44-In vitro activity of phospholipase A 2 and of peptides from Crotalus durissus terrificus venom against amastigote and promastigote forms of Leishmania (L) infantum chagasi

In vitro activity of phospholipase A ₂ and of peptides from Crotalus durissus terrificus venom against amastigote and promastigote forms of Leishmania (L.) infantum chagasi

Gustavo A. C. Barros¹, Andreia V. Pereira¹, Luciana C. Barros², Airton Lourenço Jr², Sueli A. Calvi¹, Lucilene D. Santos¹², Benedito Barraviera¹², Rui Seabra Ferreira Jr¹²³

1 Department of Tropical Diseases, Botucatu Medical School, São Paulo State University (UNESP – Univ Estadual Paulista), Botucatu, SP, Brazil

2 Center for the Study of Venoms and Venomous Animals (CEVAP), São Paulo State University (UNESP – Univ Estadual Paulista), Botucatu, SP, Brazil

3 CEVAP/UNESP, Caixa Postal 577, Fazenda Experimental Lageado, Rua José Barbosa de Barros, 1780, Botucatu, 18610-307, SP, Brasil

Background

American visceral leishmaniasis is caused by the intracellular parasite Leishmania (L.) infantum chagasi, and transmitted by the sand fly Lutzomyia longipalpis. Since treatment is based on classical chemotherapeutics with significant side effects, the search for new drugs remains the greatest global challenge. Thus, this in vitro study aimed to evaluate the leishmanicidal effect of Crotalus durissus terrificus venom fractions on promastigote and amastigote forms of Leishmania (L.) infantum chagasi.

Methods

Phospholipase A ₂ (PLA ₂ ) and a pool of peptide fraction (<3 kDa) were purified from Crotalusvenom. Furthermore, promastigotes and peritoneal macrophages of mice infected by amastigotes were exposed to serial dilutions of the PLA ₂ and peptides at intervals varying between 1.5625 μg/mL and 200 μg/mL. Both showed activity against promastigotes that varied according to the tested concentration and the time of incubation (24, 48 and 72 h).

Results

MTT assay for promastigotes showed IC ₅₀ of 52.07 μg/mL for PLA₂ and 16.98 μg/mL for the peptide fraction of the venom. The cytotoxicity assessment in peritoneal macrophages showed IC₅₀ of 98 μg/mL and 16.98 μg/mL for PLA ₂ and peptide by MTT assay, respectively. In peritoneal macrophages infected by Leishmania (L.) infantum chagasiamastigotes, the PLA ₂ stimulated growth of parasites, and at higher doses reduced growth by 23 %. The peptide fraction prevented 43 % of the intracellular parasite growth at a dose of 16.98 μg/mL, demonstrating the toxicity of this dose to macrophages. Both fractions stimulated H ₂ O ₂ production by macrophages but only PLA ₂ was able to stimulate NO production.

Conclusion

We have demonstrated the in vitro leishmanicidal activity of the PLA₂ and peptide fraction of Crotalus venom. The results encourage further studies to describe the metabolic pathways involved in cell death, as well as the prospecting of molecules with antiparasitic activity present in the peptide fraction of Crotalus durissus terrificus venom.

Key words: PLA₂; Peptides; Crotalus dutissus terrificus; Venom; Leishmanicidal activity

All experiments followed the ethical principles for animal experimentation adopted by the Brazilian College of Animal Experimentation (COBEA) and were approved by the Ethics Committee for Animal Experimentation at the Botucatu Medical School, UNESP (CEEA 890-2011).

Received: March 23, 2015; Revised: November 18, 2015; Accepted: November 24, 2015

GACB and AVP carried out the experimental protocols. LCB and AIJ performed the purification procedures. SAC and LDS analyzed data. BB and RSFJ designed experiments and wrote and revised the manuscript. RSFJ supervised the research work. All authors read and approved the final manuscript.