Lucifensin

Recently, we identified a new insect defensin, named lucifensin that is secreted/excreted by the blowfly Lucilia sericata larvae into a wound as a disinfectant during the medicinal process known as maggot therapy. Here, we report the total chemical synthesis of this peptide of 40 amino acid residues and three intramolecular disulfide bridges by using three different protocols. Oxidative folding of linear peptide yielded a peptide with a pattern of disulfide bridges identical to that of native lucifensin. The synthetic lucifensin was active against Gram-positive bacteria and was not hemolytic. We synthesized three lucifensin analogues that are cyclized through one native disulfide bridge in different positions and having the remaining four cysteines substituted by alanine. Only the analogue cyclized through a Cys16-Cys36 disulfide bridge showed weak antimicrobial activity. Truncating lucifensin at the N-terminal by ten amino acid residues resulted in a drop in antimicrobial activity. Linear lucifensin having all six cysteine residues alkylated was inactive. Circular dichroism spectra measured in the presence of α-helix-promoting compounds showed different patterns for lucifensin and its analogues. Transmission electron microscopy revealed that Bacillus subtilis treatment with lucifensin induced significant changes in its envelope.

Lucilia sericata, one of the most common species of the Calliphoridae family, is found in large numbers around droppings, garbage and carcasses. This fly species is important in medicine, forensics and veterinary medicine. The larvae of the parasite are important both in veterinary medicine and in combating of the animal diseases, as they cause significant losses in animal production. Since they are one of the first fly colonies to settle on corpses, they can also be used in determining the time of death in the field of forensic medicine. L.sericata larvae used in Maggot debridement treatment (MDT) which is a treatment method with fly larvae, help wound healing by destroying necrotic tissues and infectious agents in wounds. While the larvae protect themselves from polymicrobial flora with the proteins they secrete; at the same time, they make an interesting contribution to wound healing with these molecules secreted. One of the most important molecules discovered in recent years is lucimycin which has an antifungal effect. In addition, lucifensin and chymotrypsin secretions have gained importance in recent years due to their antibacterial effects and especially their effects on resistant gram-negative and positive bacteria. There is a need for the discovery of the molecules that can be alternative in the treatment of non-healing wounds or that can be applied together with existing antibiotics. It is necessary to investigate the antimicrobial characterization of the compounds involved in maggot therapy and their mechanisms. The aim of this study was to clone, molecular characterization and analysis of the antigenic structures of lucifensin and chymotrypsin genes, which are important defensin molecules secreted by L.sericata larvae used in MDT. Primarily, the cultivation of L.sericata colonies to be used in molecular studies were performed. Later, RNA isolation and cDNA synthesis from larvae were carried out. Lucifensin and chymotrypsin genes were individually inserted into the pJet1.2 plasmid by cloning reactions. The presence of the recombinant plasmid was confirmed by PCR screening and DNA sequence analysis methods in all steps. Nucleotide and amino acid based molecular characterizations of these two genes, which are important larval components in wound treatment, have been made. Antigenic regions and three-dimensional structures of the proteins were obtained. The isolate numbered MT495795 of the L.sericata lucifensin gene and the isolate numbered MT495794 of the chymotrypsin gene were registered to GenBank. This data reported for the first time in the Republic of Turkey will contribute to the literature. From the beginning of the 20th century until the discovery of the antibiotics, MDT was applied especially on soldiers but did not find much application area after the discovery of the antibiotics. Drug resistance, which is the most important problem encountered in the treatment of the wounds today, has led to the recall of MDT and its mechanism of action. In this study the data, obtained will constitute a source for the multidisciplinary studies of the scientists from different fields on the discovery and applicability of the important moleculesin the treatment of the wounds.

A novel homolog of insect defensin, designated lucifensin II (Lucilia cuprina Wiedemann [Diptera: Calliphoridae] defensin), was purified from hemolymph extract from larvae of the blowfly L. cuprina. The full-length primary sequence of this peptide of 40 amino acid residues and three intramolecular disulfide bridges was determined by electrospray ionization-orbitrap mass spectrometry and Edman degradation and is almost identical to the previously identified sequence of lucifensin (Lucilia sericata Meigen defensin). Lucifensin II sequence differs from that of lucifensin by only one amino acid residue, that is, by isoleucine instead of valine at position 11. The presence of lucifensin II also was detected in the extracts of other larval tissues, such as gut, salivary glands, fat body, and whole body extract.