Identification and characterization of a phospholipase A2 from the venom of the Saw-scaled viper: Novel bactericidal and membrane damaging activities.
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 Echis 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 (15 kD 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 μg/ml) for bactericidal activity of a wide variety of human pathogens against Burkholderia pseudomallei (KHW&TES), Enterobacter aerogenes, Escherichia 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 μg/ml against (B. pseudomallei, KHW) and MICs >30 μg/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 μg/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.
Perumal Samy
Ramar
R
Venom and Toxin Research Programm, Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597. rperumalsamy@yahoo.co.uk
Gopalakrishnakone
P
P
Bow
Ho
H
Puspharaj
Peter N
PN
Chow
Vincent T K
VT
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000302:isolation & purification* / Q000378:metabolism / Q000633:toxicity; D001921:Brain / Q000187:drug effects / Q000473:pathology; D016957:Burkholderia pseudomallei / Q000187:drug effects / Q000648:ultrastructure; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D021902:Enterobacter aerogenes / Q000187:drug effects / Q000648:ultrastructure; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000473:pathology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008826:Microbial Sensitivity Tests; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D054467:Phospholipases A2 / Q000302:isolation & purification* / Q000378:metabolism / Q000633:toxicity; D017386:Sequence Homology, Amino Acid; D012867:Skin / Q000187:drug effects / Q000473:pathology; D014757:Viper Venoms / Q000235:genetics / Q000302:isolation & purification* / Q000633:toxicity; D017819:Viperidae / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D014757:Viper Venoms; D054467:Phospholipases A2