Erdoğan Emrah, Karaca Serkan
Erciyes University Faculty of Medicine, Department of Medical Parasitology, Kayseri, Turkey.
Mikrobiyol Bul. 2021 Jan;55(1):81-90. doi: 10.5578/mb.20175.
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.
丝光绿蝇是丽蝇科最常见的物种之一,大量存在于粪便、垃圾和尸体周围。这种苍蝇在医学、法医学和兽医学中都很重要。该寄生虫的幼虫在兽医学和防治动物疾病方面都很重要,因为它们会给动物生产造成重大损失。由于它们是最早在尸体上定居的蝇类群体之一,所以在法医学领域也可用于确定死亡时间。丝光绿蝇幼虫用于蛆虫清创疗法(MDT),这是一种利用蝇幼虫的治疗方法,通过破坏伤口中的坏死组织和传染源来促进伤口愈合。幼虫通过分泌蛋白质来保护自己免受多种微生物菌群的侵害;与此同时,它们分泌的这些分子对伤口愈合也有有趣的促进作用。近年来发现的最重要的分子之一是具有抗真菌作用的丝光霉素。此外,由于其抗菌作用,尤其是对耐药革兰氏阴性菌和阳性菌的作用,近年来丝光绿蝇防御素和胰凝乳蛋白酶的分泌也变得很重要。需要发现可用于治疗不愈合伤口或可与现有抗生素联合应用的替代分子。有必要研究蛆虫疗法中涉及的化合物的抗菌特性及其作用机制。本研究的目的是克隆、分子表征和分析用于蛆虫清创疗法的丝光绿蝇幼虫分泌的重要防御素分子丝光绿蝇防御素和胰凝乳蛋白酶基因的抗原结构。首先,进行了用于分子研究的丝光绿蝇群体的培养。随后,从幼虫中进行RNA分离和cDNA合成。通过克隆反应将丝光绿蝇防御素和胰凝乳蛋白酶基因分别插入pJet1.2质粒。在所有步骤中,通过PCR筛选和DNA序列分析方法确认重组质粒的存在。对这两个在伤口治疗中重要的幼虫成分基因进行了基于核苷酸和氨基酸的分子表征。获得了蛋白质的抗原区域和三维结构。丝光绿蝇丝光绿蝇防御素基因的分离株编号MT495795和胰凝乳蛋白酶基因的分离株编号MT495794已提交至GenBank。这一在土耳其共和国首次报道的数据将为文献做出贡献。从20世纪初到抗生素被发现之前,蛆虫清创疗法尤其应用于士兵,但在抗生素被发现后没有得到太多应用领域。耐药性是当今伤口治疗中遇到的最重要问题,这导致了蛆虫清创疗法及其作用机制的重新受到关注。在本研究中获得的数据将为不同领域的科学家在伤口治疗中重要分子的发现和适用性方面的多学科研究提供一个来源。
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