Purification of PaTx-II from the Venom of the Australian King Brown Snake and Characterization of Its Antimicrobial and Wound Healing Activities

Ramar Perumal Samy; Stephen P Mackessy; Alagarmalai Jeyasankar; Mano Ranjana Ponraj; Octavio Luiz Franco; Matthew A Cooper; Matheswaran Kandasamy; Tapan Kumar Mohanta; Jebasingh Bhagavathsingh; Sakthivel Vaiyapuri

doi:10.3390/ijms24054359

Purification of PaTx-II from the Venom of the Australian King Brown Snake and Characterization of Its Antimicrobial and Wound Healing Activities

Int J Mol Sci. 2023 Feb 22;24(5):4359. doi: 10.3390/ijms24054359.

Affiliations

¹ Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, NUHS, MD10, 4 Medical Drive, Singapore 117594, Singapore.
² School of Biological Sciences, Campus Box 92, College of Natural and Health Sciences, University of Northern Colorado, 501 20th St., Greeley, CO 80639, USA.
³ Department of Zoology, Government Arts College (Autonomous), Coimbatore 641018, Tamil Nadu, India.
⁴ Medicinal Organic Chemistry Laboratory, Department of Applied Chemistry, Karunya Institute of Technology and Sciences, Coimbatore 641114, Tamil Nadu, India.
⁵ Centro de Análises Proteômicas e Bioquímicas, Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, UCB, Brasilia 71966-700, DF, Brazil.
⁶ Sinova Biotech, Programa de Pós-Graduação em Biotecnologia, Universidade Católica Dom Bosco, Campo Grande 79117-900, MS, Brazil.
⁷ Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072, Australia.
⁸ Radcliffee Department of Medicine, University of Oxford, Oxford OX3 9DU, UK.
⁹ Natural and Medical Sciences Research Center, University of Nizwa, Nizwa 611, Oman.
¹⁰ HPLC Mass Facility, Department of Applied Chemistry, Karunya Institute of Technology and Sciences, Coimbatore 641114, Tamil Nadu, India.
¹¹ School of Pharmacy, University of Reading, Reading RG6 6UB, UK.

Abstract

Infections caused by multi-drug-resistant (MDR) bacteria are a global threat to human health. As venoms are the source of biochemically diverse bioactive proteins and peptides, we investigated the antimicrobial activity and murine skin infection model-based wound healing efficacy of a 13 kDa protein. The active component PaTx-II was isolated from the venom of Pseudechis australis (Australian King Brown or Mulga Snake). PaTx-II inhibited the growth of Gram-positive bacteria in vitro, with moderate potency (MICs of 25 µM) observed against S. aureus, E. aerogenes, and P. vulgaris. The antibiotic activity of PaTx-II was associated with the disruption of membrane integrity, pore formation, and lysis of bacterial cells, as evidenced by scanning and transmission microscopy. However, these effects were not observed with mammalian cells, and PaTx-II exhibited minimal cytotoxicity (CC₅₀ > 1000 µM) toward skin/lung cells. Antimicrobial efficacy was then determined using a murine model of S. aureus skin infection. Topical application of PaTx-II (0.5 mg/kg) cleared S. aureus with concomitant increased vascularization and re-epithelialization, promoting wound healing. As small proteins and peptides can possess immunomodulatory effects to enhance microbial clearance, cytokines and collagen from the wound tissue samples were analyzed by immunoblots and immunoassays. The amounts of type I collagen in PaTx-II-treated sites were elevated compared to the vehicle controls, suggesting a potential role for collagen in facilitating the maturation of the dermal matrix during wound healing. Levels of the proinflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and interleukin-10 (IL-10), factors known to promote neovascularization, were substantially reduced by PaTx-II treatment. Further studies that characterize the contributions towards efficacy imparted by in vitro antimicrobial and immunomodulatory activity with PaTx-II are warranted.

Keywords: Australian king brown snake venom; PaTx-II; Pseudechis australis; antibiotic; antimicrobial; fusidic acid ointment; wound.

MeSH terms

Animals
Anti-Infective Agents* / pharmacology
Australia
Cnidarian Venoms* / pharmacology
Collagen / pharmacology
Colubridae*
Cytokines / pharmacology
Humans
Mammals
Mice
Peptides / pharmacology
Staphylococcus aureus
Wound Healing

Substances

parasicyonis toxin
Anti-Infective Agents
Cnidarian Venoms
Collagen
Peptides
Cytokines

Grants and funding

This project was funded by Proof of Concept (POC), Economic Development Board (EDB) grant WBS No: R-181000110141, Singapore. The authors also would like to thank the Medical Research Council, UK (reference: MR/W019353/1) for their funding support.