title: Characterization and regulation of expression of an antifungal peptide from hemolymph of an insect, Manduca sexta.
abstract: Insects secrete antimicrobial peptides as part of the innate immune response. Most antimicrobial peptides from insects have antibacterial but not antifungal activity. We have characterized an antifungal peptide, diapausin-1 from hemolymph of a lepidopteran insect, Manduca sexta (tobacco hornworm). Diapausin-1 was isolated by size exclusion chromatography from hemolymph plasma of larvae that were previously injected with a yeast, Saccharomyces cerevisiae. Fractions containing activity against S. cerevisiae were analyzed by SDS-PAGE and MALDI-TOF MS/MS and found to contain a 45-residue peptide that was encoded by sequences identified in M. sexta transcriptome and genome databases. A cDNA for diapausin-1 was cloned from cDNA prepared from fat body RNA. Diapausin-1 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 diapausin-1, each with 6 conserved Cys residues. Diapausin-1 was produced as a recombinant protein in Escherichia coli. Purified recombinant diapausin-1 was active against S. cerevisiae, with IC50 of 12 μM, but had no detectable activity against bacteria. Spores of some plant fungal pathogens treated with diapausin-1 had curled germination tubes or reduced and branched hyphal growth. Diapausin-1 mRNA level in fat body strongly increased after larvae were injected with yeast or with Micrococcus luteus. In addition, diapausin-1 mRNA levels increased in midgut and fat body at the wandering larval stage prior to pupation, suggesting developmental regulation of the gene. Our results indicate that synthesis of diapausin-1 is part of an antifungal innate immune response to infection in M. sexta.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506, USA.
authors.forename: Qasim
authors.initials: Q
authors.lastname: Al Souhail
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506, USA.
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Hiromasa
authors.affiliation: LOEWE Center for Insect Biotechnology and Bioresources, Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Giessen Winchesterstrasse 2, 35394 Giessen, Germany.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Rahnamaeian
authors.affiliation: Department of Plant Pathology, Kansas State University, Manhattan, KS 66506, USA.
authors.forename: Martha C
authors.initials: MC
authors.lastname: Giraldo
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506, USA.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Takahashi
authors.affiliation: Department of Plant Pathology, Kansas State University, Manhattan, KS 66506, USA.
authors.forename: Barbara
authors.initials: B
authors.lastname: Valent
authors.affiliation: LOEWE Center for Insect Biotechnology and Bioresources, Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Giessen Winchesterstrasse 2, 35394 Giessen, Germany; Institute for Insect Biotechnology, Justus-Liebig-University of Giessen, Heinrich-Buff-Ring 26-32, 39592 Giessen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Vilcinskas
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506, USA. Electronic address: kanost@ksu.edu.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Kanost
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000378:metabolism*; D000946:Antigens, Fungal / Q000276:immunology; D003001:Cloning, Molecular; D005216:Fat Body / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D016908:Gram-Positive Bacterial Infections / Q000276:immunology*; D006458:Hemolymph / Q000378:metabolism; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D007814:Larva; D018402:Manduca / Q000276:immunology*; D013058:Mass Spectrometry; D016982:Micrococcus luteus / Q000276:immunology*; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000276:immunology*
chemical_list: D000935:Antifungal Agents; D000946:Antigens, Fungal; D019476:Insect Proteins; D010455:Peptides
keywords: Antifungal; Hemolymph; Immunity; Insect; Yeast