Citation

Samy RP, Gopalakrishnakone P, Bow H, Peter P, Chow VTK. Biochimie 2010; 92(12): 1854-1866.

Affiliation

Venom and Toxin Research Programm, Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore - 117597; Singapore Eye Research Institute, 9 Hospital Drive Block C, SoN building Singapore 117597.

Copyright

(Copyright © 2010, Elsevier Publishing)

DOI

10.1016/j.biochi.2010.07.012

PMID

20723574

Abstract

Phospholipase A(2) (PLA(2)), a common toxic component of snake venom, has been implicated in various pharmacological effects. In this study, a basic myotoxic PLA(2), named EcTx-I was isolated from E. carinatus snake venom by using gel filtration on Superdex G-75, and reverse phase HPLC on C18 and C8 Sepharose columns. PLA(2), EcTx-I was 13,861.72 molecular weight as estimated by MALDI-TOF (15kD by SDS-PAGE), and consisted of 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with basic myotoxic PLA(2)s from other snake venoms. The purified PLA(2) EcTx-I was evaluated (250 mug/ml) for bactericidal activity of a wide variety of human pathogens against Burkholderia pseudomallei (KHW&TES), Enterobacter aerogenes, Eschericia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus. EcTx-I showed strong antibacterial activity against B. pseudomallei (KHW) and E. aerogenes among the tested bacteria. Other Gram-negative and Gram-positive bacteria showed only a moderate effect. However, the Gram-positive bacterium E. aerogenes failed to show any effect on EcTx-I protein at tested doses. The most significant bacteriostatic and bactericidal effect of EcTx-I was observed at MICs of >15 mug/ml against (B. pseudomallei, KHW) and MICs >30 mug/ml against E. aerogenes. Mechanisms of bactericidal and membrane damaging effects were proved by ultra-structural analysis. EcTx-I was able to induce cytotoxicity on THP-1 cells in vitro as well as lethality in BALB/c mice. EcTx-I also induced mild myotoxic effects on mouse skin, but was devoid of hemolytic effects on human erythrocytes up to 500 mug/ml. It is shown that the toxic effect induced by E. carinatus venom is due to the presence of myotoxic PLA(2) (EcTx-I). The result also corroborates the hypothesis of an association between toxic and enzymatic domains. In conclusion, EcTx-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.

Language: en