Targeting Trypanosome Cation Pumps and Channels

人类非洲锥虫病（HAT，昏睡病）是一种被忽视但致命的媒介传播疾病 布氏锥虫Trypanosoma brucei ssp.（T. brucei）。6000万人有感染HAT的风险， 在撒哈拉以南的非洲地区，每年报告有5万例新病例和相同数量的死亡病例。唯一的 市场上可用的抗锥虫药物毒性极强， 治疗患者的脑病。理想的抗锥虫药物将靶向重要的生理 过程和/或非变体寄生虫衍生的分子，而不会不利地影响人类宿主。我们 其他研究表明，T.布氏杆菌为4- 比宿主细胞外环境中遇到的低10个数量级，并且寄生虫需要有效的 维持[Ca2 +]\稳态，存活和增殖的机制。在我们之前的拨款中， 我们鉴定并表征了两种关键的质膜样阳离子泵（ATP酶; TBCA 1和TBCA 2） 利用T.并产生抗体以将其免疫定位在血液和昆虫中 阶段寄生虫。我们使用瞬时RNAi技术确定了它们在昆虫和血液阶段寄生虫中的功能作用 抑制技术和合成抑制剂分析。我们随后构建了重组抗泵 基于一种新的细菌幽灵疫苗输送技术的疫苗，由莫尔豪斯学院开发， 药物，部分保护小鼠免受寄生虫的攻击。我们的研究结果表明，TBCA 1 类似于真菌的K +/Na +-ATP酶，而TBCA 2是一种类似于质膜的Ca 2 + ATP酶。RNAi 抑制这些靶标导致寄生虫死亡率增加。此外，我们通过抑制测定 研究表明T.布氏杆菌不仅受Ca~（2+）ATP酶的调节， 钙离子通道由于通过RNAi抑制靶向Ca2+泵增加了寄生虫死亡率， 用TBCA 2接种显著降低了感染小鼠的寄生虫血症和存活率，我们提出， 靶向关键阳离子泵（TBCA 1和TBCA 2）以及L型Ca2+通道， 合成的抑制剂药物或疫苗将足以抑制T.布鲁塞伊和提供完整的 对T.布氏杆菌感染在这个竞争性的续约提案中，我们更进一步， 假设锥虫利用阳离子泵和通道来介导 哺乳动物宿主血液以及靶向特异性药物同时抑制和 接种疫苗可以预防T。布氏杆菌的增殖和发育。提出了两个具体目标： 具体目标1.我们将在T.布鲁氏菌和 确定其在Ca2+稳态中的作用。具体目标2。我们将构建并测试各种Ca2 + 在新的基于细菌血影的疫苗系统中作为抗原的泵/通道基因构建体 感染T。并测定其对T.布氏杆菌感染我们的长期 我们的目标是在我们既定的原则证明的基础上，开发和提供一种新型的小型 能够抑制T. 布氏杆菌在发展过程中。我们还计划通过此应用程序生成足够的数据，以申请 RQ-1型赠款，用于资助未来的抗锥虫药物研究。

Human African Trypanosomiasis (HAT, sleeping sickness) is a neglected but fatal vector borne disease caused by Trypanosoma brucei ssp. (T. brucei). Sixty million people are at risk of infection with HAT, and 50,000 new case's and an equal number of deaths are reported annually in subsaharan Africa. The only available anti-trypanosomal drugs on the market are extremely toxic, and result in post-treatment encephalopathy in treated patients. The ideal anti-trypanosomal agents will target vital physiological processes and/or non-variant parasite-derived molecules without adversely affecting the human host. We and others have shown that that cytosolic calcium ion concentration [Ca2+ ]i in blood stages of T. brucei is 4- 10 orders of magnitude below that encountered in host extracellular milieu and that parasites require effective mechanisms to maintain [Ca2+ ]\ homeostasis, survival, and proliferation in their host. In our previous grant, we identified and characterized two key plasma-membrane-like cation pumps (ATPases; TBCA1 and TBCA2) utilized by T. brucei for survival and generated antibodies to immunolocalize them in bloodstage and insect stage parasites. We determined their functional role in insect and bloodstage parasites using transient RNAi inhibition technology and synthetic inhibitor assays. We subsequently constructed recombinant anti-pump vaccines based on a novel bacterial ghost vaccine delivery technology, developed at Morehouse School of Medicine, which partially protected against parasite challenge in mice. Our results revealed that TBCA1 resembled a fungal K+/Na+-ATPase while TBCA2 was a plasma-membrane-like Ca2+ ATPase. RNAi inhibition of these targets resulted in increased parasite mortality. Furthermore, we determined by inhibition studies that Ca2+ homeostasis in T. brucei is not only regulated by the Ca2+ ATPases but also by L-type calcium ion channels. Since targeting the Ca2+ pumps by RNAi inhibition increased parasite mortality and vaccination with TBCA2 significantly reduced parasitemia and survival of infected mice, we propose that targeting the key cation pumps (TBCA1 and TBCA2) as well as L-type Ca2+ channels together with either synthetic inhibitor drugs or vaccines, will be sufficient to inhibit proliferation of T. brucei and provide complete protection against T. brucei infection. In this competitive renewal proposal, we have gone a step further to hypothesize that trypanosomes utilize cation pumps and channels to mediate establishment in mammalian host blood and that simultaneous inhibition by target specific drugs and blocking by vaccination will prevent T. brucei proliferation and development. Two specific aims are proposed: In Specific aim 1. we will functionally characterize and localize the L-type Ca2+ channel in T. brucei and determine its role in Ca2+ homeostasis. In Specific aim 2. we will construct and test various Ca2+ pump/channel gene constructs as antigens in a novel bacterial ghost based vaccine system against infections by T. brucei and determine their levels of protection against T. brucei infection. Our longterm goal is to build on our established proof of principle to develop and deliver a novel class of small molecule drugs and/or immunotherapeutics capable of inhibiting the essential Ca2+ pumps and channels of T. brucei during development. We also plan to generate enough data, through this application, to apply for an RQ-1 type grant to fund future studies on anti-trypanosome drugs.