Poster 127 - Antimicrobial peptides screening of the skin secretion from the frog Leptodactylus

Poster 127. Congresso da Sociedade Brasileira de Toxinologia, 8., Symposium of the Pan American Section of the International Society on Toxinology, 8., 2004, Angra dos Reis, Brasil. Abstracts... J. Venom. Anim. Toxins incl.Trop. Dis., 2004, 10, 3, p.486.

Antimicrobial Peptides Screening Of The Skin Secretion From The Frog Leptodactylus labyrinthicus

^{1,2Nascimento, A.S.; ^1,2Zanotta, L.C.; ²Fontes, W.; ²Sousa, M.V.; ¹Schwartz, E.F. and ^1,2Castro, M.S.}

1 Laboratório de Toxinologia, Departamento de Ciências Fisiológicas/IB, Universidade de Brasília, Brasília/DF; 2 Laboratório de Bioquímica e Química de Proteínas - Centro Brasileiro de Serviços e Pesquisas em Proteínas, Departamento de Biologia Celular/IB, Universidade de Brasília, Brasília/DF.

Antimicrobial peptides have been widely isolated over the past 17 years from frog skin secretions. These peptides exert their antimicrobial properties by binding preferentially microbial membranes causing membrane disruption and their therapeutic applications in human health have been explored. Antimicrobial peptides are active in low concentrations and it has been observed that these peptides induce less microbial resistance than other xenobiotics. In the present work we performed a screening for antimicrobial peptides of the skin secretion from the frog Leptodactyluslabyrinthicus. Adult specimens of L. labyrinthicus were collected in Brasilia/DF. The skin secretion was obtained by mild electric stimulation and lyophilized. Aliquots of 200 mL (45 mg/mL) of crude secretion were fractionated using a C18 column (Vydac 218TP1010). All fractions were manually collected and freeze-dried. The antimicrobial activity was monitored against Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923). using liquid growth inhibition assay. The HPLC fractions were dissolved in 60 mL of Milli Q water and 50 mL of each fraction were used to test antibacterial activity. These aliquots were incubated with 50 mL of bacterial cells (approximately 1 x 10⁵ bacteria/mL) for 24 hours at 37°C. Bacterial growth was monitored at 595 nm. Thirteen fractions showed antimicrobial activity against S. aureus and 9 nine fractions showed activity against E. coli. Two fractions showed activity against both bacteria. These active fractions were analyzed by MALDI-TOF (Reflex IV, Bruker) and, at least, four different peptides were identified by their molecular masses (1071 Da, 1727 Da, 1784 Da, 2063 Da). Posterior stages include fully characterization of each active peptide. This study evidences the presence of a large number of fractions with antimicrobial activity on the skin secretion of the frog L. labyrinthicus, besides previously isolated labyrinthins.