Characterization of antimicrobial peptides in skin secretions from discrete populations of Lithobates chiricahuensis (Ranidae) from central and southern Arizona

► Characterization of six antimicrobial peptides present in skin secretions of populations of the leopard frog Lithobates chiricahuensis from distinct ranges in southern and central Arizona. ► Characterization of two peptides (brevinin-1CHc and palustrin-2CHa) present only in secretions of frogs from the southern range. ► Identification of esculentin-2CHa as a potent, broad spectrum antimicrobial peptide with therapeutic potential. ► Phylogenetic analysis of the North American leopard frogs based upon the primary structures of brevinin-1 peptides. Populations of the Chiricahua leopard frog Lithobates chiricahuensis (Ranidae) occupying regions in southern Arizona (southern range) are morphologically distinct from those from the Mogollon Rim of central Arizona (northern range) and a comparison of DNA sequences of mitochondrial genes has suggested that they may represent separate species. Peptidomic analysis of norepinephrine-stimulated skin secretions has led to the identification of six peptides with antimicrobial activity in samples from specimens from both groups. The primary structure of the peptides (esculentin-2CHa, ranatuerin-2CHa, -CHb, and -CHc, and brevinin-1CHa and -CHb) isolated from both southern and northern range frogs are identical consistent with the proposal that the two populations are conspecific. However, palustrin-2CHa and the atypical brevinin-1CHc (FFPTIAG***LTKLFCA ITKKC), containing a five amino acid residue deletion, were identified only in secretions from southern range specimens. Consequently, there is some support for the proposal that the two populations are closely related but separate species but this support is relatively weak. Esculentin-2CHa (GFSSIFRGVAKFASKGLG KDLAKLGVDLVACKISKQC) displayed the highest antimicrobial potency (MIC ≤ 10 μM) against a variety of microorganisms and was only moderately hemolytic (LC 50 = 150 μM). Cladistic analysis based upon the primary structures of brevinin-1 peptides indicates a close phylogenetic relationship between L. chiricahuensis, L. onca, and L. yavapaiensis.