Lebocin-1/2 precursor

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.

The innate immune response of insects includes induced expression of genes encoding a variety of antimicrobial peptides. The signaling pathways that stimulate this gene expression have been well characterized by genetic analysis in Drosophila melanogaster, but are not well understood in most other insect species. One such pathway involves proteolytic activation of a cytokine called Spätzle, which functions in dorsal-ventral patterning in early embryonic development and in the antimicrobial immune response in larvae and adults. We have investigated the function of Spätzle in a lepidopteran insect, Manduca sexta, in which hemolymph proteinases activated during immune responses have been characterized biochemically. Two cDNA isoforms for M. sexta Spätzle-1 differ because of alternative splicing, resulting in a 10 amino acid residue insertion in the pro-region of proSpätzle-1B that is not present in proSpätzle-1A. The proSpätzle-1A cDNA encodes a 32.7 kDa polypeptide that is 23% and 44% identical to D. melanogaster and Bombyx mori Spätzle-1, respectively. Recombinant proSpätzle-1A was a disulfide-linked homodimer. M. sexta hemolymph proteinase 8 cleaved proSpätzle-1A to release Spätzle-C108, a dimer of the C-terminal 108 residue cystine-knot domain. Injection of Spätzle-C108, but not proSpätzle-1A, into larvae stimulated expression of several antimicrobial peptides and proteins, including attacin-1, cecropin-6, moricin, lysozyme, and the immunoglobulin domain protein hemolin, but did not significantly affect the expression of two bacteria-inducible pattern recognition proteins, immulectin-2 and beta-1,3-glucan recognition protein-2. The results of this and other recent studies support a model for a pathway in which the clip-domain proteinase pro-hemolymph proteinase 6 becomes activated in plasma upon exposure to Gram-negative or Gram-positive bacteria or to beta-1,3-glucan. Hemolymph proteinase 6 then activates pro-hemolymph proteinase 8, which in turn activates Spätzle-1. The resulting Spätzle-C108 dimer is likely to function as a ligand to activate a Toll pathway in M. sexta as a response to a wide variety of microbial challenges, stimulating a broad response to infection. Structured digital abstract * MINT-7295125: Spätzle 1A (uniprotkb:C8BMD1) and Spätzle 1A (uniprotkb:C8BMD1) bind (MI:0407) by comigration in gel electrophoresis (MI:0807).

Insects produce a group of antimicrobial peptides (AMPs) in response to microbial infections. Most AMPs are synthesized as inactive precursors/pro-proteins and require proteolytic processing to generate small active peptides. Here we report identification and functional analysis of two lebocin-related proteins (Leb-B and Leb-C) from the tobacco hornworm, Manduca sexta. The mRNA levels of Leb-B and Leb-C increased significantly in larval fat body and hemocytes after injection of Escherichia coli, Micrococcus luteus and Saccharomyces cerevisiae. Western blotting using rabbit polyclonal antibody to Leb-B showed accumulation of large protein(s) and small peptide(s) in larval hemolymph after microbial injection. This result and the presence of RXXR motifs in the deduced amino acid sequences led to our postulation that Leb-B/C may be inactive precursors that are processed in larval hemolymph to generate short active peptides. To test this hypothesis, we expressed and purified full-length and various fragments of Leb-B and Leb-C as thioredoxin (TRX) fusion proteins. We found that fusion proteins could be cleaved by induced larval plasma, and the cleavage sites were determined by protein sequencing. Antibacterial activity of peptide fragments was also verified using synthetic peptides, and active M. sexta lebocin peptides were located at the N-termini of Leb-B/C, which are different from Bombyx mori lebocins 1-4 that are located close to the C-termini. In addition, we found that synthetic Leb-B(22-48) peptide not only had higher antibacterial activity but also caused agglutination of E. coli cells. Our results provide valuable information for studying processing of lebocin precursors in lepidopteran insects.