RNAi dynamics in Juvenile Fasciola spp. Liver flukes reveals the persistence of gene silencing in vitro.
RNAi dynamics in Juvenile Fasciola spp. Liver flukes reveals the persistence of gene silencing in vitro.
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幼年片形吸虫属的 RNAi 动态。肝吸虫揭示了体外基因沉默的持续性。
DOI：
10.1371/journal.pntd.0003185
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发表时间：
2014-09
影响因子：
3.8
通讯作者：
Maule AG
中科院分区：
文献类型：
--
作者：
McVeigh P;McCammick EM;McCusker P;Morphew RM;Mousley A;Abidi A;Saifullah KM;Muthusamy R;Gopalakrishnan R;Spithill TW;Dalton JP;Brophy PM;Marks NJ;Maule AG
片形吸虫属肝吸虫在人类和动物中引起恶性疾病。虽然目前的控制是不可持续的，由于驱虫药耐药性，基因沉默（RNA干扰，RNAi）有可能有助于新的治疗靶点的功能验证。已有报道肝片吸虫幼虫对双链RNA诱导的RNAi的敏感性。为了利用这一点，我们探测RNAi动力学，稳定性和持久性，目的是建立一个强大的平台，在肝吸虫的反向遗传学。我们描述了一种市售肝吸虫株（美国太平洋西北野生株）的标准化RNAi方案的开发，通过7个毒力基因的稳健转录沉默进行验证，并对3个毒力基因进行了深入的实验优化：组织蛋白酶L（FheCatL）和B（FheCatB）半胱氨酸蛋白酶，以及σ类谷胱甘肽转移酶（FheσGST）。在两种F.肝片形吸虫和姜片形吸虫幼虫在暴露于长（200-320 nt）dsRNA或27 nt短干扰（si）RNA后可实现。虽然青少年是高度RNAi敏感的，他们表现出较慢的转录和蛋白质敲除动力学比以前报道的。在短至4小时暴露于触发物（靶标依赖性）后可检测到敲低，并且在所有情况下，在长时间dsRNA暴露后沉默持续≥25天。通过混合多个长dsRNA组合沉默三个靶标是类似有效的。尽管有深刻的转录抑制，我们发现在蛋白质抑制发生之前有一个显著的时间滞后; FheσGST和FheCatL蛋白质抑制分别仅在9天和21天后才可检测到。尽管敲低动力学存在显著变化，但我们发现短暂暴露于长dsRNA或siRNA会触发肝吸虫NEJ中的稳健RNAi转录和持久性，从而支持开发用于对照靶标验证的多通量表型筛选。RNAi在吸虫中的持久性鼓励使用在感染前暴露于RNAi触发物的蠕虫进行基因功能的体内研究。RNA干扰（RNAi）是一种选择性沉默（或减少表达）mRNA转录本的方法，这种方法可用于询问基因和蛋白质的功能，并通过研究沉默特定基因对寄生虫生存或行为的影响来验证驱虫药物或疫苗的潜在靶标。这项研究的重点是肝吸虫寄生虫，它会导致严重的疾病，在人类和动物。我们只有少数几种药物可以治疗这些感染，而吸虫对这些感染正在产生抗药性，而且还没有开发出抗吸虫疫苗。迫切需要治疗和控制肝吸虫寄生虫的新选择，而RNAi是开发此类治疗的有力工具。该研究开发了一套用于在幼年肝吸虫中触发RNAi的简单方法，其显示尽管可以在所有测试的靶标上容易地实现稳健的转录抑制，但蛋白质抑制仅在靶标特异性滞后期（可能与蛋白质半衰期相关）之后发生，这在当前体外维持条件下可能需要>25天。这些发现对于旨在将RNAi应用于肝吸虫生物学研究和靶标验证的研究人员来说非常重要。
Fasciola spp. liver fluke cause pernicious disease in humans and animals. Whilst current control is unsustainable due to anthelmintic resistance, gene silencing (RNA interference, RNAi) has the potential to contribute to functional validation of new therapeutic targets. The susceptibility of juvenile Fasciola hepatica to double stranded (ds)RNA-induced RNAi has been reported. To exploit this we probe RNAi dynamics, penetrance and persistence with the aim of building a robust platform for reverse genetics in liver fluke. We describe development of standardised RNAi protocols for a commercially-available liver fluke strain (the US Pacific North West Wild Strain), validated via robust transcriptional silencing of seven virulence genes, with in-depth experimental optimisation of three: cathepsin L (FheCatL) and B (FheCatB) cysteine proteases, and a σ-class glutathione transferase (FheσGST). Robust transcriptional silencing of targets in both F. hepatica and Fasciola gigantica juveniles is achievable following exposure to long (200–320 nt) dsRNAs or 27 nt short interfering (si)RNAs. Although juveniles are highly RNAi-susceptible, they display slower transcript and protein knockdown dynamics than those reported previously. Knockdown was detectable following as little as 4h exposure to trigger (target-dependent) and in all cases silencing persisted for ≥25 days following long dsRNA exposure. Combinatorial silencing of three targets by mixing multiple long dsRNAs was similarly efficient. Despite profound transcriptional suppression, we found a significant time-lag before the occurrence of protein suppression; FheσGST and FheCatL protein suppression were only detectable after 9 and 21 days, respectively. In spite of marked variation in knockdown dynamics, we find that a transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence in liver fluke NEJs supporting the development of multiple-throughput phenotypic screens for control target validation. RNAi persistence in fluke encourages in vivo studies on gene function using worms exposed to RNAi-triggers prior to infection. RNA interference (RNAi) is a method for selectively silencing (or reducing expression of) mRNA transcripts, an approach which can be used to interrogate the function of genes and proteins, and enables the validation of potential targets for anthelmintic drugs or vaccines, by investigating the impact of silencing a particular gene on parasite survival or behaviour. This study focuses on liver fluke parasites, which cause serious disease in both humans and animals. We have only a handful of drugs with which to treat these infections, to which flukes are developing resistance, and no anti-fluke vaccines have yet been developed. New options for treatment and control of liver fluke parasites are sorely needed, and RNAi is a powerful tool in the development of such treatments. This study developed a set of simple methods for triggering RNAi in juvenile liver fluke, which show that although robust transcriptional suppression can be readily achieved across all targets tested, protein suppression occurs only after a target-specific lag period (likely related to protein half-life), which may require >25 days under current in vitro maintenance conditions. These findings are important for researchers aiming to employ RNAi in investigations of liver fluke biology and target validation.
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