Cys-loop ligand-gated chloride channels from Dirofilaria immitis: Biological function and pharmacology

原丝虫是一种蚊子传播的寄生线虫，主要感染家养和野狗，是一种叫做心丝虫的使人衰弱的疾病的病原体。在过去的二十年里，对心丝虫的唯一预防性治疗是来自大环内酯家族的药物（也称为驱虫药），主要针对一种称为谷氨酸门控氯通道的胱氨酸环受体。然而，现在有报道称这种寄生虫在某些地区对大环内酯类药物产生耐药性。因此，开发新的驱虫剂的需求和机会越来越大。配体门控氯通道（lgcc）的cys-loop家族仍然是未来驱虫剂发展的一个有吸引力的目标。这些受体来自一个大的五聚体（即5个亚基）配体门控离子通道家族，在寄生线虫的神经系统中起重要作用。它们已被证明在几个生物过程中发挥关键作用，如运动、喂养和繁殖。在D. immitis基因组中，大约有24个基因编码该家族的亚基，其中大多数尚未被表征。此外，我们还不清楚这些受体在牙周炎的运动等各种生物过程中所起的作用。该提案将作为该病原体的cys-loop LGCC家族的第一个全面研究。本研究的一个组成部分将包括评估微丝虫（幼虫期）和成虫中受体基因的转录水平。这将有助于利用针对各种受体基因的RNAi进行进一步的表征，并使用Worminator系统进行表型分析，该系统量化了响应生物活性分子的蠕虫运动变化。进一步表征D. immitis cys-loop受体将通过我们的爪蟾表达系统完成，并使用2电极电压钳电生理学进行表征。电生理学将提供各种受体的药理学（即药物）特征的详细信息，并将是帮助确定以前未知受体的天然配体的关键。总的来说，这项研究将揭示lgcc在D. immitis生物学中的作用的新细节，并可能确定新的有希望的驱虫靶点。该项目将为高素质人才提供良好的培训机会，因为它不仅可以提高受训人员的技术/研究技能，还可以使他们从分子到生物体水平对特定生物系统有深入的了解。此外，我们预计本研究提供的对寄生线虫的RNAi的进一步发展将对寄生虫学界做出巨大贡献。最后，由于气候变化，加拿大心丝虫传播的风险增加，这项研究将极大地有利于该国应对这种病原体的承诺。

Dirofilaria immitis is a mosquito-transmitted parasitic nematode that primarily infects domestic and wild dogs and is the causative agent of the debilitating disease called heartworm. For the past two decades the only preventative treatments for heartworm are drugs (also called anthelmintics) from the macrocyclic lactone family which primarily target a type of cys-loop receptor called the glutamate-gated chloride channels. However, there are now reports of resistance of this parasite to macrocyclic lactone drugs in certain regions. Therefore, there is a growing need and opportunity for the development of new anthelmintics. The cys-loop family of ligand-gated chloride channels (LGCCs) remains an attractive target for the development of future anthelmintics. These receptors are from a large family of pentameric (i.e. 5 subunits) ligand-gated ion channels that play an important role in the nervous system of parasitic nematodes. They have been shown to play key roles in several biological processes such as movement, feeding and reproduction. In the D. immitis genome there are about 24 genes that encode subunits from this family and most have yet to be characterized. Moreover, we still do not know the role these receptors play in various biological process in D. immitis such as movement. This proposal will serve as the first comprehensive study of the cys-loop LGCC family from this pathogen. A component of this study will involve an evaluation of transcript levels of receptor genes in microfilaria (larval stage) and adult worms. This will help shape further characterization using RNAi targeting various receptor genes with analysis of phenotype using the Worminator system which quantifies changes in worm movement in response to bioactive molecules. Further characterization of D. immitis cys-loop receptors will be completed through our Xenopus expression system for characterization using 2-electrode voltage clamp electrophysiology. Electrophysiology will provide details on the pharmacological (ie drug) profile of various receptors and will be key in helping to identify the natural ligand for previously unknown receptors. Overall this research will uncover new details on the the role of LGCCs in the biology of D. immitis and possibly identify new promising targets for anthelmintic development.  The project will provide excellent training opportunities for highly-qualified personnel as it will not only enhance the technical/research skills of the trainees but will provide them with a deep understanding of a particular biological system from the molecular to organism level.  In addition, we anticipate that further developments in RNAi for parasitic nematodes provided by this research will contribute immensely to the parasitology community.  Finally, since there is an increased risk of transmission of heartworm in Canada due to climate change, this research will greatly benefit this country's commitment to tackling this pathogen.