Viperatoxin-II: A novel viper venom protein as an effective bactericidal agent

Ramar Perumal Samy; Bradley G Stiles; Arunachalam Chinnathambi; M E Zayed; Sulaiman Ali Alharbi; Octavio Luiz Franco; Edward G Rowan; Alan Prem Kumar; Lina H K Lim; Gautam Sethi

doi:10.1016/j.fob.2015.10.004

Viperatoxin-II: A novel viper venom protein as an effective bactericidal agent

FEBS Open Bio. 2015 Oct 23:5:928-41. doi: 10.1016/j.fob.2015.10.004. eCollection 2015.

Affiliations

¹ Venom and Toxin Research Programme, Department of Anatomy, Yong Loo Lin School of Medicine, National University Health System (NUHS), National University of Singapore, Singapore 117597; Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, NUHS, National University of Singapore, Singapore 117597; Department of Physiology, NUS Immunology Programme, Centre for Life Sciences, Yong Loo Lin School of Medicine, NUHS, National University of Singapore, Singapore 117456.
² Integrated Toxicology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702-5011, USA; Department of Biology, Wilson College, 1015 Philadelphia Avenue, Chambersburg, Pennsylvania 17201, USA.
³ Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia.
⁴ Universidade Católica de Brasília, Centro de Análises Proteômicas e Bioquímicas, Pós-Graduação em Ciências Genômicas e Biotecnologia UCB, Brasília-DF, Brazil; S-Inova Biotech, Pos-Graduação em Biotecnologia, Universidade Catolica Dom Bosco, Campo Grande, MS, Brazil.
⁵ Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0RE, United Kingdom.
⁶ Department of Pharmacology, Yong Loo Lin School of Medicine, National University Singapore, Singapore 117600; Cancer Science Institute, National University of Singapore, Singapore 117599; School of Biomedical Sciences, Curtin University, Western Australia, Australia; Department of Biological Sciences, University of North Texas, Denton, TX, USA.
⁷ Department of Physiology, NUS Immunology Programme, Centre for Life Sciences, Yong Loo Lin School of Medicine, NUHS, National University of Singapore, Singapore 117456.
⁸ Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia; Department of Pharmacology, Yong Loo Lin School of Medicine, National University Singapore, Singapore 117600; Cancer Science Institute, National University of Singapore, Singapore 117599.

Abstract

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have become a rising threat to public health. There is an urgent need for development of promising new therapeutic agents against drug resistant bacteria like S. aureus. This report discusses purification and characterization of proteins from Indian Russell's viper snake venom. Novel 15-kDa proteins called "Viperatoxin" (VipTx-I and VipTx-II) were extracted from the whole venom and evaluated using in vitro antimicrobial experiments. The N-terminal amino acid sequence of "Viperatoxin" showed high sequence homology to daboiatoxin isolated from the same venom and also matched phospholipase A2 (PLA2) enzymes isolated from other snake venoms. In an in vitro plate assay, VipTx-II but not VipTx-I showed strong antimicrobial effects against S. aureus and Burkholderia pseudomallei (KHW & TES), Proteus vulgaris and P. mirabilis. The VipTx-II was further tested by a broth-dilution assay at 100-3.1 μg/ml concentrations. The most potent bactericidal effect was found at the lowest dilutions (MICs of 6.25 μg/ml) against B. pseudomallei, S. aureus and P. vulgaris (MICs of 12.25 μg/ml). Electron microscopic investigation revealed that the protein-induced bactericidal potency was closely associated with pore formation and membrane damage, even at the lowest concentrations (<20 μg/ml). The toxin caused a low level of cytotoxic effects as observed in human (THP-1) cells at higher concentrations. Molecular weight determinations of VipTx-II by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one major, along with a few minor bands. The results indicate that VipTx-II plays a significant role in bactericidal and membrane damaging effects in vitro. Non-cytotoxic properties on human cells highlight it as a promising candidate for further evaluation of antimicrobial potential in vivo.

Keywords: Bactericidal; Daboia russelli russelli; MALDI-TOF/MS, matrix-assisted laser desorption ionization-time of flight/mass spectrometer; MDR, multi-drug resistant; MH, Mueller Hinton; MICs, minimum inhibitory concentrations; MRSA, methicillin-resistant Staphylococcus aureus; MTXs, myotoxins; PLA2, phospholipase A2; Phospholipase A2; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TS, Tryptic Soya; VipTx-I and VipTx-II, viperatoxins I and II; Viperatoxin-I; Viperatoxin-II.