Purification, characterization and bactericidal activities of basic phospholipase A2 from the venom of Agkistrodon halys (Chinese pallas).
Agkistrodon snake venoms contain a variety of phospholipases (PLA2), some of which are myotoxic. In this study, we used reverse-phase HPLC to purify PLA2 from the venom of Agkistrodon halys. The enzyme named as AgkTx-II, a basic Asp49 PLA2, has a molecular masses of 13,869.05. The amino acid sequence and molecular mass of AgkTx-II was identical to those of an Asp49 basic myotoxic PLA2 previously isolated from this venom. Antibacterial activities were tested by susceptibility and broth-dilution assays. AgkTx-II exerted a potent antibacterial activity against Staphylococcus aureus, Proteus vulgaris, Proteus mirabilis, and Burkholderia pseudomallei. The MIC values of AgkTx-II ranged between 85 and 2.76microM and was most effective against S. aureus, P. vulgaris, P. mirabilis (MIC of 21.25microM) and B. pseudomallei (MIC of 10.25microM). This AgkTx-II rapidly killed S. aureus, P. vulgaris and B. pseudomallei in a dose-dependent manner. The effect of the AgkTx-II on bacterial membranes was evaluated by scanning and transmission electron microscopy. AgkTx-II caused morphological alterations apparent on their cellular surfaces, suggesting a killing mechanism based on membrane permeabilization and damage. Cytotoxicity was measured by XTT tetrazolium (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) and lactate dehydrogenase (LDH) assays using U-937 cells (monocytes). The AgkTx-II did not affect cell viability up to 500microM concentrations but cell death was evident at 1000microM concentration after 24 and 48h. Furthermore, the repeated exposure of AgkTx-II (2-14microM) treated mice showed different tissue alterations, mainly at the brain and kidney; the toxicological potential of AgkTx-II remains to be elucidated. The AgkTx-II exhibits no hemolytic action even at high doses (10-100microM) in human erythrocytes. However, the AgkTx-II is believed to exert its bactericidal effect by permeabilizing the bacterial membrane by forming pores. In addition, the basic PLA2 AgkTx-II displays a bactericidal effect, which may be either dependent or independent of catalysis.
Perumal Samy
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R
Venom and Toxin Research Programme, Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597.
Gopalakrishnakone
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P
Ho
Bow
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Chow
Vincent T K
VT
D017836:Agkistrodon / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D017124:Conserved Sequence; D003435:Crotalid Venoms / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D050296:Microbial Viability / Q000187:drug effects; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D054467:Phospholipases A2 / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D016415:Sequence Alignment; D020298:U937 Cells
C031442:Agkistrodon venoms; D000900:Anti-Bacterial Agents; D003435:Crotalid Venoms; D054467:Phospholipases A2