Novel In Vitro and In Vivo Bioluminescent Assays of Giardia Cellular Functioning

 描述（由申请人提供）：贾第虫是一种广泛存在的人畜共患肠道寄生虫，是人类十大寄生虫之一，可引起严重的急性和慢性腹泻病。贾第虫是一个全球性的健康问题，但基本的生物学问题仍然存在，我们对体内感染动力学和发病机制知之甚少。最近的抗心血管药物开发策略集中于大化合物库的高通量筛选以鉴定新的候选药物，但成功有限。除代谢外，鞭毛运动、附着和细胞分裂对宿主定殖也至关重要。针对这些过程的治疗可以干扰滋养体定植和增殖，减少感染的长度或严重程度，并减少脱落囊肿的数量。在R21阶段，我们将开发更有效的药物靶点识别和验证工具。具体来说，我们将创建基于生物标记的体外和体内测定，以监测和量化特定的细胞过程。然后，我们将用生物报告菌株感染小鼠，并使用非侵入性体内光学成像来测定感染过程中寄生虫的细胞过程（目的1）。为了帮助目标识别，我们还将开发和使用基于CRISPR的基因组编辑方法，以在贾第虫中创建敲除和敲低突变体（Aim 2）。使用我们在R21阶段开发的方法，我们将评估和优先考虑细胞过程和可能的药物靶点。具体而言，我们将使用生物报告菌株在关键寄生过程中产生突变体，以测定体外（Aim 3）和体内（Aim 4）的突变表型。使用新的遗传工具（目标2），我们将在运动，细胞分裂，代谢或包囊中创建几个突变体。突变体将用于感染小鼠，我们将使用我们的光学成像测定来评估寄生虫增殖和包囊化的时间和空间缺陷（目的4）。这些方法对于鉴定药物靶点、评价抗心内膜炎候选药物的作用以及验证候选药物的疗效和毒性至关重要。

 DESCRIPTION (provided by applicant): Giardia is a widespread zoonotic intestinal parasite and is one of the ten major parasites of humans, causing significant acute and chronic diarrheal disease. Giardia is a global health concern, yet fundamental biological questions remain, and we know little about in vivo infection dynamics and pathogenesis. Recent anti-giardial drug development strategies that have focused on high throughput screening of large compound libraries to identify new candidate drugs have met with limited success. In addition to metabolism, flagellar motility, attachment and cell division are critical for host colonization. Therapies that target these processes could interfere with trophozoite colonization and proliferation, reducing the length or severity of the infection, and reducing the number of shed cysts. In the R21 phase we will develop more effective tools for drug target identification and validation. Specifically, we will create bioreporter-based in vitro and in vivo assays to monitor and quantify specific cellular processes. We will then infect mice with bioreporter strains and use non-invasive in vivo optical imaging to assay parasite cellular processes over the course of an infection (Aim 1). To aid in target identification, we also will develop and use CRISPR-based methods of genome editing to create knockout and knockdown mutants in Giardia (Aim 2). Using methods we have developed in the R21 phase, we will assess and prioritize cellular processes and possible drug targets. Specifically, we will create mutants in key parasitic processes using bioreporter strains to assay mutant phenotypes in vitro (Aim 3) and in vivo (Aim 4). Using new genetic tools (Aim 2), we will create several mutants in motility, cell division, metabolism or encystation. Mutants will be used to infect mice, and we will use our optical imaging assays to evaluate temporal and spatial defects in parasite proliferation and encystation (Aim 4). These methods are essential for identifying drug targets, evaluating the effects of anti-giardial drug candidates, and for validating candidate efficacy and toxicity.