Reptilian Beta-defensins

Beta-defensins are a family of small cationic antimicrobial peptides (AMPs) that are strongly conserved throughout Eukaryotes. Their discovery was associated with broad spectrum antimicrobial activity, but it has since been determined that this family of proteins have more diverse functions, including roles such as a chemokine for macrophages and determining coat colour in dogs. In Reptiles, the innate immune response is the principal system for clearing invading pathogenic organisms. Beta-defensins are a major component of the innate immune response, yet their biological function is largely unknown. It is likely that their antimicrobial activity mimics that of their mammalian counterparts. Due to little research within reptilian beta-defensins many questions remain to be answered. Cluster characterisation, gene polymorphisms and defensin gene repertoire along with evolutionary mechanisms, conservation of synteny and physical properties of these peptides needs to be elucidated. This work identified novel gene clusters in 3 snake, 3 lizard, and 2 turtle/tortoise genomes as well as additional analysis on 2 Crocodylia species. Bioinformatic discovery through techniques such BLAST and annotation analysis reveals that these genes reside in single cluster. Conservation of synteny is observed within these clusters and in all species the cluster was flanked on one side by Cathepsin B (CTSB) and the other side by either, Exportin 1 (XPO1) in the Squamates and by Translocation Associated Membrane Protein 2 (TRAM2) in Crocodylia and the turtles/tortoises. Throughout reptilians, gene homology is observed with genes residing nearest CTSB being most obvious and through gene annotation and splice site prediction a two-exon organisation is the predominant gene structure of these beta-defensins. Their physical properties suggest that they are mostly cationic, with a few detected exceptions. The dS/dN ratio of synonymous and nonsynonymous substitutions on a gene level suggests that there is conservation in the first exon, which encodes a signal peptide and more positive Darwinian selection in the second exon which encodes the active AMP suggesting an ‘arms race’ between host and pathogen. Site wise evolutionary analyses on the second exon using HyPhy shows a common beta-defensin cysteine motif which is undergoing purifying selection and residues between these, showing positive selection. All this demonstrates that these are an evolving group of immune genes. In addition to this, a streamlined, cost-effective methodology has been developed to express, purify, and study these peptides to aid the investigation into their physical properties. This can provide information as to whether these novel antimicrobials have a much-needed use in future therapeutics or medicines.