RNA interference targeting cathepsin L and Z-like cysteine proteases of Onchocerca volvulus confirmed their essential function during L3 molting
RNA interference targeting cathepsin L and Z-like cysteine proteases of Onchocerca volvulus confirmed their essential function during L3 molting
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DOI：
10.1016/j.molbiopara.2004.08.003
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发表时间：
2004-12-01
影响因子：
1.5
通讯作者：
Hashmi, S
中科院分区：
文献类型：
--
作者：
Lustigmana, S;Zhang, J;Hashmi, S
我们描述了成功地使用RNA干扰(RNAi)来研究人类丝虫寄生虫螺旋体第三期幼虫(L3)的基因功能。我们针对两个特异的基因产物，组织蛋白L(Ov-cpl)和组织蛋白酶Z-样(Ov-cpZ)半胱氨酸蛋白酶，被认为在草鱼L3蜕皮过程中起作用。我们发现与编码成熟酶的Cp1或CPZ区域相对应的荧光标记的Cy(TM)3-dsRNA可以进入幼虫。0.5 mg·ml~(-1)Cpl过夜处理后，幼虫的蜕皮率分别比正常对照幼虫降低92%和86%。结果表明，虽然幼虫在蜕皮的最后阶段开始蜕皮，但蜕皮受到抑制。这种影响是基因特异性的，因为在Cpl或CPZ dsRNA存在下不蜕皮的幼虫分别表达另一种半胱氨酸蛋白酶CPZ和CPL。使用针对每种酶的抗体的免疫电子显微镜证实了这一点。我们的研究最终证实了这两种酶对卷叶绿僵菌L3到第四期幼虫的蜕皮是必不可少的。我们还证实，酶的活性是特定于蜕皮过程中发生的变化的，在蜕皮过程中，1-3天，即角质层之间的分离正在进行。通过丝虫基因组计划鉴定的丰富的L3和蜕皮L3基因，其中许多虽然没有归属功能，但被确定为疫苗候选或潜在的药物靶标，RNAi在草鱼L3中的发展可能有助于进一步研究。(C)2004爱思唯尔B.V保留所有权利。
We describe the successful use of RNA interference (RNAi) to investigate gene function in the human filarial parasite Onchocerca volvulus third-stage larvae (L3). We targeted two specific gene products, the O. volvulus cathepsin L (Ov-CPL) and cathepsin Z-like (Ov-CPZ) cysteine proteases, which were proposed to function during O. volvulus L3 molting. We show that fluorescent-labeled Cy(TM)3-dsRNA corresponding to cpl or cpz regions encoding the mature enzymes can enter the larvae. The molting rate of larvae treated overnight with 0.5 mg ml(-1) cpl was reduced by 92% and 86% in comparison to normal control worms. It appeared that although the larvae started the molting process the last stage of molting, ecdysis was inhibited. The effect was gene specific, as larvae that did not molt in the presence of cpl or cpz dsRNA expressed the other cysteine protease, CPZ and CPL, respectively. This was confirmed by immunoelectron microscopy using antibodies directed against each enzyme. Our present study validate conclusively that both enzymes are essential for the molting of O. volvulus L3 to fourth-stage larvae. We also confirmed that the activity of the enzymes is specific to the changes that occur during the molting process on days 1-3, when the separation between the cuticles is in progress. The development of RNAi in O. volvulus L3 could further help study many of the abundant L3 and molting L3 genes identified through the filarial genome project, many of which, although have no attributed function, were identified as vaccine candidates or potential drug targets. (C) 2004 Elsevier B.V All rights reserved.