Crocodilians are ancient reptiles that have adapted throughout evolution to inhabit pathogen‑rich environments. Despite commonly receiving wounds, the likelihood of developing a systemic infection is rare for these animals, indicating a potent immune system. Defensins, a class of cysteine‑rich cationic host defence peptides, contribute to the innate immunity of all eukaryotes. These peptides, which permeabilise microbial cell membranes through the direct binding of negatively charged phospholipids, have been well characterised in humans¹ and plants², however, the defensins of reptiles are poorly understood.

In this study³, to better define the structure-function of crocodilian defensins, Crocodylus porosus (saltwater crocodile) β-defensin 13 (CpoBD13) was recombinantly expressed in the methylotrophic yeast Pichia pastoris. CpoBD13 was shown to inhibit the growth of the pathogenic fungus Candida albicans through the permeabilisation of the plasma membrane. Phospholipid-binding experiments revealed that CpoBD13 specifically bound the membrane lipid phosphatidic acid (PA). The protein structure of CpoBD13 in complex with PA was determined using X-ray crystallography and revealed that protein-lipid interactions were mediated by arginine and histidine residues. Membrane permeabilisation assays at a range of physiologically relevant pH levels showed that the antifungal activity of CpoBD13 was greater at pH <6.0 due to the increase in charge, and therefore the affinity for PA, accredited to the protonation of the peptide’s histidine residues.

These results indicate that the membrane-targeting mechanism, established in the studies of human and plant defensins, has been evolutionarily conserved in the crocodilian defensin CpoBD13. This study has also uncovered that CpoBD13’s ability to bind PA and permeabilise fungal cell membranes is regulated by changes in pH, an ability which has not been observed in previous defensin studies. Collectively, these data may be helpful in the development and peptide engineering of future defensin‑based antifungal therapeutics.