Antimicrobial peptides from Rana [Lithobates] catesbeiana: Gene structure and bioinformatic identification of novel forms from tadpoles

Antimicrobial peptides (AMPs) exhibit broad-spectrum antimicrobial activity, and have promise as new therapeutic agents. While the adult North American bullfrog (Rana [Lithobates] catesbeiana) is a prolific source of high-potency AMPs, the aquatic tadpole represents a relatively untapped source for new AMP discovery. The recent publication of the bullfrog genome and transcriptomic resources provides an opportune bridge between known AMPs and bioinformatics-based AMP discovery. The objective of the present study was to identify novel AMPs with therapeutic potential using a combined bioinformatics and wet lab-based approach. In the present study, we identified seven novel AMP precursor-encoding transcripts expressed in the tadpole. Comparison of their amino acid sequences with known AMPs revealed evidence of mature peptide sequence conservation with variation in the prepro sequence. Two mature peptide sequences were unique and demonstrated bacteriostatic and bactericidal activity against Mycobacteria but not Gram-negative or Gram-positive bacteria. Nine known and seven novel AMP-encoding transcripts were detected in premetamorphic tadpole back skin, olfactory epithelium, liver, and/or tail fin. Treatment of tadpoles with 10 nM 3,5,3′-triiodothyronine for 48 h did not affect transcript abundance in the back skin, and had limited impact on these transcripts in the other three tissues. Gene mapping revealed considerable diversity in size (1.6–15 kbp) and exon number (one to four) of AMP-encoding genes with clear evidence of alternative splicing leading to both prepro and mature amino acid sequence diversity. These findings verify the accuracy and utility of the bullfrog genome assembly, and set a firm foundation for bioinformatics-based AMP discovery.