Purificação e análise da atividade antimicrobiana do peptídeo sintético des-His16-LyeTx I, análogo de um peptídeo obtido da peçonha da aranha Lycosa erythrognatha

Abstract

One avenue for research on new antimicrobial proteins and peptides is of animal origin. The spider venoms are complex mixtures of bioactive molecules including peptides, proteins, salts, amino acids, polyamines, and others. These molecules act on different biological targets and arouse interest in its potential for biotechnological and therapeutic application. Our group (Santos et al, 2009) isolated and characterized the cationic antimicrobial peptide LyeTx I, from the venom of the spider Lycosa erythrognatha. Based on the knowledge of its sequence this and other related peptides have been obtained by chemical synthesis using the methodology of Fmoc / t-butyl. One of these peptides, called des-His16-LyeTx I has a histidine residue at least compared to LyeTx1 and the N-terminal acetylated. This study evaluated the antimicrobial activity of this synthetic peptide, by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12223, Pseudomonas aeruginosas ATCC 27853, Propionibacterium acnes ATCC 6919, Fusobacterium necrophorum ATCC 25286, Bacteroides fragilis ATCC 25285, Peptostreptococcus anaerobius ATCC 27337, Cryptococcus neoformans ATCC 90112, Candida albicans ATCC 90028, Aspergillus fumigatus ATCC 16913 e Paracoccidioides brasiliensis: Pb01, Pb 18 e B339. After chemical synthesis, the peptide des-His16-LyeTx1 was purified by high performance reversed phase column (RP-HPLC) with linear gradient of 0.1% TFA (v / v) acetonitrile, and lyophilized. His mass was determined by mass spectrometry The MIC was determined by the microdilution peptide, at concentrations between 1-128 g / ml for bacteria and according to the concentrations determined by CLSI (Clinical and Laboratory Standards Institute) standard samples for fungi and yeasts. The reading was based on the observation by the naked eye from turbidity. For CBM, aliquots from wells where no growth was detected were cultivated. There was obtained 14 mg of the peptide with a high degree of purity. The mass of the peptide was 2.738 Kd determined by mass spectrometer. The MIC was 8 g / mL for samples of E. coli, P. aeruginosa and P. anaerobius. To S. aureus, S. epidermidis and P. acnes, the determined value was 4 g / mL in tests with B. fragilis the result obtained was 16 g / mL. In experiments with bacterial samples was observed VIII equivalence between the MIC and MBC, suggesting bactericidal activity of the peptide des-His16-LyeTx I. Of all the tested strains, only F. necrophorum was not sensitive to the peptide, for their antimicrobial activity. For yeast samples, the values for MIC were 16 g / mL for C. neoformans, 64 g / mL for C. albicans and A. fumigatus and P. brasiliensis there was no activity. The data suggest great potential for biotechnological des Hys-Lye Tx1 and opens perspectives for studies aimed at increasing its characterization including verifying possible toxicity, or other effects for the human being.