Characterization, regulation and biophysical studies of immune-related peptides from Manduca sexta
Year: 2016
Type: dissertation
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.Biophysical analysis showed that diapausin-1 binds to the β-1,3 glucan component of the S. cerevisiae cell wall.A second insect peptide investigated in this project was M.sexta stress-response peptide 1(SRP1), an immune-related peptide upregulated under different stress conditions including immune-challenge.Preliminary results for NMR structure determination are presented.Most of the amino acid residue spin systems were assigned, and we determined the connectivities of many amino residues as a first step to solve the NMR structure.The circular dichroism spectrum of SRP1 indicates that the peptide lacks alpha-helical structure and may contain beta strands and turns.
Authors: Al souhail, Qasim Mohammed
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2411062967