Moricin-like peptide A

An antibacterial peptide was isolated from a lepidopteran insect, Spodoptera litura. The molecular mass of this peptide was determined to be 4489.55 by matrix assisted laser desorption/ionization-time of flight mass (MALDI-TOF MS) spectrometry. The peptide consists of 42 amino acids and the sequence has 69-98% identity to those of moricin-related peptides, antibacterial peptides from lepidopetran insects. Thus, the peptide was designated S. litura (Sl) moricin. Sl moricin showed a broad antibacterial spectrum against Gram-positive and negative bacteria. Sl moricin gene was inducible by bacterial injection and expressed tissue-specifically in the fat body and hemocytes. Furthermore, the solution structure of Sl moricin was determined by two-dimensional (2D) 1H-nuclear magnetic resonance (NMR) spectroscopy and hybrid distance geometry-simulated annealing calculation. The tertiary structure revealed a long alpha-helix containing eight turns along nearly the full length of the peptide like that of moricin, confirming that Sl moricin is a new moricin-like antibacterial peptide. These results suggest that moricin is present not only in B. mori but also in other lepidopteran insects forming a gene family.

Screening for components with antifungal activity in the hemolymph of immune-stimulated Galleria mellonella larvae led to the identification of four novel moricin-like peptides (A, B, C3 and D). Subsequently, eight moricin-like peptide genes (A, B, C1-5 and D) were isolated and shown to code for seven unique peptides (mature C4 and C5 are identical). These genes contained single introns which varied from 180 to 1090bp. The moricin-like peptides were particularly active against filamentous fungi, preventing the growth of Fusarium graminearum at 3 microg/ml, and were also active against yeasts, gram positive bacteria and gram negative bacteria. Searches of the databases identified 30 moricin-like peptide genes which code for 23 unique mature peptides, all belonging to the Lepidoptera (moths and butterflies). The first comprehensive phylogenetic analysis of the moricin-like peptides suggested that they fall into two basic classes which diverged a long time ago. The peptides have since diversified extensively through a high level of gene duplication within species, as seen in G. mellonella and Bombyx mori. The restriction of moricin-like peptides to the Lepidoptera combined with their potent antifungal activity suggests that this diverse peptide family may play a role in the defence response of moths and butterflies.

The complete antimicrobial peptide repertoire of Galleria mellonella was investigated for the first time by LC/MS. Combining data from separate trypsin, Glu-C and Asp-N digests of immune hemolymph allowed detection of 18 known or putative G. mellonella antimicrobial peptides or proteins, namely lysozyme, moricin-like peptides (5), cecropins (2), gloverin, Gm proline-rich peptide 1, Gm proline-rich peptide 2, Gm anionic peptide 1 (P1-like), Gm anionic peptide 2, galiomicin, gallerimycin, inducible serine protease inhibitor 2, 6tox and heliocin-like peptide. Six of these were previously known only as nucleotide sequences, so this study provides the first evidence for expression of these genes. LC/MS data also provided insight into the expression and processing of the antimicrobial Gm proline-rich peptide 1. The gene for this peptide was isolated and shown to be unique to moths and to have an unusually long precursor region (495 bp). The precursor region contained other proline-rich peptides and LC/MS data suggested that these were being specifically processed and were present in hemolymph at very high levels. This study shows that G. mellonella can concurrently release an impressive array of at least 18 known or putative antimicrobial peptides from 10 families to defend itself against invading microbes.