RESUMO
Chagas disease, leishmaniasis, and malaria are major parasitic diseases disproportionately affecting the underprivileged population in developing nations. Finding new, alternative anti-parasitic compounds to treat these diseases is crucial because of the limited number of options currently available, the side effects they cause, the need for long treatment courses, and the emergence of drug-resistant parasites. Anti-microbial peptides (AMPs) derived from amphibian skin secretions are small bioactive molecules capable of lysing the cell membrane of pathogens while having low toxicity against human cells. Here, we report the anti-parasitic activity of five AMPs derived from skin secretions of three Ecuadorian frogs: cruzioseptin-1, cruzioseptin-4 (CZS-4), and cruzioseptin-16 from Cruziohyla calcarifer; dermaseptin-SP2 from Agalychnis spurrelli; and pictuseptin-1 from Boana picturata. These five AMPs were chemically synthesized. Initially, the hemolytic activity of CZS-4 and its minimal inhibitory concentration against Escherichia coli, Staphylococcus aureus, and Candida albicans were determined. Subsequently, the cytotoxicity of the synthetic AMPs against mammalian cells and their anti-parasitic activity against Leishmania mexicana promastigotes, erythrocytic stages of Plasmodium falciparum and mammalian stages of Trypanosoma cruzi were evaluated in vitro. The five AMPs displayed activity against the pathogens studied, with different levels of cytotoxicity against mammalian cells. In silico molecular docking analysis suggests this bioactivity may occur via pore formation in the plasma membrane, resulting in microbial lysis. CZS-4 displayed anti-bacterial, anti-fungal, and anti-parasitic activities with low cytotoxicity against mammalian cells. Further studies about this promising AMP are required to gain a better understanding of its activity.IMPORTANCEChagas disease, malaria, and leishmaniasis are major tropical diseases that cause extensive morbidity and mortality, for which available treatment options are unsatisfactory because of limited efficacy and side effects. Frog skin secretions contain molecules with anti-microbial properties known as anti-microbial peptides. We synthesized five peptides derived from the skin secretions of different species of tropical frogs and tested them against cultures of the causative agents of these three diseases, parasites known as Trypanosoma cruzi, Plasmodium falciparum, and Leishmania mexicana. All the different synthetic peptides studied showed activity against one of more of the parasites. Peptide cruzioseptin-4 is of special interest since it displayed intense activity against parasites while being innocuous against cultured mammalian cells, which indicates it does not simply hold general toxic properties; rather, its activity is specific against the parasites.
Assuntos
Anuros , Leishmania mexicana , Plasmodium falciparum , Pele , Trypanosoma cruzi , Animais , Trypanosoma cruzi/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Humanos , Leishmania mexicana/efeitos dos fármacos , Pele/parasitologia , Pele/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos/química , Proteínas de Anfíbios/farmacologia , Proteínas de Anfíbios/química , Equador , Doença de Chagas/tratamento farmacológicoRESUMO
This study investigated the morphology of Rhinella crucifer cutaneous glands, as well as the protein/peptide profiles and bioactivities of body gland secretions (BGS) and parotoid macrogland secretions (PS). The parotoid as well as dorsal and ventral skin fragments of male and female individuals were processed for histological analysis. The protein and peptide profiles of male and female gland secretions were evaluated. Male secretions were also assessed for proteolytic, trypsin inhibiting, hemagglutinating, hemolytic, antimicrobial, and anticoagulant activities. The R. crucifer skin structure presented protuberances that are clearly visible and formed by the integument, which has cutaneous glands throughout the body. An average of 438 and 333 glands were identified in males in females, respectively. No significant differences were observed in the distribution of glands across the body as well as for area and perimeter of glands. Differences were observed in protein composition between the PS and BGS from males and females, and secretions from animals collected from undisturbed and anthropogenically disturbed areas. Proteins with similarities to catalase and elongation factor 1-alpha were detected in the PS. Zymography revealed proteolytic activity in both male BGS and PS. Male BGS showed antibacterial activity against Enterococcus faecalis and Escherichia coli and anticoagulant activity, being able to prolong prothrombin time by 6.34-fold and activated partial thromboplastin time by 2.17-fold. Finally, male PS and BGS caused a maximum hemolysis degree of 1.4%. The data showed that the cutaneous secretions of R. crucifer are potentially promising for biotechnological prospecting.
Assuntos
Bufonidae , Pele , Animais , Masculino , Feminino , Bufonidae/metabolismo , Pele/metabolismo , Pele/química , Glândulas Exócrinas/metabolismo , Secreções Corporais/química , Proteínas de Anfíbios/metabolismo , Proteínas de Anfíbios/farmacologiaRESUMO
In recent years, antimicrobial peptides isolated from amphibian toxins have gained attention as new multifunctional drugs interacting with different molecular targets. We aimed to rationally design a new peptide from temporin-PTa. Hp-MAP3 (NH2-LLKKVLALLKKVL-COOH), net charge (+4), hydrophobicity (0.69), the content of hydrophobic residues (69%), and hydrophobic moment (0.73). For the construction of the analog peptide, the physicochemical characteristics were reorganized into hydrophilic and hydrophobic residues with the addition of lysines and leucines. The minimum inhibitory concentration was 2.7 to 43 µM against the growth of Gram-negative and positive bacteria, and the potential for biofilm eradication was 173.2 µM. Within 20 min, the peptide Hp-MAP3 (10.8 µM) prompted 100% of the damage to E. coli cells. At 43.3 µM, eliminated 100% of S. aureus within 5 min. The effects against yeast species of the Candida genus ranged from 5.4 to 86.6 µM. Hp-MAP3 presents cytotoxic activity against tumor HeLa at a concentration of 21.6 µM with an IC50 of 10.4 µM. Furthermore, the peptide showed hemolytic activity against murine erythrocytes. Structural studies carried out by circular dichroism showed that Hp-MAP3, while in the presence of 50% trifluoroethanol or SDS, an α-helix secondary structure. Finally, Amphipathic Hp-MAP3 building an important model for the design of new multifunctional molecules.
Assuntos
Proteínas de Anfíbios , Peptídeos Catiônicos Antimicrobianos , Animais , Humanos , Camundongos , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Proteínas de Anfíbios/farmacologia , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Dicroísmo Circular , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Ranidae , Staphylococcus aureus/efeitos dos fármacosRESUMO
Antimicrobial peptides (AMPs) constitute part of a broad range of bioactive compounds present on diverse organisms, including frogs. Peptides, produced in the granular glands of amphibian skin, constitute a component of their innate immune response, providing protection against pathogenic microorganisms. In this work, two novel cruzioseptins peptides, cruzioseptin-16 and -17, extracted from the splendid leaf frog Cruziohyla calcarifer are presented. These peptides were identified using molecular cloning and tandem mass spectrometry. Later, peptides were synthetized using solid-phase peptide synthesis, and their minimal inhibitory concentration and haemolytic activity were tested. Furthermore, these two cruzioseptins plus three previously reported (CZS-1, CZS-2, CZS-3) were computationally characterized. Results show that cruzioseptins are 21-23 residues long alpha helical cationic peptides, with antimicrobial activity against E. coli, S. aureus, and C. albicans and low haemolytic effect. Docking results agree with the principal action mechanism of cationic AMPs that goes through cell membrane disruption due to electrostatic interactions between cationic residues in the cruzioseptins and negative phosphate groups in the pathogen cell membrane. An action mechanism through enzymes inhibition was also tried, but no conclusive results about this mechanism were obtained.
Assuntos
Proteínas de Anfíbios , Peptídeos Antimicrobianos , Candida albicans/crescimento & desenvolvimento , Escherichia coli/crescimento & desenvolvimento , Staphylococcus aureus/crescimento & desenvolvimento , Proteínas de Anfíbios/química , Proteínas de Anfíbios/isolamento & purificação , Proteínas de Anfíbios/farmacologia , Animais , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/isolamento & purificação , Peptídeos Antimicrobianos/farmacologia , RanidaeRESUMO
The objectives of this study were to examine the physicochemical and structural properties of peptide derivatives of dermaseptin S4, investigate their detrimental effects on red blood and sperm cells and ascertain their antibacterial potency to control bacterial contaminants in fresh bovine semen. The dermaseptin S4 peptide derivatives used in this study were K4S4, S4(5-28), S4(5-28)a, K20S4(5-28), K4S4(1-16)a, K4S4(1-15)a and K4S4(1-15). Peptides K4S4, S4(5-28)a, K20S4(5-28), K4S4(1-15)a and K4S4(1-16)a, with a higher positive charge, were the most potent against the bacterial strains tested, with the lowest minimum inhibitory concentration (MIC), whereas S4(5-28) and K4S4(1-15), with a lower positive charge, showed the highest MIC (p < .01). Haemolysis percentage depended on peptide concentration (p < .01). The K4S4 was the most powerful haemolytic peptide, showing the highest haemolysis percentage at all peptide concentrations (p < .01). In contrast, S4(5-28), S4(5-28)a, K20S4(5-28) and K4S4(1-15) were not able to produce 50% cell lysis up to 100 µM (p < .01). All peptides reduced sperm motility in a dose-dependent manner when used in concentrations from 16 to 64 µM (p < .01). The highest reduction was seen due to K4S4 activity, and the lowest reductions of sperm motility were observed due to K4S4(1-16)a and K4S4(1-15)a activity (p < .01). Hence, we can conclude that K4S4(1-16)a and K4S4(1-15)a at a concentration of approximately 15 µM are the most promising peptides as antibacterial agents in fresh bovine semen, because at this concentration, they showed the most potent antibacterial activity against evaluated strains without significant effects on haemolysis or a reduction in sperm motility.
Assuntos
Proteínas de Anfíbios/farmacologia , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Espermatozoides/efeitos dos fármacos , Proteínas de Anfíbios/química , Animais , Peptídeos Catiônicos Antimicrobianos/química , Bactérias/efeitos dos fármacos , Bovinos , Eritrócitos/efeitos dos fármacos , Masculino , Testes de Sensibilidade Microbiana/veterinária , Sêmen , Motilidade dos Espermatozoides/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
In recent years, the number of new antimicrobial drugs launched on the market has decreased considerably even though there has been an increase in the number of resistant microbial strains. Thus, antimicrobial resistance has become a serious public health problem. Amphibian skin secretions are a rich source of host defense peptides, which generally are cationic and hydrophobic molecules, with a broad-spectrum of activity. In this study, one novel multifunctional defense peptide was isolated from the skin secretion of the Chaco tree frog, Boana raniceps. Figainin 2 (1FLGAILKIGHALAKTVLPMVTNAFKPKQ28) is cationic and hydrophobic, adopts an α-helical structure in 50% (v/v) trifluoroethanol (TFE), and is thermally stable. This peptide exhibited activity against Gram-negative and Gram-positive pathogenic bacteria arboviruses, T. cruzi epimastigotes; however, it did not show activity against yeasts. Figainin 2 also showed antiproliferative activity on cancer cells, is moderately active on human erythrocytes, and activates the oxidative burst in human neutrophils.
Assuntos
Proteínas de Anfíbios/metabolismo , Anuros/metabolismo , Defensinas/metabolismo , Pele/metabolismo , Proteínas de Anfíbios/química , Proteínas de Anfíbios/farmacologia , Animais , Arbovírus/efeitos dos fármacos , Bactérias/efeitos dos fármacos , Candida/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Defensinas/química , Defensinas/farmacologia , Hemólise/efeitos dos fármacos , Humanos , Neutrófilos/efeitos dos fármacos , Conformação Proteica em alfa-Hélice , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Lipoxygenases are non-heme iron-containing lipid dioxygenases enzymes that catalyze the hydroperoxidation of lipids. The Mexican axolotl (Ambystoma mexicanum) is a prominent source of the enzyme with a regeneration capacity in limbs. It has been shown that transfected human osteosarcoma and keratinocyte cells with epidermal lipoxygenase (LOXe) have an increased rate of cell migration. In the present study, LOXe, a peripheral membrane protein, was produced in Escherichia coli. The enzyme was purified using different detergents, anionic solutions, and gel filtration chromatography. Kinetic assay of the enzyme activity was carried out by the spectroscopy method using arachidonic acid as a substrate. Finally, the enzyme was characterized and its growth effect on human fibroblast cells was examined by MTT viability assay. Enzyme kinetic parameters including Km of 90.4 µM and Vmax of 2.63 IU were determined for LOXe. The enzyme with 0.1 nM end concentration promoted the growth of 5000 cells/well human fibroblast cells up to 11% (P < 0.01). In the present study, we introduce an E. coli expression system to produce an excessive amount of soluble LOXe and the efficient purification method to provide a soluble and active form of LOXe that is effective in stimulating human fibroblast cell proliferation.
Assuntos
Proteínas de Anfíbios , Proliferação de Células/efeitos dos fármacos , Fibroblastos/metabolismo , Lipoxigenases , Ambystoma mexicanum , Proteínas de Anfíbios/biossíntese , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/isolamento & purificação , Proteínas de Anfíbios/farmacologia , Animais , Epiderme , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Fibroblastos/citologia , Humanos , Lipoxigenases/biossíntese , Lipoxigenases/genética , Lipoxigenases/isolamento & purificação , Lipoxigenases/farmacologia , Masculino , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologiaRESUMO
In this research, we present a preliminary computational study of four Dermaseptin-related peptides from the skin exudate of the gliding tree frog Agalychnis spurrelli. Experimentally, the amino acid sequence of these peptides was elucidated through molecular cloning and tandem mass spectrometry and synthetic peptides were assayed against E. coli, S. aureus, and C. albicans to determine their antimicrobial properties. With the sequences on hand, a computational study of the structures was carried out, obtaining their physicochemical properties, secondary structure, and their similarity to other known peptides. A molecular docking study of these peptides was also performed against cell membrane and several enzymes are known to be vital for the organisms. Results showed that Dermaseptin-related peptides are α-helical cationic peptides with an isoelectric point above 9.70 and a positive charge of physiological pH. Introducing theses peptides in a database, it was determined that their identity compared with known peptides range from 36 to 82% meaning these four Dermaseptins are novel peptides. This preliminary study of molecular docking suggests the mechanism of action of this peptide is not given by the inhibition of essential enzymatic pathways, but by cell lysis. Graphical abstract.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Simulação de Acoplamento Molecular , Proteínas de Anfíbios/química , Proteínas de Anfíbios/metabolismo , Proteínas de Anfíbios/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Anuros , Candida albicans/efeitos dos fármacos , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Estrutura Secundária de Proteína , Staphylococcus aureus/efeitos dos fármacosRESUMO
Inflammation is a natural defense mechanism of the immune system; however, when unregulated, it can lead to chronic illness. Glucocorticoids are the most commonly used agents to effectively treat inflammatory conditions, including autoimmune diseases, however these substances can trigger a number of side effects. Thus, viable alternatives to the use of these drugs would be advantageous. In this study, we have analyzed the anti-inflammatory profile of three synthetic peptides first identified in skin secretion of the tree frog Hypsiboas raniceps. Structural characterization was performed using NMR spectroscopy and Mass Spectrometry, and the peptides were tested in vitro in RAW 264.7 cells and in vivo in Balb/c mice for their functional properties. The samples did not show a significant antimicrobial profile. NMR spectroscopy indicated that AC12 (ACFLTRLGTYVC) has a disulfide bond between C2 and C11 and a ß-sheet-turn-ß-sheet conformation in aqueous solution. This peptide showed no cytotoxic effect in mammalian cells and it was the most effective in reducing anti-inflammatory markers, such as NO, TNF-α and IL-12. Peptide DK16 (DKERPICSNTFRGRKC) demonstrated anti-inflammatory properties in vitro, while RC11 (RCFRRRGKLTC) significantly altered the cell viability in RAW 264.7 but was shown to be safe in Balb/c erythrocytes. Our results indicate that, of the three peptides studied, AC12 is the most efficient in reducing anti-inflammatory markers, and it could be a potential agent for the treatment of inflammatory diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Anuros/metabolismo , Inflamação/tratamento farmacológico , Peptídeos/farmacologia , Pele/metabolismo , Proteínas de Anfíbios/química , Proteínas de Anfíbios/metabolismo , Proteínas de Anfíbios/farmacologia , Animais , Anti-Infecciosos/farmacologia , Biomarcadores , Interleucina-12 , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Células RAW 264.7 , Pele/química , Fator de Necrose Tumoral alfaRESUMO
Cutaneous secretions produced by amphibians of the family Bufonidae are rich sources of bioactive compounds that can be useful as new chemical templates for agrochemicals. In crop protection, the use of elicitors to induce responses offers the prospect of durable, broad-spectrum disease control using the plant's own resistance. Therefore, we evaluated the potential of methanolic extracts of cutaneous secretions of two species of amphibians of the family Bufonidae found in the Amazon biome-Rhaebo guttatus (species 1) and Rhinella marina (species 2)-in the synthesis of phytoalexins in soybean cotyledons, bean hypocotyls, and sorghum mesocotyls. Additionally, changes in the enzyme activity of ß-1,3-glucanase, peroxidase (POX), and polyphenol oxidase (PPO) and in the total protein content of soybean cotyledons were determined. In the soybean cultivar 'TMG 132 RR', our results indicated that the methanolic extract of R. guttatus cutaneous secretions suppressed glyceollin synthesis and ß-1,3-glucanase activity and increased POX and PPO activities at higher concentrations and total protein content at a concentration of 0.2 mg/mL. On the other hand, the methanolic extract of R. marina cutaneous secretions induced glyceollin synthesis in the soybean cultivars 'TMG 132 RR' and 'Monsoy 8372 IPRO' at 0.1-0.2 mg/mL and 0.2 mg/mL, respectively. The methanolic extract of R. marina cutaneous secretions also increased the specific activity of POX and PPO in 'Monsoy 8372 IPRO' and 'TMG 132 RR', respectively, and decreased the activity of ß-1,3-glucanases in 'Monsoy 8372 IPRO'. At 0.3 mg/mL, it stimulated phaseolin synthesis. The extracts did not express bioactivity in the synthesis of deoxyanthocyanidins in sorghum mesocotyls. The study in soybean suggests that the bioactivity in defense responses is influenced by cultivar genotypes. Therefore, these results provide evidence that extracts of cutaneous secretions of these amphibians species may contribute to the bioactivity of defense metabolites in plants.