Host-directed Therapeutics for Amebiasis

 描述（由申请方提供）：假设：阿米巴胞饮期间人K+通道的激活是细胞死亡的因果介质。初步数据：K+通道被确定在一个新的正向遗传筛选宿主因子所需的溶组织内阿米巴的刺胞杀死。关键的初步实验数据也支持K+通道的重要性，包括：（1）E。溶组织菌在杀死靶细胞之前激活K+通道;和（2） 抑制K+通道保护细胞免受阿米巴杀伤。方法：我们将通过分析特定的结肠K+通道作为人类肠道阿米巴病模型中潜在的药物靶点来扩展这一发现。我们将在人类类肠中测试高置信度候选K+通道基因的CRISPR-Cas9缺失。基因缺失研究将与肠道中的药理学抑制剂测试配对，目的是为K+通道抑制剂作为新型疗法提供原理证明。这些研究的成功完成将确定人类K+通道是否是阿米巴病的新的宿主导向的可药物靶点。显著性：E.溶组织病是一种在全球范围内发病率和死亡率都很高的疟疾，尤其是在发展中国家的儿童中。侵袭性疾病的药物种类单一，毒性和耐药性的出现是临床关注的问题。高致病性自由生活阿米巴（Naegleria fowleri，阿米巴属，和Balamuthia mandrillaris）也通过胞啃作用杀死人类细胞，因此我们旨在预防肠道阿米巴病中胞啃死亡的研究也可以为自由生活的阿米巴提供治疗。该提案的创新方面是：将人类K+通道确定为潜在宿主治疗靶点的正向遗传筛选;关注阿米巴嗜咬细胞杀伤，这是寄生性大肠杆菌共享的独特细胞过程。histolytica和自由生活的阿米巴;并通过使用尖端技术（人类结肠类肠模型和经验证的CRISPR基因缺失）。工作的环境是威廉·A。弗吉尼亚大学佩特里实验室。Petri博士在阿米巴病研究领域已经有25年的领导地位，他的实验室是一个令人兴奋和严谨的环境，用于肠道感染的研究。有7名博士后和5名研究生，独立但互补的项目。该实验室包含所有必要的资源和专业知识，以完成高影响力的研究，并非常重视合作和思想和技术的共享，以及研究员和研究生的独立性和职业发展。每周的实验室会议和每日的非正式讨论（由实验室内的Petri博士办公室提供便利）有助于促进正在进行的研究计划。

 DESCRIPTION (provided by applicant): Hypothesis: Activation of human K+ channels during amebic trogocytosis is the causal mediator of cell death. Preliminary data: K+ channels were identified in a novel forward genetic screen for host factors required for trogocytic killing by Entamoeba histolytica. Key preliminary experimental data also support the importance of K+ channels including: (1) E. histolytica activated K+ channels prior to killing target cells; and (2) inhibition of K+ channels protected cells from amebic killing. Approach: We will extend this discovery by analyzing specific colonic K+ channels as potential drug targets in the human enteroid model of amebiasis. We will test CRISPR-Cas9 deletions of high confidence candidate K+ channels genes in human enteroids. Gene deletion studies will be paired with pharmacologic inhibitor testing in enteroids with the goal of providing proof-of-principle for K+ channel inhibitrs as novel therapeutics. Successful completion of these studies will determine if human K+ channels are novel host-directed, druggable targets for amebiasis. Significance: E. histolytica is a diarrheal disease with significant global morbidity and mortality, especially in children in the developing world. There is a single class of drugs for invasive disease and toxicity and the emergence of resistance are clinical concerns. The highly pathogenic free-living amebae (Naegleria fowleri, Acanthamoeba sp., and Balamuthia mandrillaris) also kill human cells via trogocytosis, therefore our studies aiming to prevent trogocytic death in enteric amebiasis, may also offer therapy for the free-living amebae. Innovative aspects of the proposal are: the forward genetic screen that identified human K+ channels as a potential host therapeutic target; the focus on amebic trogocytic killing, a unique cellular process shared by parasitic E. histolytica and the free-living amebae; and by the use of cutting edge technology (a human colonic enteroid model and validated CRISPR gene deletions). The environment for the work is Dr. William A. Petri's laboratory at The University of Virginia. Dr. Petri has been a leader in the fied of amebiasis research for 25 years and his laboratory is an intellectually exciting and rigorous environment for the study of enteric infections. There are 7 postdoctoral fellows and 5 graduate students, with independent yet complementary projects. The laboratory contains all the necessary resources and expertise to accomplish high-impact research and great emphasis is placed on collaboration and sharing of ideas and techniques, as well as independence and career development for the fellows and graduate students. Weekly laboratory meetings and daily informal discussions (facilitated by the presence of Dr. Petri's office within the laboratory serve to promote the ongoing research program.