1. Purification of peptides with differential cytolytic activities from the skin secretions of the Central American frog, Lithobates vaillanti (Ranidae)
J Michael Conlon, Haider Raza, Laurent Coquet, Thierry Jouenne, Jérôme Leprince, Hubert Vaudry, Jay D King Comp Biochem Physiol C Toxicol Pharmacol. 2009 Aug;150(2):150-4. doi: 10.1016/j.cbpc.2009.04.003. Epub 2009 Apr 18.
Peptide-based defenses of ranid frogs from Mexico and Central America have been studied in much less detail than those from North America. Peptides belonging to the brevinin-1 (5 peptides), palustrin-2 (1 peptide), and ranatuerin-2 (3 peptides) families were isolated from norepinephrine-stimulated skin secretions of the Costa Rican frog, Lithobates vaillanti (Ranidae) and characterized structurally. Brevinin-1VLa (FLGAIAGVAAKFLPKVFCFITKKC) and brevinin-1VLc (FLPVIASVAAKVLPK VFCFITKKC) showed particularly high growth-inhibitory potency (MIC < or =3 microM) against a Gram-positive microorganism Staphylococcus aureus and the opportunistic yeast pathogen Candida albicans and potent cytolytic activity (LC(50)< or =8 microM) against both human erythrocytes and HepG2 hepatoma-derived cells. The peptides were also active against a Gram-negative microorganism Escherichia coli (MIC< or =50 microM). Substitutions in brevinin-1VLd (Lys(11) --> Asn) and brevinin-1VLe (Lys(11) --> Ser) that decrease cationicity result in loss of activity against E. coli. Ranatuerin-2VLb (GIMDTIKGAAKDLAGQLLDKLKCKITKC) showed relatively weak antimicrobial activity (MIC> or =75 microM) but selective cytolytic activity against HepG2 tumor cells (LC(50)=30 microM) compared with erythrocytes (LC(50)>200 microM). In addition, a dodecapeptide (RICYAMWIPYPC) were isolated from the secretions that were devoid of antimicrobial activity. This component contains an Ala-Met bond that constitutes the scissile bond in the selective elastase inhibitor, elafin but the peptide did not inhibit pancreatic elastase at concentrations up to 100 microM.
2. Antimicrobial peptides from the skin secretions of the South-East Asian frog Hylarana erythraea (Ranidae)
Nadia Al-Ghaferi, Jolanta Kolodziejek, Norbert Nowotny, Laurent Coquet, Thierry Jouenne, Jérôme Leprince, Hubert Vaudry, Jay D King, J Michael Conlon Peptides. 2010 Apr;31(4):548-54. doi: 10.1016/j.peptides.2009.12.013. Epub 2009 Dec 14.
Peptidomic analysis of norepinephrine-stimulated skin secretions of the South-East Asian frog Hylarana erythraea (formerly Rana erythraea partim) has led to the identification of multiple peptides with antimicrobial activity. Structural characterization of the peptides demonstrated that they belong to the brevinin-1 (3), brevinin-2 (2), esculentin-2 (4), and temporin (1) families. The values in parentheses indicate the number of paralogs. In addition, a peptide (GVIKSVLKGVAKTVALG ML.NH(2)) was isolated that shows some structural similarity to the brevinin-2-related peptides (B2RP) previously isolated from North American frogs of the genus Lithobates. A synthetic replicate of the species B2RP showed broad-spectrum growth inhibitory activity against reference strains of Escherichia coli (MIC=12.5 microM), Staphylococcus aureus (MIC=12.5 microM) and Candida albicans (MIC=50 microM) and was active against multidrug-resistant clinical isolates of Acetinobacter baumannii (MIC in the range 6-12.5 microM). The hemolytic activity of the peptide was relatively low (LC(50)=280 microM). Phylogenetic analysis based upon the amino acid sequences of 47 brevinin-2 peptides from 17 Asian species belonging to the family Ranidae provides support for the placement of H. erythraea in the genus Hylarana.
3. Primary structures of skin antimicrobial peptides indicate a close, but not conspecific, phylogenetic relationship between the leopard frogs Lithobates onca and Lithobates yavapaiensis (Ranidae)
J Michael Conlon, Laurent Coquet, Jérôme Leprince, Thierry Jouenne, Hubert Vaudry, Jay D King Comp Biochem Physiol C Toxicol Pharmacol. 2010 Apr;151(3):313-7. doi: 10.1016/j.cbpc.2009.12.004. Epub 2009 Dec 31.
The phylogenetic relationship between the relict leopard frog Lithobates (Rana) onca (Cope, 1875) and the lowland leopard frog Lithobates (Rana) yavapaiensis (Platz and Frost, 1984) is unclear. Chromatographic analysis of norepinephrine-stimulated skin secretions from L. onca led to the identification of six peptides with antimicrobial activity. Determination of their primary structures indicated that four of the peptides were identical to brevinin-1Ya, brevinin-1Yb, brevinin-1Yc and ranatuerin-2Ya previously isolated from skin secretions of L. yavapaiensis. However, a peptide belonging to the temporin family (temporin-ONa: FLPTFGKILSGLF.NH(2)) and an atypical member of the ranatuerin-2 family containing a C-terminal cyclic heptapeptide domain (ranatuerin-2ONa: GLMDTVKNAAKNLAGQMLDKLKCKITGSC) were isolated from the L. onca secretions but were not present in the L. yavapaiensis secretions. Ranatuerin-2ONa inhibited the growth of Escherichia coli (MIC=50muM) and Candida albicans (MIC=100muM ) and showed hemolytic activity (LC(50)=90muM) but was inactive against Staphylococcus aureus. The data indicate a close phylogenetic relationship between L. onca and L. yavapaiensis but suggest that they are not conspecific species.
