INDUCTION OF ANTIMICROBIAL PEPTIDE SYNTHESIS IN Leptodactylus pentadactylus (ANURA, AMPHIBIA) SKIN, BY MECHANICAL, CHEMICAL, AND MICROBIAL STIMULUS

FONTENELE-BERTO R. (1), CORDEIRO-SOUSA J. (1), OLIVEIRA R. G. (1), REBOUÇAS JR J. P. (1), HONÓRIO JR J.E.R. (2), OLINDA A. C. C. (1), CARVALHO I. F. (1), ADED DA SILVA P. (1), FONTENELE-CARDI N. C. F. (4), PIMENTA D. C. (3), CARDI B. A. (1,2), CARVALHO K. M.(1)

(1) Laboratório de Toxinologia e Farmacologia Molecular, UECE; (2) Laboratório de Animais Peçonhentos, UECE; (3) Centro de Toxinologia Aplicada (CAT/CEPID – FAPESP), Instituto Butantan; (4) Hospital São José de Doenças Infecciosas, Ceará.

The anuran granular skin glands synthesize and store polypeptides with a broad spectrum of antimicrobial activity that play an important role in the innate immunity and constitute the first-line defense against invading pathogens. The aim of this work was verify the influence of mechanical, chemical and microbial stimuli in the production of antimicrobial peptides in the skin of the anuran L. pentadactylus. Thus, adult specimens had remained in captivity under space and nutricional conditions adjusted separately and submitted to different treatments, such as: i) light cuts in the skin, ii) subcutaneous injections of incomplete Freud Adjuvant (200 ml), iii) subcutaneous injections of Candida sp. suspensions (200 ml), and iv) subcutaneous injections of Pseudomonas aeruginosa (200 ml). After 3, 7 and 14 days the animals were submitted to electric stimulation (2-7V) for extraction of the cutaneous secretions, that were collected, lyophilized, weighed and kept -20ºC. Aliquots of these samples were purified by HPLC using a C18 column (25X250mm), eluted with 0-80% linear gradient of acetonitrile at a flow of 0,8ml/min. The analysis of the chromatographic profiles with all the treatments used shows a significant increment of the peaks at the region correspondent to elution with 30-50% of acetonitrile. The analysis of mass spectrometry of these fractions indicated the presence of peptides with molecular mass of 1.500 to 25.000 Da, presenting antimicrobial activity against Pseudomonas aeruginosa. These results suggest that L. pentadactylus cutaneous secretory machinary is activate in response to aggressive stimuli by inespecific manner, in agreement with the innate immune pathway on these animals.

KEYWORDS: Leptodactylus, peptides, stimulation

FINANCIAL SUPPORT: FUNCAP, CAPES, CNPq

CORRESPONDENCE TO: carvalhokris@gmail.com