title: Viperatoxin-II: A novel viper venom protein as an effective bactericidal agent.
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.
authors.affiliation: 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.
authors.forename: Ramar Perumal
authors.initials: RP
authors.lastname: Samy
authors.affiliation: 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.
authors.forename: Bradley G
authors.initials: BG
authors.lastname: Stiles
authors.affiliation: Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia.
authors.forename: Arunachalam
authors.initials: A
authors.lastname: Chinnathambi
authors.affiliation: Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia.
authors.forename: M E
authors.initials: ME
authors.lastname: Zayed
authors.affiliation: Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia.
authors.forename: Sulaiman Ali
authors.initials: SA
authors.lastname: Alharbi
authors.affiliation: 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.
authors.forename: Octavio Luiz
authors.initials: OL
authors.lastname: Franco
authors.affiliation: Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0RE, United Kingdom.
authors.forename: Edward G
authors.initials: EG
authors.lastname: Rowan
authors.affiliation: 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.
authors.forename: Alan Prem
authors.initials: AP
authors.lastname: Kumar
authors.affiliation: Department of Physiology, NUS Immunology Programme, Centre for Life Sciences, Yong Loo Lin School of Medicine, NUHS, National University of Singapore, Singapore 117456.
authors.forename: Lina H K
authors.initials: LH
authors.lastname: Lim
authors.affiliation: 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.
authors.forename: Gautam
authors.initials: G
authors.lastname: Sethi
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