RESUMO
Turgencin A, a potent antimicrobial peptide isolated from the Arctic sea squirt Synoicum turgens, consists of 36 amino acid residues and three disulfide bridges, making it challenging to synthesize. The aim of the present study was to develop a truncated peptide with an antimicrobial drug lead potential based on turgencin A. The experiments consisted of: (1) sequence analysis and prediction of antimicrobial potential of truncated 10-mer sequences; (2) synthesis and antimicrobial screening of a lead peptide devoid of the cysteine residues; (3) optimization of in vitro antimicrobial activity of the lead peptide using an amino acid replacement strategy; and (4) screening the synthesized peptides for cytotoxic activities. In silico analysis of turgencin A using various prediction software indicated an internal, cationic 10-mer sequence to be putatively antimicrobial. The synthesized truncated lead peptide displayed weak antimicrobial activity. However, by following a systematic amino acid replacement strategy, a modified peptide was developed that retained the potency of the original peptide. The optimized peptide StAMP-9 displayed bactericidal activity, with minimal inhibitory concentrations of 7.8 µg/mL against Staphylococcus aureus and 3.9 µg/mL against Escherichia coli, and no cytotoxic effects against mammalian cells. Preliminary experiments indicate the bacterial membranes as immediate and primary targets.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Produtos Biológicos/química , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Sequência de Aminoácidos/genética , Animais , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Organismos Aquáticos/genética , Produtos Biológicos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Humanos , Testes de Sensibilidade Microbiana , Proteínas Citotóxicas Formadoras de Poros/síntese química , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Análise de Sequência de Proteína , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidadeRESUMO
A Gram-stain-negative, facultatively anaerobic Vibrio strain, designated NFH.MB010T, was isolated from an epidermal lesion on the test (hard shell skeleton) of a green sea urchin (Strongylocentrotus droebachiensis) collected from northern Norway. Cells of strain NFH.MB010T were rod shaped and motile by means of a single, long polar flagellum. Growth was observed at 1-5% NaCl (w/v) and at 4 °C, but not above 28 °C. Phylogenetic analyses based on eight-gene multilocus sequence analysis (16S rRNA, atpA, gyrB, mreB, pyrH, recA, rpoA and rpoD) suggested novelty at the species level. In silico DNA-DNA hybridization and orthologous average nucleotide identity estimates showed percentage genomic resemblances to its closest relative, Vibrio splendidus, that were well below the established same species threshold values. Phenotypically, utilization of glycogen and gentiobiose, inability of acetoin production, and undetectable valine arylamidase and trypsin activity discriminated strain NFH.MB010T from the closely related reference strains. Protein spectra generated by maldi-tof mass spectrometry further consolidated the species level uniqueness of strain NFH.MB010T. Based on the described polyphasic approach, strain NFH.MB010T therefore appears as a novel species within the Splendidus clade of the genus Vibrio, and the name Vibrio echinoideorum sp. nov. is proposed, with NFH.MB010T (=DSM 107264T=LMG 30656T) as the type strain.