Antimicrobial peptides expressed in medicinal maggots of the blow fly Lucilia sericata show combinatorial activity against bacteria.

The larvae of the common green bottle fly (Lucilia sericata) produce antibacterial secretions that have a therapeutic effect on chronic and nonhealing wounds. Recent developments in insect biotechnology have made it possible to use these larvae as a source of novel anti-infectives. Here, we report the application of next-generation RNA sequencing (RNA-Seq) to characterize the transcriptomes of the larval glands, crop, and gut, which contribute to the synthesis of antimicrobial peptides (AMPs) and proteins secreted into wounds. Our data confirm that L. sericata larvae have adapted in order to colonize microbially contaminated habitats, such as carrion and necrotic wounds, and are protected against infection by a diverse spectrum of AMPs. L. sericata AMPs include not only lucifensin and lucimycin but also novel attacins, cecropins, diptericins, proline-rich peptides, and sarcotoxins. We identified 47 genes encoding putative AMPs and produced 23 as synthetic analogs, among which some displayed activities against a broad spectrum of microbial pathogens, including Pseudomonas aeruginosa, Proteus vulgaris, and Enterococcus faecalis. Against Escherichia coli (Gram negative) and Micrococcus luteus (Gram positive), we found mostly additive effects but also synergistic activity when selected AMPs were tested in combination. The AMPs that are easy to synthesize are currently being produced in bulk to allow their evaluation as novel anti-infectives that can be formulated in hydrogels to produce therapeutic wound dressings and adhesive bandages.

Lucimycin, an antifungal peptide from the therapeutic maggot of the common green bottle fly Lucilia sericata.

We report the identification, cloning, heterologous expression and functional characterization of a novel antifungal peptide named lucimycin from the common green bottle fly Lucilia sericata. The lucimycin cDNA was isolated from a library of genes induced during the innate immune response in L. sericata larvae, which are used as therapeutic maggots. The peptide comprises 77 amino acid residues with a molecular mass of 8.2 kDa and a pI of 6.6. It is predicted to contain a zinc-binding motif and to form a random coil, lacking ß-sheets or other secondary structures. Lucimycin was active against fungi from the phyla Ascomycota, Basidiomycota and Zygomycota, in addition to the oomycete Phytophtora parasitica, but it was inactive against bacteria. A mutant version of lucimycin, lacking the four C-terminal amino acid residues, displayed 40-fold lower activity. The activity of lucimycin against a number of highly-destructive plant pathogens could be exploited to produce transgenic crops that are resistant against fungal diseases.

The therapeutic potential of the insect metalloproteinase inhibitor against infections caused by Pseudomonas aeruginosa.

OBJECTIVES: The objective of this study was to investigate the therapeutic potential of the insect metalloproteinase inhibitor (IMPI) from Galleria mellonella, the only known specific inhibitor of M4 metalloproteinases. METHODS: The fusion protein IMPI-GST (glutathione-S-transferase) was produced by fermentation in Escherichia coli and was tested for its ability to inhibit the proteolytic activity of the M4 metalloproteinases thermolysin and Pseudomonas elastase (PE), the latter a key virulence factor of the wound-associated and antibiotic-resistant pathogen Pseudomonas aeruginosa. We also tested the ability of IMPI to inhibit the secretome (Sec) of a P. aeruginosa strain obtained from a wound. KEY FINDINGS: We found that IMPI-GST inhibited thermolysin and PE in vitro and increased the viability of human keratinocytes exposed to Sec by inhibiting detachment caused by changes in cytoskeletal morphology. IMPI-GST also improved the cell migration rate in an in vitro wound assay and reduced the severity of necrosis caused by Sec in an ex vivo porcine wound model. CONCLUSIONS: The inhibition of virulence factors is a novel therapeutic approach against antibiotic resistant bacteria. Our results indicate that IMPI is a promising drug candidate for the treatment of P. aeruginosa infections.

A Jonah-like chymotrypsin from the therapeutic maggot Lucilia sericata plays a role in wound debridement and coagulation.

Lucilia sericata larvae are used in maggot debridement therapy, a traditional wound healing approach that has recently been approved for the treatment of chronic wounds. Maggot excretion products (MEP) contain many different proteases that promote disinfection, debridement and the acceleration of wound healing, e.g. by activating the host contact phase/intrinsic pathway of coagulation. In order to characterise relevant procoagulant proteases, we analysed MEP and identified a chymotrypsin-like serine protease with similarities to Jonah proteases from Drosophila melanogaster and a chymotrypsin from Lucilia cuprina. A recombinant form of the L. sericata Jonah chymotrypsin was produced in Escherichia coli. The activated enzyme (Jonahm) had a pH optimum of 8.0 and a temperature optimum of 37 °C, based on the cleavage of the chromogenic peptide s-7388 and casein. Jonahm reduced the clotting time of human plasma even in the absence of the endogenous protease kallikrein, factor XI or factor XII and digested the extracellular matrix proteins fibronectin, laminin and collagen IV, suggesting a potential mechanism of wound debridement. Based on these characteristics, the novel L. sericata chymotrypsin-like serine protease appears to be an ideal candidate for the development of topical drugs for wound healing applications.

Two c-type lysozymes boost the innate immune system of the invasive ladybird Harmonia axyridis.

The invasive ladybird beetle Harmonia axyridis has a two-layered immune system, featuring the constitutive production of the low-molecular-mass antimicrobial compound harmonine and the inducible production of a broad range of antimicrobial peptides (AMPs). Here we show that the immune system also features two c-type lysozymes, the acidic c-lys3 (pI = 5.46) and the basic c-lys4 (pI = 8.18). The injection of bacteria into H.axyridis boosted c-lys4 gene expression 8-fold in the gut, whereas the c-lys3 gene was expressed at comparable levels in both naïve and challenged beetles. Both c-lys3 and c-lys4 were expressed in Pichia pastoris and the bacteriolytic activity of the recombinant proteins was found to be calcium-dependent with pH maxima of 6.0 and 6.5, respectively. In a Bacillus subtilis growth inhibition assay, the antimicrobial activity of harmonine and two highly-inducible H.axyridis AMPs (coleoptericins) was potentiated in the presence of c-lys4 but not c-lys3, resulting in 4-fold (harmonine) and up to 16-fold (AMP) lower minimum inhibitory concentrations. Our results suggest that two structurally and functionally distinct lysozymes contribute to innate immune responses of H.axyridis and augment the harmonine and AMP components of the immune response.