A new temporin with antibacterial activity and cytotoxicity from the skin secretion of Lithobates palmipes (Spix, 1824) (Amphibia: Ranidae) from Brazilian Atlantic Forest

Abstract

This work investigated the peptide profile of skin secretion from Lithobates palmipes collected from the Brazilian Atlantic Forest. The secretion was submitted to reversed phase high-performance liquid chromatography (RP-HPLC) and the fractions were screened for antibacterial activity. RP-HPLC resulted in the separation of several peaks, among which 10 showed antibacterial activity and contained peptides of the ranatuerin, brevinin and temporin families. Fraction 6 was resubmitted to RP-HPLC and a novel peptide from temporin family (temporin-PMb) had its primary structure determined. Temporin-PMb and non-amidated temporin-PMb were synthesized, purified, and evaluated for antibacterial activity, hemolytic activity and cytotoxicity to keratinocytes and cancer cells. Temporin-PMb was active against Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa as well as against methicilin-resistant S. aureus (MRSA) and Acinetobacter baumannii. It was cytotoxic to human cervical adenocarcinoma cells (HeLa) and human mammary adenocarcinoma cells (MCF7) with IC₅₀ of 32.4 and 24.1 μM, respectively. It was also toxic to human keratinocytes (HaCaT; IC₅₀ of 25.0 μM) and showed hemolytic activity. The non-amidated form showed low hemolytic activity and lower HaCaT toxicity, but was only effective against E. coli, S. aureus MRSA, and A. baumanii. In conclusion, Atlantic Forest L. palmipes skin secretion contained different bioactive peptides, including a novel temporin with antibacterial effect and cytotoxicity towards human cancer cells. The amide group was responsible for the activities of the wild-type temporin-PMb. Peptide engineering studies are encouraged aiming at minimizing unwanted effects.