Le Clec'h Winka, Anderson Timothy J C, Chevalier Frédéric D
Department of Genetics, Texas Biomedical Research Institute, P.O. Box 760549, San Antonio, TX, 78245, USA.
Parasit Vectors. 2016 Jan 22;9:32. doi: 10.1186/s13071-016-1319-6.
Biomphalaria snails are the intermediate host of the blood fluke Schistosoma mansoni, which infect more than 67 million people in tropical areas. Phenoloxidase enzymes (POs), including tyrosinases, catecholases, and laccases, are known to play a role in the immune defenses of arthropods, but the PO activity present in Biomphalaria spp. hemolymph has not been characterized. This study was designed to characterize substrate specificity and reaction optima of PO activity in Biomphalaria spp. hemolymph as a starting point to understand the role of this important invertebrate enzyme activity in snail biology and snail-schistosome interactions.
We used spectrophotometric assays with 3 specific substrates (L-tyrosine for tyrosinase, L-DOPA for catecholase, and PPD for laccase) and diethylthiocarbarmate (DETC) as specific PO inhibitor to characterize PO activity in the hemolymph of uninfected snails from two Biomphalaria species, and to determine the impact of the parasite Schistosoma mansoni on the PO activity of its B. glabrata vector.
We identified laccase activity in hemolymph from uninfected B. glabrata and B. alexandrina. For both species, the activity was optimal at 45 °C and pH 8.5, and located in the plasma. The K m and V max of PO enzymes are 1.45 mM and 0.024 OD.min(-1) for B. glabrata, and 1.19 mM and 0.025 OD.min(-1) for B. alexandrina. When the snail vector is parasitized by S. mansoni, we observed a sharp reduction in laccase activity seven weeks after snail infection.
We employed a highly specific spectrophotometric assay using PPD substrate which allows accurate measurement of laccase activity in Biomphalaria spp. hemolymph. We also demonstrated a strong impact of the parasite S. mansoni on laccase activity in the snail host.
双脐螺是曼氏血吸虫的中间宿主，在热带地区，该寄生虫感染人数超过6700万。酚氧化酶（POs），包括酪氨酸酶、儿茶酚酶和漆酶，已知在节肢动物的免疫防御中发挥作用，但双脐螺血淋巴中的PO活性尚未得到表征。本研究旨在表征双脐螺血淋巴中PO活性的底物特异性和反应最适条件，以此为起点，了解这种重要的无脊椎动物酶活性在蜗牛生物学以及蜗牛-血吸虫相互作用中的作用。
我们使用分光光度法，以3种特定底物（酪氨酸用于酪氨酸酶、L-多巴用于儿茶酚酶、对苯二酚用于漆酶）和二乙氨基二硫代甲酸盐（DETC）作为特定的PO抑制剂，来表征两种双脐螺未感染蜗牛血淋巴中的PO活性，并确定寄生虫曼氏血吸虫对其光滑双脐螺载体的PO活性的影响。
我们在未感染的光滑双脐螺和亚历山大双脐螺的血淋巴中鉴定出漆酶活性。对于这两个物种，该活性在45℃和pH 8.5时最佳，且存在于血浆中。光滑双脐螺PO酶的K m和V max分别为1.45 mM和0.024 OD.min(-1)，亚历山大双脐螺的分别为1.19 mM和0.025 OD.min(-1)。当蜗牛载体被曼氏血吸虫寄生时，我们观察到蜗牛感染7周后漆酶活性急剧下降。
我们采用了一种使用对苯二酚底物的高特异性分光光度法，该方法能够准确测量双脐螺血淋巴中的漆酶活性。我们还证明了寄生虫曼氏血吸虫对蜗牛宿主中漆酶活性有强烈影响。
