RESUMO
Ureases from different biological sources display non-ureolytic properties that contribute to plant defense, in addition to their classical enzymatic urea hydrolysis. Antifungal and entomotoxic effects were demonstrated for Jaburetox, an intrinsically disordered polypeptide derived from jack bean (Canavalia ensiformis) urease. Here we describe the properties of Soyuretox, a polypeptide derived from soybean (Glycine max) ubiquitous urease. Soyuretox was fungitoxic to Candida albicans, leading to the production of reactive oxygen species. Soyuretox further induced aggregation of Rhodnius prolixus hemocytes, indicating an interference on the insect immune response. No relevant toxicity of Soyuretox to zebrafish larvae was observed. These data suggest the presence of antifungal and entomotoxic portions of the amino acid sequences encompassing both Soyuretox and Jaburetox, despite their small sequence identity. Nuclear Magnetic Resonance (NMR) and circular dichroism (CD) spectroscopic data revealed that Soyuretox, in analogy with Jaburetox, possesses an intrinsic and largely disordered nature. Some folding is observed upon interaction of Soyuretox with sodium dodecyl sulfate (SDS) micelles, taken here as models for membranes. This observation suggests the possibility for this protein to modify its secondary structure upon interaction with the cells of the affected organisms, leading to alterations of membrane integrity. Altogether, Soyuretox can be considered a promising biopesticide for use in plant protection.
Assuntos
Agentes de Controle Biológico/farmacologia , Glycine max/enzimologia , Peptídeos/farmacologia , Urease/química , Animais , Agentes de Controle Biológico/química , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Dicroísmo Circular , Hemócitos/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Dinâmica Molecular , Peptídeos/química , Proteínas de Plantas/química , Dobramento de Proteína , Espécies Reativas de Oxigênio/metabolismo , Rhodnius/efeitos dos fármacosRESUMO
Antimicrobial peptides (AMPs) are promising candidates for the development of future antibiotics. In an attempt to increase the efficacy of therapeutic AMPs, computer-based design methods appear as a reliable strategy. In this study, we evaluated the antimicrobial efficiency and mechanism of action of a novel designed AMP named PaDBS1R1, previously designed by means of the Joker algorithm, using a fragment of the ribosomal protein L39E from the archaeon Pyrobaculum aerophilum as a template. PaDBS1R1 displayed low micromolar broad-spectrum antimicrobial activity against Gram-negative (MIC of 1.5⯵M) and Gram-positive (MIC of 3⯵M) bacteria, including carbapenem-resistant Klebsiella pneumoniae (MIC of 6.25⯵M) and methicillin-resistant Staphylococcus aureus (MIC of 12.5⯵M), without cytotoxicity towards HEK-293 cells. In addition, membrane permeabilization and depolarization assays, combined with time-kill studies and FEG-SEM imaging, indicated a fast membrane permeation and further leakage of intracellular content. Biophysical studies with lipid vesicles show a preference of PaDBS1R1 for Gram-negative bacteria-like membranes. We investigated the three-dimensional structure of PaDBS1R1 by CD and NMR analyses. Our results suggest that PaDBS1R1 adopts an amphipathic α-helix upon interacting with hydrophobic environments, after an initial electrostatic interaction with negative charges, suggesting a membrane lytic effect. This study reveals that PaDBS1R1 has potential application in antibiotic therapy.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/efeitos dos fármacos , Antibacterianos/farmacologia , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Dicroísmo Circular , Bactérias Gram-Negativas , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Luz , Lipídeos/química , Espectroscopia de Ressonância Magnética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Micelas , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Conformação Proteica em alfa-Hélice , Espalhamento de RadiaçãoRESUMO
A new antimicrobial peptide, herein named Stigmurin, was selected based on a transcriptomic analysis of the Brazilian yellow scorpion Tityus stigmurus venom gland, an underexplored source for toxic peptides with possible biotechnological applications. Stigmurin was investigated in silico, by circular dichroism (CD) spectroscopy, and in vitro. The CD spectra suggested that this peptide interacts with membranes, changing its conformation in the presence of an amphipathic environment, with predominance of random coil and beta-sheet structures. Stigmurin exhibited antibacterial and antifungal activity, with minimal inhibitory concentrations ranging from 8.7 to 69.5µM. It was also showed that Stigmurin is toxic against SiHa and Vero E6 cell lines. The results suggest that Stigmurin can be considered a potential anti-infective drug.