Identification, Characterization, Immunolocalization, and Biological Activity of Lucilin Peptide.

Maggots from the Lucilia sp. genus are used for debridement of infected and necrotic wounds. Broad-spectrum antimicrobial activity has been described in the excretion/secretions (ES1) of these larvae. This study identifies the genetic sequence of a cecropin-like antimicrobial peptide from Lucilia eximia. Total RNA was extracted and used for PCR-RACE amplification of a cecropin, the native peptide was immunolocalized in the tissues and secretions of the larvae, and a synthetic analog was used to explore its antimicrobial, cytotoxic, LPS neutralizing and wound-healing activities in vitro. The genetic cDNA sequence of a cecropin-like antimicrobial peptide in L. eximia called "Lucilin" was amplified, corresponding to 63 aa completed protein and 40 aa mature peptide; the structure of the mature peptide was predicted as an α-helix. The peptide was immunolocalized in the salivary glands, fat body, the ES, and hemolymph of the maggots. Lucilin synthetic peptide analog was active against E. coli DH10B with a MIC2 of 7.8 µg/mL, E. coli extended spectrum b-lactamase (ESBL) (MIC: 15.6 µg/mL), and Enterobacter cloacae (MIC: 125 µg/mL), but it was not active against Pseudomonas aeruginosa and Staphylococcus epidermidis; and had no cytotoxic or hemolytic activity. It showed immunomodulatory activity against human peripheral blood mononuclear cells (PBMCs) stimulated with LPS, reducing the TNF-α production when treated at 17 µg/mL and induces cell migration of Hacat at 5 and 50 µg/mL. Lucilin is a cecropin-like peptide from L. eximia with antimicrobial activity against Gram negative bacteria and immunomodulatory activities, decreasing the TNF-α production in PBMCs and inducing cellular migration in human keratinocytes.

Distribution and characteristics of ABFs, cecropins, nemapores, and lysozymes in nematodes.

Several groups of antimicrobial effector molecules have been identified in nematodes, but most studies have been limited to Caenorhabditis elegans and, to a lesser extent, Ascaris suum. Although these two species are not closely related, they are not representative of overall nematode diversity. This study utilized available sequence information to investigate whether four groups of antimicrobial effectors (defensin-like antibacterial factors [ABFs], cecropins, saposin domain-containing proteins, and lysozymes) are components of an archetypal nematode immune system or more narrowly restricted. Saposin domain-containing proteins (caenopores in C. elegans) and lysozymes were widely distributed and found in most taxa, but likely have digestive as well as defensive functions. ABFs were widely distributed in fewer taxa, suggesting selective loss in some lineages. In contrast, cecropins were identified in only three related species, suggesting acquisition of this effector molecule in their common ancestor.

[Study on the interaction mechanism of antimicrobial peptide Cecropin-XJ in Xinjiang silkworm and Staphylococcus aureus DNA by spectra].

Nowadays many of antimicrobial peptides have been extensively studied in order to elucidate their antimicrobial mode of action. Much of the research focused on mechanisms of cytoplasmic membrane disruption has been proposed for antimicrobial peptides, but it is not known whether their antimicrobial mode of action is due to their effects on bacterial chromosome. To obtain more information about the possible mechanisms, Cecropin-XJ, a kind of antimicrobial peptide from Xinjiang silkworm, was used as subject and prepared by purification of Pichia yeast fermentation containing cecropin-XJ gene expression product. Subsequently, ultraviolet absorption spectra were employed to investigate whether this antibacterial function is due to Cecropin-XJ works on S. aureus DNA in vitro. The increase in absorbance of DNA samples at 260nm due to the addition of Cecropin-XJ was measured. It is called hyperchromicity of DNA, which can provide a direct measure of the degree of base-pair unstacking. The unstacking results in the loss of duplex helix, and then leads to the duplex helix becoming relaxing. At the same time, the interaction mode was studied by using ethidium bromide (EB) as an extrinsic fluorescence probe. With the addition of Cecropin-XJ, the intensity of intrinsic fluorescence absorbance of DNA at 307 nm increases greatly. It is suggested that the addition of Cecropin-XJ may unstack base-paire of DNA exposing the fluorescent amino acide, leading to the enhancing of DNA fluorescence intensity. Subsequently the competition between Cecropin-XJ and EB to combine with DNA was found. It is suggested that the style was groove binding and intercalation in the interaction of Cecropin-XJ and double helix DNA. Furthermore, in this study, the binding constant and binding number of Cecropin-XJ complex with DNA were determined. There were different binding constant and number of EB complex with DNA because of the the addition of Cecropin-XJ. It is showed that the interaction of Cecropin-XJ and DNA was based on intercalation or non-intercalation. Meanwhile, these results help explain the molecule mechanism of antimicrobial peptide from the interaction style and structure characteristic of Cecropin-XJ and S. aureus DNA. These findings could contribute to further investigation on the mechanism of action of Cecropin-XJ.