Structural and Biological Characterization of Antimicrobial Peptides from Rana Sylvatica and Kassina Senegalensis

Gene-encoded antimicrobial peptides are an important component of host defense in both invertebrates and vertebrates, ranging from insects to mammals. Although these peptides are usually isolated in high concentrations, they possess varying potencies against microorganisms. In this study, three peptides were isolated from two different frog families, eight peptide analogs were synthesized to improve antimicrobial activity, and two peptides were co-incubated for synergistic effects. Two peptides, termed kassinatuerins 1 and 2, from the skin of the African running frog, Kassina senegalensis, and one peptide, termed brevinin 1SY (SY for sylvatica), from the skin of the wood frog, Rana sylvatica, have been isolated in pure form and characterized both structurally and biologically. Of these three peptides, kassinatuerins 1 and 2 contain a C-terminally a-amidated residue and fall into a class of antimicrobial peptides that has not been identified before. Both kassinatuerin 1 (Gly-Phe-Met-Lys-Tyr-Ile-Gly-Pro-Leu-Ile10- Pro-His-Ala-Val-Lys-Ala-IIe-Ser-Asp-Leu20-Ile.NH2) and kassinatuerin 2 (Phe-Ile-Gln- Tyr-Leu-AIa-Pro-Leu-Ile-Prol0-His-Ala-Val-Lys-Ala-Ile-Ser-Asp-Leu-Ile20.NH2) were isolated in high yield, 75 nmol/g tissue and 96 nmol/g tissue, respectively.|Kassinatuerin 1 showed antimicrobial activity against the gram-positive bacterium, Staphylococcus aureus, the gram-negative bacterium, Escherichia coli and the yeast, Candida albicans whereas kassinatuerin 2 did not. Additionally, kassinatuerin 1 hemolyzed (concentration of peptide producing 50% hemolysis, HC50 = 54 pM) human red blood cells. Brevinin 1SY (Phe-Leu-Pro-Val-Val-Ala-Gly-Leu-Ala-Alalü-Lys-Val- Leu-Pro-Ser-lle-lle-Cys-Ala-Val20-Thr-Lys-Lys-Cys) contains an intramolecular disulfide bridge that forms a heptapeptide ring. In this study, brevinin 1SY synthesis was induced when the animal was in an environment that promoted the growth of microorganisms consistent with a role in the animal’s defense strategy. This peptide is similar to brevinins previously isolated from Rana breviptxiaporsa, Rana esculenta, and Rana sphenocephala. It was the only detectable antimicrobial peptide in the skin of R. sylvatica and inhibited growth of S. aureus (minimum inhibitory concentration, MIC = 7 |iM) and E. coli (MIC = 45 }iM).|The eight kassinatuerin 1 analogs synthesized in this study were designed to increase antimicrobial activity and decrease hemolytic activity. Kassinatuerin 1 inhibited growth of S. aureus (MIC = 8 (iM), E. coli (MIC = 4 uM), and C. albicans (MIC = 70 |iM). Kassinatuerin 1 analogs containing a carboxyl terminus instead of the C-terminally a-amidated residue displayed decreased potency and hemolytic activity.|The MICs of these kassinatuerin 1 analogs paired with secondary structure predictions for kassinatuerin 1 supports increased cationicity in hydrophilic regions of the peptide increases antimicrobial activity while maintaining the peptide’s a-helicity. At the same time, hemolytic activity of the eight kassinatuerin 1 analogs increased as the cationicity in hydrophilic regions of the peptide increased.|Kassinatuerin 2 lacks antimicrobial activity up to 225 p.M, but when co-incubated with kassinatuerin 1, the MICs of kassinatuerin 1 display up to 800-fold and up to 4-fold less than kassinatuerin 1 alone against S. aureus and E. coli, respectively. The speculated mechanism of action for these peptides is that kassinatuerin 1 facilitates binding to the bacterial membrane and kassinatuerin 2 enhances the permeability of the membrane.

Grantor dc:publisher

Creighton University

Year dc:date.issued

2005

Author dc:creator

• Matutte, Beverly

Advisor dc:contributor.advisor

• Smith, David

Statement dc:rights

• Copyright is retained by the Author. A non-exclusive distribution right is granted to Creighton University and to ProQuest following the publishing model selected above.

Language dc:language.iso

en_US

Handle dc:identifier.uri

http://hdl.handle.net/10504/55718

OAI identifier oai:identifier

oai:cdr.creighton.edu:10504/55718