Host-directed Therapeutics for Amebiasis

阿米巴病的宿主导向治疗

• 批准号: 9247887
• 负责人: Chelsea S. Marie
• 金额: $ 19.75万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9247887
• 关键词: Acanthamoeba; Acute; Amebiasis; Amebic colitis; Amoeba genus; Apical; Balamuthia mandrillaris; Biological Assay; CRISPR/Cas technology; Cell Death; Cell physiology; Cells; Cessation of life; Child; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Colon; Data; Development; Disease; Drug Targeting; Entamoeba histolytica; Enteral; Environment; Epithelial; Epithelium; Event; Gene Deletion; Genes; Genetic Screening; Goals; Human; In Vitro; Infection; Inflammation; Integration Host Factors; Intestines; Invaded; Ion Transport; Ions; Knock-out; Laboratories; Light; Measurement; Measures; Mediating; Mediator of activation protein; Modeling; Molecular; Morbidity - disease rate; Naegleria fowleri; Nature; Parasites; Parasitic Diseases; Pathogenicity; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Physiological; Postdoctoral Fellow; Potassium; Potassium Channel; Predisposition; Prevention; RNA Interference; Research; Resistance; Resources; Role; Stem cells; Surface; Susceptibility Gene; Techniques; Technology; Testing; Therapeutic; Tissues; Toxic effect; Universities; Virginia; Virulence; base; career development; cell killing; chemokine; collaborative environment; cytokine; cytotoxicity; drug testing; efficacy testing; experimental study; genetic variant; genome editing; genome wide association study; ginsenoside M1; graduate student; inhibitor/antagonist; innovation; insight; killings; knock-down; meetings; microbial; monolayer; mortality; new therapeutic target; novel; novel therapeutics; prevent; programs; public health relevance; response; success; targeted treatment; therapeutic target

 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.

 描述(由申请人提供)：假设：在阿米巴巨噬细胞增多症期间人的K+通道的激活是细胞死亡的原因中介。初步数据：在一种新的正向遗传筛查中发现了K+通道，这是溶解组织内阿米巴杀死滋养细胞所需的宿主因素。关键的初步实验数据也支持K+通道的重要性，包括：(1)溶解组织杆菌在杀死靶细胞之前激活K+通道；以及(2) 抑制K+通道可保护细胞免受阿米巴杀伤。方法：我们将通过分析人类肠样阿米巴病模型中特定的结肠K+通道作为潜在的药物靶点来扩展这一发现。我们将测试CRISPR-Cas9删除高置信度候选K+通道基因。基因缺失研究将与肠道类药物的药物抑制剂测试相结合，目的是为K+通道抑制剂作为新的治疗药物提供原则证据。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、可用药的靶点。意义：溶组织埃希氏菌是一种腹泻疾病，具有显著的全球发病率和死亡率，尤其是在发展中国家的儿童中。侵袭性疾病和毒性只有一类药物，出现耐药性是临床关注的问题。高致病性的自由生活阿米巴(Naegleria fowleri、棘阿米巴和曼德拉巴拉穆斯)也通过巨噬细胞增多症杀死人类细胞，因此我们的研究旨在防止肠道阿米巴病中的巨噬细胞死亡，也可能为自由生活的阿米巴提供治疗。该提案的创新方面包括：将人类K+通道确定为潜在宿主治疗目标的正向遗传筛查；重点放在阿米巴滋养细胞杀伤上，这是寄生的溶组织埃希菌和自由生活的阿米巴共同的一种独特的细胞过程；以及使用尖端技术(人类结肠肠样模型和经过验证的CRISPR基因缺失)。这项工作的环境是威廉·A·佩里博士在弗吉尼亚大学的实验室。25年来，佩里博士一直是阿米巴病研究领域的领导者，他的实验室为肠道感染的研究提供了一个令人兴奋和严格的环境。有7名博士后研究员和5名研究生，项目独立但相互补充。该实验室拥有完成高影响力研究的所有必要资源和专业知识，并非常重视合作和分享想法和技术，以及研究员和研究生的独立和职业发展。每周的实验室会议和每天的非正式讨论(由实验室内的佩里博士的办公室提供便利)有助于促进正在进行的研究计划。