Moricin-like peptide D

Background: Galleria mellonella larvae were infected with conidia of Aspergillus fumigatus and the cellular and humoral immune responses of larvae to the pathogen were characterized as invasive aspergillosis developed. Results: At 2 h post-infection there was an increase in hemocyte density to 7.43 ± 0.50 × 106/ml from 0.98 ± 0.08 × 106/ml at 0 h. Hemocytes from larvae immune primed for 6 h with heat killed A. fumigatus conidia displayed superior anti-fungal activity. Examination of the spread of the fungus by Cryo-imaging and fluorescent microscopy revealed dissemination of the fungus through the larvae by 6 h and the formation of distinct nodules in tissue. By 24 h a range of nodules were visible at the site of infection and at sites distant from that indicating invasion of tissue. Proteomic analysis of larvae infected with viable conidia for 6 h demonstrated an increase in the abundance of gustatory receptor candidate 25 (37 fold), gloverin-like protein (14 fold), cecropin-A (11 fold). At 24 h post-infection gustatory receptor candidate 25 (126 fold), moricin-like peptide D (33 fold) and muscle protein 20-like protein (12 fold) were increased in abundance. Proteins decreased in abundance included fibrohexamerin (13 fold) and dimeric dihydrodiol dehydrogenase (8 fold). Conclusion: The results presented here indicate that G. mellonella larvae may be a convenient model for studying the stages in the development of invasive aspergillosis and may offer an insight into this process in mammals.

The work presents identification of antimicrobial peptides and proteins (AMPs) in the hemolymph of Galleria mellonella larvae infected with two Pseudomonas aeruginosa strains (ATCC 27,853 and PA18), differing in the profile of secreted proteases. The insects were immunized with bacteria cultivated in rich (LB) and minimal (M9) media, which resulted in appearance of a similar broad set of AMPs in the hemolymph. Among them, 13 peptides and proteins were identified, i.e. proline-rich peptides 1 and 2, lebocin-like anionic peptide 1 and anionic peptide 2, defensin/galiomicin, cecropin, cecropin D-like peptide, apolipophoricin, gallerimycin, moricin-like peptide B, lysozyme, apolipophorin III, and superoxide dismutase. Bacterial strain- and/or medium-dependent changes in the level of proline-rich peptide 1, anionic peptide 1 and 2, moricin-like peptide B, cecropin D-like and gallerimycin were observed. The analysis of the expression of genes encoding cecropin, gallerimycin, and galiomicin indicated that they were differently affected by the bacterial strain but mainly by the medium used for bacterial culture. The highest expression was found for the LB medium. In addition to the antibacterial and antifungal activity, proteolytic activity was detected in the hemolymph of the P. aeruginosa-infected insects. Based on these results and those presented in our previous reports, it can be postulated that the appearance of AMPs in G. mellonella hemolymph can be triggered not only by P. aeruginosa pathogen associated molecular patterns (PAMPs) but also by bacterial extracellular proteases secreted during infection. However, although there were no qualitative differences in the set of AMPs depending on the P. aeruginosa strain and medium, differences in the level of particular AMPs synthesized in response to the bacteria used were observed.

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.