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

 描述(申请人提供)：贾第鞭毛虫是一种分布广泛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疾病。贾第鞭毛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。最近的抗贾第鞭毛虫药物开发策略侧重于对大型化合物库进行高通量筛选，以确定新的候选药物，但收效甚微。除了新陈代谢，鞭毛的运动、附着和细胞分裂对寄主的定植也是至关重要的。针对这些过程的治疗可能会干扰滋养体的定植和增殖，缩短感染的长度或严重程度，并减少脱落的包囊数量。在R21阶段，我们将开发更有效的药物靶标识别和验证工具。具体地说，我们将创建基于生物报告程序的体外和体内测试，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分析感染过程中寄生虫的细胞过程(目标1)。为了帮助识别目标，我们还将开发和使用基于CRISPR的基因组编辑方法来创建贾第虫的基因敲除和基因敲除突变(目标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。具体地说，我们将在关键的寄生过程中创建突变株，使用生物报告菌株在体外(Aim 3)和体内(Aim 4)检测突变表型。使用新的遗传工具(目标2)，我们将在运动、细胞分裂、新陈代谢或囊化方面创造几个突变。突变将被用来感染小鼠，我们将使用我们的光学成像分析来评估寄生虫增殖和囊化过程中的时间和空间缺陷(目标4)。这些方法对于确定药物靶点、评估抗贾第鞭毛虫候选药物的效果以及验证候选药物的有效性和毒性是必不可少的。

 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.