Characterization of hemolymph phenoloxidase activity in two Biomphalaria snail species and impact of Schistosoma mansoni infection.
Characterization of hemolymph phenoloxidase activity in two Biomphalaria snail species and impact of Schistosoma mansoni infection.
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DOI：
10.1186/s13071-016-1319-6
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发表时间：
2016-01-22
影响因子：
3.2
通讯作者：
Chevalier FD
中科院分区：
文献类型：
--
作者：
Le Clec'h W;Anderson TJ;Chevalier FD
生物钉螺是曼氏血吸虫的中间宿主，在热带地区有超过6700万人感染这种血吸虫。酚氧化酶(POS)包括酪氨酸酶、儿茶酚酶和漆酶，在节肢动物的免疫防御中发挥作用，但在Biomphalaria spp.中存在PO活性。血淋巴的特征还没有确定。本研究旨在研究Biomphalaria spp.中PO活性的底物专一性和最适反应条件。以血淋巴为起点，了解这种重要的无脊椎动物酶活性在钉螺生物学和钉螺-血吸虫相互作用中的作用。用L酪氨酸底物、L多巴底物和PPD底物(漆酶底物)和二乙基硫代氨基甲酸酯(DETC)作为专一性PO抑制剂，用分光光度法测定了两种生物软体未感染钉螺血淋巴中PO活性，并研究了曼氏血吸虫寄生虫对其光面芽孢杆菌载体PO活性的影响。我们鉴定了未感染光滑假单胞菌和亚历山大假单胞菌的血淋巴中的漆酶活性。两种酶的最适温度为45℃，最适pH为8.5，且均位于血浆中。PO酶的Km和Vmax分别为1.45OmM和0.024 OD.min-1，亚历山大藻为1.19OmM和0.025 OD.min-1。当钉螺媒介被曼氏链球菌寄生时，我们观察到在钉螺感染7周后漆酶活性急剧下降。我们采用了一种使用PPD底物的高特异性分光光度分析方法，该方法可以准确地测量Biomphalaria spp.血淋巴。我们还展示了寄生虫S.mansoni对钉螺宿主漆酶活性的强烈影响。本文的在线版本(doi：10.1186/s13071-016-1319-6)包含补充材料，授权用户可以使用。
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 Km and Vmax 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. The online version of this article (doi:10.1186/s13071-016-1319-6) contains supplementary material, which is available to authorized users.