Antimicrobial peptides isolated from skin secretions of the diploid frog, Xenopus tropicalis (Pipidae) Mohamed F. Ali a , AnaMaria Soto b , Floyd C. Knoop c , J. Michael Conlon a ; * a Department of Biomedical Sciences, Creighton University Medical School, Omaha, NE 68178-0405, USA b Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA c Department of Medical Microbiology and Immunology, Creighton University Medical School, Omaha, NE 68178, USA Received 16 July 2001; accepted 30 August 2001 Abstract Seven peptides (XT-1^XT-7) with antimicrobial activity were isolated from norepinephrine-stimulated skin secretions of the diploid clawed frog, Xenopus tropicalis. Structural characterization of the peptides demonstrated that amino acid sequence similarity to antimicrobial peptides previously isolated from Xenopus laevis was low, suggesting that the species are not closely related phylogenetically. Peptides XT-5 and XT-3 are probably the orthologs of X. laevis peptide glycine-leucine amide (PGL a ) and the N-terminal spacer region of prolevitide, respectively. XT-1, XT-6 and XT-7 show limited structural similarity to the spacer region of X. laevis procaeruleins and the paralogs XT-2 and XT-4 are similar to corresponding regions of proxenopsin. Orthologs of the magainins were not identified. The C-terminally K-amidated peptide XT-7 (GLLGPLLKIAAKVGSNLL.NH 2 ) showed the lowest minimum inhibitory concentrations against reference microorgan- isms (Staphylococcus aureus 5 WM, Escherichia coli 5 WM, and Candida albicans 40 WM) and was also active against clinical isolates of methicillin-resistant S. aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus group C, Shigella sonnei, Pseudomonas aeruginosa and Enterobacter cloacae. The peptide was, however, hemolytic against human erythrocytes (50% lysis at 70 WM). Circular dichroism studies showed that XT-7 has a random structure in aqueous solution, pH 7.0 but adopts an K-helical conformation in the presence of 50% trifluoroethanol. Decreasing the cationicity of XT-7 either by replacement of the C-terminal CONH 2 group by COOH or by deletion of the Lys 8 residue produced analogs with greatly ( s 10-fold) decreased antimicrobial potencies. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Amphibian skin ; Antimicrobial ; Proxenopsin ; Procaerulein ; Prolevitide ; Peptide glycine-leucine amide 1. Introduction The emergence of pathogenic microorganisms with resistance to commonly used antibiotics has necessi- tated a search for new sources of antimicrobial drugs [1]. The fact that certain nosocomial (hospital ac- quired) infections are already resistant to all avail- able antibiotics, and therefore essentially untreatable, dramatically demonstrates the need for completely new types of antimicrobial agents to which the bac- teria have not developed resistance [2]. Bactericidal and fungicidal peptides synthesized in the skins of certain frogs represent a promising source of such potential therapeutic agents [3^5]. The skin secretions of the African clawed frog, Xenopus laevis, contain 0167-4838 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII:S0167-4838(01)00272-2 Abbreviations : MIC, minimum inhibitory concentration * Corresponding author. Fax : 402-280-2690. E-mail address : jmconlon@creighton.edu (J.M. Conlon). Biochimica et Biophysica Acta 1550 (2001) 81^89