Crocodile defensin (CpoBD13) antifungal activity via pH-dependent phospholipid targeting and membrane disruption

Abstract

Crocodilians are an order of ancient reptiles that thrive in pathogen-rich environments. The ability to inhabit these harsh environments is indicative of a resilient innate immune system. Defensins, a family of cysteine-rich cationic host defence peptides, are a major component of the innate immune systems of all plant and animal species, however crocodilian defensins are poorly characterised. We now show that the saltwater crocodile defensin CpoBD13 harbors potent antifungal activity that is mediated by a pH-dependent membrane-targeting action. CpoBD13 binds the phospholipid phosphatidic acid (PA) to form a large helical oligomeric complex, with specific histidine residues mediating PA binding. The utilisation of histidine residues for PA engagement allows CpoBD13 to exhibit differential activity at a range of environmental pH values, where CpoBD13 is optimally active in an acidic environment.

Fig. 1. CpoBD13 is a histidine-rich β-defensin that permeabilises C. albicans in a pH-dependent manner.

a Full-length peptide sequence of CpoBD13. Connecting lines represent disulphide bonds between cysteine residues. b Sequence alignment of CpoBD13 with its close homologues A. sinensis (Chinese alligator) β-defensin 13 (AsBD13, GenBank accession code AUG31287.1) and Gallus gallus (chicken) avian β-defensin 13 (AvBD13, NCBI RefSeq NP_001001780.1). c Fungal growth inhibition assay of C. albicans treated with CpoBD13 over a 24 h period. d Membrane permeabilisation of C. albicans after a 30 min treatment with CpoBD13 as determined by the uptake of propidium iodide. e MTT and MTS cell viability assay of human primary (AHDF and HUVEC) and tumourigenic (HeLa, PC3 and U937) cells treated with a range of CpoBD13 concentrations after 48 h. f Net charge of CpoBD13 at pH 5–9 highlights a sharp decrease in positive charge at pH greater than 6.0. The theoretical net charge of the peptide was calculated using a simplified method based on the pK_a of free amino acids. g Membrane permeabilisation of C. albicans treated with CpoBD13 in conditions buffered to pH 5.5, 6.5 and 7.5. Data in (c–e & g) represent mean ± SEM, n = 3. h Live microscopy of C. albicans treated with 20 µM CpoBD13 conjugated to the fluorophore BODIPY FL EDA (green) in the presence of PI (orange). Images are representative of three independent experiments. i Live microscopy of the accumulation of CpoBD13-BODIPY (20 µM, green) at the plasma membrane of C. albicans cells. Scale bars represent 10 µm. Images are representative of three independent experiments. (c–g) Source data are provided as a Source Data file.

Fig. 2. CpoBD13 binds and preferentially lyses liposomes containing the anionic phospholipid phosphatidic acid.

a Immunodetection of CpoBD13-HA overlayed on a PIP Strip shows specific binding to PA. b Liposome pulldown comparing the binding of CpoBD13 to PC only and PC:PA (95:5 molar ratio) liposomes. Bound (B, black) and Unbound (U, white) fractions were subjected to SDS-PAGE analysis and colloidal Coomassie staining. The intensities of the bands were calculated as a fraction of a 1 µg loading control (LC) using ImageJ software. Data represent mean ± SEM, n = 3. *P < 0.05, two-tailed unpaired t test. c Liposome lysis of calcein-encapsulated PC only (white) and PC:PA (black) liposomes by 20 µM CpoBD13 over 30 min. Lysis was calculated as a fraction of a 100% lysis control using 0.1% Triton X-100. Data represent mean ± SEM, n = 3. ***P = 0.0009, two-way ANOVA. d Chemical crosslinking of CpoBD13 using BS(PEG)₅ in the presence of various concentrations of PA followed by SDS-PAGE analysis and colloidal Coomassie staining. Image is representative of three independent experiments. e TEM images of CpoBD13 alone, PA alone or CpoBD13:PA complexes (scale bars represent 1 µm). Images are representative of two independent experiments. (a–d) Source data are provided as a Source Data file.

Fig. 3. Crystal structure of the CpoBD13:PA complex.

a Three-dimensional structure of CpoBD13 determined by X-ray crystallography superimposed with HBD-2 (PDB ID 1FD3). Zoomed in region shows the substitution of K36 (a crucial residue for membranolytic activity) in HBD-2 for H35 in CpoBD13. b Four CpoBD13 chains are found in the asymmetric unit (white, cyan, steel blue and magenta cartoon), bound to four PA molecules shown as green sticks. c Type I and d Type II PA binding sites. Key interacting residues are shown as sticks. Hydrogen bonds or ionic interactions are marked as black dashed lines. Schematic representation of the e Type I and f Type II PA binding sites. g CpoBD13:PA complex forms a single-stranded left-handed helix. The coil shown comprises the content of four asymmetric units. The location of a single type I PA binding site is boxed in black.

Fig. 4. Structure–function analysis of the interaction between CpoBD13 and PA.

Data represent ± SEM, n = 3. All statistical evaluations are comparisons to the corresponding CpoBD13 wild type sample. ns, not significant, *P < 0.05, **P < 0.01, ****P < 0.0001 two-tailed unpaired t test. Exact P values are stated above their respective graphs. a Fungal growth inhibition of C. albicans treated with wild type or mutant CpoBD13 over 24 h. b Calculated IC₅₀ values of the fungal growth curves. # The IC₅₀ of CpoBD13 (H21A) could not be determined within the assessed concentration range (0–10 µM). c Membrane permeabilisation of C. albicans treated with wild type or mutant CpoBD13 for 30 min as determined by the uptake of PI. d Membrane permeabilisation of C. albicans in pH buffered conditions (pH 5.5 = solid line, pH 7.5 = dashed line) treated with wild type or mutant CpoBD13 for 30 min. e Liposome pulldown quantification of the bound fractions of PC only (white) and 95:5 molar ratio PC:PA (black) liposomes incubated with 1 µg of CpoBD13 or its mutants performed at pH 7.4 and f again at pH 5.5. g Liposome lysis of calcein-encapsulated PC only and PC:PA liposomes by 20 µM wild type or mutant CpoBD13 over 30 min. Lysis was calculated as a fraction of a 100% lysis control using 0.1% Triton X-100. h Biochemical crosslinking of wild type or mutant CpoBD13 using BS(PEG)₅ in the presence of PA followed by SDS-PAGE analysis and colloidal Coomassie staining. Image is representative of three independent experiments. (a–h) Source data are provided as a Source Data file.

Fig. 5. Effects of compound mutations in the PA-binding site of CpoBD13 on its antifungal activity.

a Sequence alignment of CpoBD13 with the designed compound mutants. Residues that differ from wild type CpoBD13 are highlighted in black. b Fungal growth inhibition of C. albicans treated with a range of CpoBD13 and CpoBD13 compound mutant concentrations over 24 h. Data represent mean ± SEM, n = 3. c Calculated IC₅₀ values of the fungal growth curves. Data represent mean ± SEM, n = 3. All statistical evaluations are comparisons to the corresponding CpoBD13 wild type sample. **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed unpaired t test. Exact P values are stated above their respective graphs. d Membrane permeabilisation of C. albicans in pH-buffered conditions (pH 5.5 = solid line, pH 7.5 = dashed line) treated with CpoBD13 (H21A/H35A) (purple) and CpoBD13 (H21R/H35R) (orange) for 30 min. Data represent mean ± SEM, n = 3. ns, not significant, two-way ANOVA. (b–d) Source data are provided as a Source Data file.