Identification of an antifungal peptide from hemolymph of an insect, Manduca sexta (976.4)

Abstract

Insects secrete antimicrobial peptides as part of the innate immunity response. These peptides are often cationic, low molecular peptides with diverse structures. Most antimicrobial peptides from insects have antibacterial but not antifungal activity. We have characterized a newly identified peptide, antifungal peptide‐1 (AFP1) from hemolymph of a lepidopteran insect, Manduca sexta (tobacco hornworm). AFP1 was isolated by size exclusion chromatography from hemolymph plasma of larvae. Fractions containing activity against the yeast, Saccharomyces cerevisiae were analyzed my SDA‐PAGE and MALDI‐TOF MS/MS and found to contain a 41 residue peptide that was encoded by sequences identified in M. sexta transcriptome and genome data bases. AFP1 is a member of the diapausin family of peptides, which includes members known to have antifungal activity. The M. sexta genome contains 14 genes with high similarity to AFP1, each with 6 conserved Cys residues. A cDNA for AFP1 was cloned from cDNA prepared from fat body RNA. AFP1 was produced as a recombinant protein in Escherichia coli. Purified natural and recombinant AFP1 were active against S. cerevisiae, with IC50 of 15 micromolar, but they had no detectable activity against bacteria. Experiments to investigate activity of AFP1 against other fungi are ongoing. AFP1 mRNA level strongly increased after larvae were injected with yeast or with Micrococcus luteus. Our results indicate that synthesis of AFP1 is part of the innate immune response to infection in M. sexta.Grant Funding Source: Supported by NIH grant GM41247.