutum L.) is an important agricultural commodity, which is attacked by several pests such as the cotton boll weevil Anthonomus grandis. Adult A. grandis feed on fruits and leaf petioles, reducing drastically the crop production. The predominance of boll weevil digestive serine proteinases has motivated inhibitor screenings in order to discover new ones with the capability to reduce the digestion process. The present study describes a novel proteinase inhibitor from chickpea seeds (Cicer arietinum L.) and its effects against A. grandis. This inhibitor, named CaTI, was purified by using affinity Red-Sepharose Cl-6B chromatography, followed by reversed-phase HPLC (Vydac C18-TP). SDS-PAGE and MALDI-TOF analyses, showed a unique monomeric protein with a mass of 12,877 Da. Purified CaTI showed significant inhibitory activity against larval cotton boll weevil serine proteinases (78%) and against bovine pancreatic trypsin (73%), when analyzed by fluorimetric assays. Although the molecular mass of CaTI corresponded to alpha-amylase/trypsin bifunctional inhibitors masses, no inhibitory activity against insect and mammalian alpha-amylases was observed. In order to observe CaTI in vivo effects, an inhibitor rich fraction was added to an artificial diet at different concentrations. At 1.5% (w/w), CaTI caused severe development delay, several deformities and a mortality rate of approximately 45%. These results suggested that CaTI could be useful in the production of transgenic cotton plants with enhanced resistance toward cotton boll weevil.
Assuntos
Cicer/química , Proteínas de Plantas/toxicidade , Sementes/química , Gorgulhos/efeitos dos fármacos , Gorgulhos/enzimologia , Agricultura/métodos , Animais , Eletroforese em Gel de Poliacrilamida , Larva/efeitos dos fármacos , Larva/enzimologia , Larva/fisiologia , Peso Molecular , Praguicidas/isolamento & purificação , Praguicidas/farmacologia , Praguicidas/toxicidade , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Inibidores da Tripsina , Gorgulhos/crescimento & desenvolvimento , Gorgulhos/fisiologia , alfa-Amilases/antagonistas & inibidoresRESUMO
Alpha-amylase inhibitors have important roles in plant defense mechanisms, particularly against insects, and several of these inhibitors have been expressed in different crops to increase their resistance to particular insects. In this work, we report the cloning and expression of a gene encoding for a new alpha-amylase inhibitor (BIII) from rye (Secale cereale) seeds. The BIII gene contains 354 nucleotides that encode for 118 amino acids sequence. A 313 bp fragment of the gene was expressed in Escherichia coli and resulted in a functional inhibitor that reduced the activity of alpha-amylases of larvae of the coleopteran pests Acanthoscelides obtectus, Zabrotess subfasciatus and Anthonomus grandis. In contrast, the inhibitor did not inhibit the activity of porcine pancreatic alpha-amylase. Although the amino acid sequence of BIII showed high identity with those of bifunctional inhibitors, the recombinant protein was unable to inhibit trypsin-like serine proteinases. The effects of recombinant BIII were evaluated in vivo against A. grandis. When first instar larvae were reared on an artificial diet containing four different concentrations of BIII, a reduction in larval weight and a mortality of 83% were observed at the highest concentration.
Assuntos
Inibidores Enzimáticos/química , Insetos , Controle Biológico de Vetores , Secale/química , alfa-Amilases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Candida albicans is a common human-pathogenic fungal species with the ability to cause several diseases including surface infections. Despite the clear difficulties of Candida control, antimicrobial peptides (AMPs) have emerged as an alternative strategy for fungal control. In this report, different concentrations of antifungal Cm-p1 (Cencritchis muricatus peptide 1) were electrospun into nanofibers for drug delivery. The nanofibers were characterized by mass spectrometry confirming the presence of the peptide on the scaffold. Atomic force microscopy and scanning electronic microscopy were used to measure the diameters, showing that Cm-p1 affects fiber morphology as well as the diameter and scaffold thickness. The Cm-p1 release behavior from the nanofibers demonstrated peptide release from 30 min to three days, leading to effective yeast control in the first 24 hours. Moreover, the biocompatibility of the fibers were evaluated through a MTS assay as well as ROS production by using a HUVEC model, showing that the fibers do not affect cell viability and only nanofibers containing 10% Cm-p1-PVA improved ROS generation. In addition, the secretion of pro-inflammatory cytokines IL-6 and TNF-α by the HUVECs was also slightly modified by the 10% Cm-p1-PVA nanofibers. In conclusion, the electrospinning technique applied here allowed for the manufacture of biodegradable biomimetic nanofibrous extracellular membranes with the ability to control fungal infection.
Assuntos
Antifúngicos , Organismos Aquáticos/química , Candida albicans/crescimento & desenvolvimento , Células Endoteliais da Veia Umbilical Humana/metabolismo , Nanofibras/química , Peptídeos , Antifúngicos/química , Antifúngicos/farmacologia , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Interleucina-6/metabolismo , Teste de Materiais/métodos , Microscopia de Força Atômica , Nanofibras/ultraestrutura , Peptídeos/química , Peptídeos/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Fourteen different cDNA fragments encoding serine proteinases were isolated by reverse transcription-PCR from cotton boll weevil (Anthonomus grandis) larvae. A large diversity between the sequences was observed, with a mean pairwise identity of 22% in the amino acid sequence. The cDNAs encompassed 11 trypsin-like sequences classifiable into three families and three chymotrypsin-like sequences belonging to a single family. Using a combination of 5' and 3' RACE, the full-length sequence was obtained for five of the cDNAs, named Agser2, Agser5, Agser6, Agser10 and Agser21. The encoded proteins included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Southern blotting analysis suggested that one or two copies of these serine proteinase genes exist in the A. grandis genome. Northern blotting analysis of Agser2 and Agser5 showed that for both genes, expression is induced upon feeding and is concentrated in the gut of larvae and adult insects. Reverse northern analysis of the 14 cDNA fragments showed that only two trypsin-like and two chymotrypsin-like were expressed at detectable levels. Under the effect of the serine proteinase inhibitors soybean Kunitz trypsin inhibitor and black-eyed pea trypsin/chymotrypsin inhibitor, expression of one of the trypsin-like sequences was upregulated while expression of the two chymotrypsin-like sequences was downregulated.
Assuntos
Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Gorgulhos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/análise , Gossypium/genética , Larva/enzimologia , Larva/genética , Dados de Sequência Molecular , Família Multigênica , Controle Biológico de Vetores/métodos , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidores da Tripsina/farmacologia , Gorgulhos/enzimologia , Gorgulhos/crescimento & desenvolvimentoRESUMO
The cotton boll weevil, Anthonomus grandis, is an economically important pest of cotton in tropical and subtropical areas of several countries in the Americas, causing severe losses due to their damage in cotton floral buds. Enzymatic assays using gut extracts from larval and adult boll weevil have demonstrated the presence of digestive serine proteinase-like activities. Furthermore, in vitro assays showed that soybean Kunitz trypsin inhibitor (SKTI) was able to inhibit these enzymes. Previously, in vivo effects of black-eyed pea trypsin chymotrypsin inhibitor (BTCI) have been demonstrated towards the boll weevil pest. Here, when neonate larvae were reared on an artificial diet containing SKTI at three different concentrations, a reduction of larval weight of up to 64% was observed for highest SKTI concentration 500 microM. The presence of SKTI caused an increase in mortality and severe deformities of larvae, pupae and adult insects. This work therefore represents the first observation of a Kunitz trypsin inhibitor active in vivo and in vitro against A. grandis. Bioassays suggested that SKTI could be used as a tool in engineering crop plants, which might exhibit increased resistance against cotton boll weevil.