Baumann Andre, Skaljac Marisa, Lehmann Rüdiger, Vilcinskas Andreas, Franta Zdenӗk
Department of Bioresources, Fraunhofer Institute for Molecular Biology and Applied Ecology, Winchesterstraße 2, 35394 Giessen, Germany.
Department of Bioresources, Fraunhofer Institute for Molecular Biology and Applied Ecology, Winchesterstraße 2, 35394 Giessen, Germany; Justus-Liebig-University of Giessen, Institute for Insect Biotechnology, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany.
Insect Biochem Mol Biol. 2017 Apr;83:44-53. doi: 10.1016/j.ibmb.2017.02.007. Epub 2017 Feb 22.
Lucilia sericata maggots are the only species currently approved for maggot debridement therapy (MDT), an alternative treatment for chronic and recalcitrant wounds. Maggots promote wound debridement, disinfection and healing by producing a complex mixture of proteins, peptides and low-molecular-weight compounds in their secretions and excretions, but the individual components are not well characterized at the molecular level. Here we investigated the purine catabolism pathway in L. sericata, focusing on the production of allantoin by Urate Oxidase (UO), which is thought to promote wound healing. We produced recombinant L. sericata UO in Escherichia coli, and characterized the properties of the pure enzyme in terms of the optimum pH (7-10) and temperature (20-25 °C), its stability, sensitivity to inhibition and ion dependency. We used quantitative RT-PCR and RNA in situ hybridization to monitor the expression of the UO gene, and we used a guinea pig anti-UO antibody to detect the native enzyme by western blot and by florescence immunohistochemistry in larval tissues. We found that L. sericata UO is exclusively present in the larval excretion organ (the Malpighian tubes) and is freely available in the cytoplasm rather than restricted to a specific subcellular compartment. Allantoin is a final product of L. sericata purine catabolism. It is produced by UO in the Malpighian tubes to remove uric acid from the hemolymph and is consequently excreted via the hindgut. Our findings confirm the hypothesis that both actively secreted molecules and excretion products contribute to the beneficial effects of MDT.
丝光绿蝇蛆是目前唯一被批准用于蛆虫清创疗法(MDT)的物种,这是一种治疗慢性和顽固性伤口的替代疗法。蛆虫通过在其分泌物和排泄物中产生蛋白质、肽和低分子量化合物的复杂混合物来促进伤口清创、消毒和愈合,但这些成分在分子水平上尚未得到很好的表征。在这里,我们研究了丝光绿蝇的嘌呤分解代谢途径,重点关注尿酸氧化酶(UO)产生尿囊素的过程,尿囊素被认为可以促进伤口愈合。我们在大肠杆菌中生产了重组丝光绿蝇UO,并从最佳pH值(7-10)和温度(20-25°C)、稳定性、抑制敏感性和离子依赖性等方面对纯酶的特性进行了表征。我们使用定量RT-PCR和RNA原位杂交来监测UO基因的表达,并使用豚鼠抗UO抗体通过蛋白质印迹法和荧光免疫组织化学法在幼虫组织中检测天然酶。我们发现丝光绿蝇UO仅存在于幼虫排泄器官(马氏管)中,并且在细胞质中是游离的,而不是局限于特定的亚细胞区室。尿囊素是丝光绿蝇嘌呤分解代谢的最终产物。它由马氏管中的UO产生,以从血淋巴中去除尿酸,随后通过后肠排出。我们的研究结果证实了这样一个假设,即主动分泌的分子和排泄产物都有助于MDT的有益效果。