Poster Presentation Lorne Infection and Immunity 2020

Identification and characterisation of crocodilian membrane-targeting host defence peptides (#163)

Scott Williams 1 , Fung Lay 1 , Kha Phan 1 , Marc Kvansakul 1 , Mark Hulett 1
  1. Biochemistry and Genetics, La Trobe Institute for Molecular Science, Melbourne, VIC, Australia

Crocodilians are an order of ancient reptiles that throughout their evolution have developed a potent immune system, allowing them to thrive in environments abundant with pathogenic microbes[1]. Defensins, a family of cysteine-rich cationic antimicrobial peptides, play an important role in eukaryotic host defence. The antimicrobial activity of defensins (such as human defensins HBD-2[2] and HBD-3[3], as well as plant defensin NaD1[4]) is mediated by permeabilising cell membranes via the direct binding of lipids, such as the phosphatidylinositol phosphates. While extensive research has been focused on the defensins of plants and humans, the defensins of reptiles are poorly understood.

In this study, to better define the structure-function of crocodilian defensins, the Crocodylus porosus (saltwater crocodile) β-defensin 25 (CpBD25) was recombinantly expressed in the methylotrophic yeast Pichia pastoris. CpBD25 was shown to inhibit the growth of the pathogenic fungus Candida albicans (IC50 ~4.5 µM) by permeabilisation of the plasma membrane. The protein structure of CpBD25 was determined using X-ray crystallography and revealed a striking similarity to HBD-2, confirming that the tertiary structure of defensins has been conserved across the evolutionary divergence of humans and crocodiles. Further investigation revealed that a number of basic residues in HBD-2, previously shown to be crucial for phosphoinositide binding[2], had been substituted with histidine in CpBD25, suggesting that CpBD25 has a unique mode of interaction with membranes that may be pH-dependent. Membrane permeabilization assays at a range of physiologically relevant pH levels showed that the membranolytic activity of CpBD25 was greater at a pH <6.0, likely due to the charge-state of its histidine residues.

These results indicate that the membrane-targeting mechanism and tertiary structure, observed in the well-characterised plant and human defensins, have been evolutionarily conserved in the crocodilian defensin CpBD25. This study has also revealed that CpBD25’s membranolytic activity is regulated by changes in pH, which has not been observed in the studies of previously characterised defensins. The potential of using defensins as novel therapeutics is attracting significant interest and these data may be useful for engineering more effective peptides towards such applications.

  1. [1] van Hoek, ML (2014) “Antimicrobial peptides in reptiles”, Pharmaceuticals, 7:723-753
  2. [2] Järvå M, Phan TK, Lay FT, Caria S, Kvansakul M and Hulett MD (2018) “Human β-defensin 2 kills Candida albicans through phosphatidylinositol 4,5-bisphosphate–mediated membrane permeabilisation”, Science Advances, 4:eaat0979.
  3. [3] Phan TK, Lay FT, Poon IK, Hind MG, Kvansakul M and Hulett MD (2018) “Human β-defensin 3 contains an oncolytic motif that binds PI(4,5)P2 to mediate tumour cell permeabilisation”, Oncotarget, 7:2054-69.
  4. [4] Poon IKH, Baxter AA, Lay FT, Mills GD, Adda CG, Payne JA, Phan TK, Ryan GF, White JA, Veneer PK, van der Weerden NL, Anderson MA, Kvansakul M and Hulett MD (2014) “Phosphoinositide-mediated oligomerization of a defensin induces cell lysis”, eLife, 3:e01808