Molecular architecture, function and biogenesis of the ventral disc in Giardia in

描述(申请人提供)：贾第鞭毛虫病是全世界急性原虫肠道感染的最常见原因，而慢性贾第鞭毛虫病是发展中国家高发病率的主要原因。由于缺乏协调一致的研究努力，贾第鞭毛虫病已被指定为世界卫生组织(WHO)忽视的疾病。肠贾第鞭毛虫是一种寄生原生生物，其致病性依赖于通过腹盘附着在肠道微绒毛上，腹盘是一种新的细胞骨架结构。相互矛盾的生物物理支持和明显缺乏分子证据阻碍了对拟议的贾第鞭毛膜附着机制的研究。这里的主要焦点是评估两个解释心脏附着的“经典”假说的支持情况：“构象变化”模型和“流体力学”模型。“构象变化”模型认为腹侧盘的构象变化导致了基于吸力的附着。我们将使用腹侧椎间盘的高分辨率冷冻电子断层扫描、活细胞成像和新的附着分析来研究可能的椎间盘构象动力学(目标1)。我们还将研究光盘相关膜联蛋白(1-giardins)在光盘构象动力学中的作用(目标2)，并使用全基因组、高通量随机GFP可视化屏幕识别和表征新的光盘相关成分(目标3)。贾第鞭毛附着体的“流体力学模型”认为，腹侧鞭毛产生一种流体动力流，使基于吸力的附着体发生作用。为了测试这一替代方案，我们将测试具有运动性缺陷的各种突变体的附着动力学(目标2)。最后，为了说明我们对视盘结构和功能的分析(目标1-3)，我们将描述细胞分裂过程中背侧视盘的生物发生和亲代视盘的分解(目标4)。有丝分裂后，两个背侧子盘组装在一起，亲代腹侧盘和正中体解体。利用可光激活的GFP标记菌株的活体成像和微管分解突变体的分析，我们将检验这一假说，即正中体作为背侧子盘的盘组件的储存库。公共卫生相关性：肠贾第鞭毛虫是人类十大寄生虫之一，目前对贾第鞭毛虫缺乏了解，导致它被指定为“被忽视的疾病”。据信，世界上六分之一的人口患有贾第鞭毛虫病。贾第鞭毛虫通过一种未知的机制附着在宿主小肠上。本文提出的研究直接探讨贾第鞭毛虫的附着机制，将为药物开发提供更多靶点。具体地说，我们研究了“腹侧盘”的功能，这是一种吸盘状结构。

DESCRIPTION (provided by applicant): Giardiasis is the most common cause of acute protozoan intestinal infection worldwide, and chronic giardiasis is a major contributor to high morbidity in developing countries. Due to the lack of concerted research efforts, giardiasis has been designated a World Health Organization (WHO) neglected disease. Giardia intestinalis is a parasitic protist, and its pathogenicity is dependent upon attachment to the intestinal microvilli via the ventral disc, a novel cytoskeletal structure. Conflicting biophysical support and a conspicuous lack of molecular evidence have hampered the investigation of proposed giardial attachment mechanisms. The primary focus here is to evaluate support for two "classic" hypotheses that explain giardial attachment: the "Conformational Change" model and the "Hydrodynamic" model. The "Conformational Change" model proposes that conformational changes of the ventral disc cause suction-based attachment. We will investigate putative disc conformational dynamics using high resolution cryoelectron tomography of the ventral disc, live cell imaging, and novel attachment assays (Aim 1). We will also examine the role of disc-associated annexins (1-giardins) in disc conformational dynamics (Aim 2), and identify and characterize novel disc-associated components using a genome-wide, high-throughput random GFP visual screen (Aim 3). The "Hydrodynamic Model" of giardial attachment posits that the ventral flagella produce a hydrodynamic current enabling suction-based attachment. To test this alternative, we will assay attachment dynamics in various mutants with motility defects (Aim 2). Lastly, to inform our analyses of disc structure and function (Aims 1-3), we will characterize dorsal disc biogenesis and parental disc disassembly during cell division (Aim 4). After mitosis, two dorsal daughter discs are assembled and the parental ventral disc and median body are disassembled. Using live imaging of photoactivatable GFP- tagged strains and analyses of microtubule disassembly mutants, we will test the hypothesis that the median body acts as a reservoir of disc components for dorsal daughter discs. PUBLIC HEALTH RELEVANCE: Giardia intestinalis is one of the ten major parasites in humans, and the current lack of understanding about Giardia has resulted in it being designated a "neglected disease". One sixth of the world's population is believed to suffer from giardiasis. Giardia attaches to the host small intestine via an undefined mechanism. The research proposed here directly investigates the attachment mechanism of Giardia, and will offer additional targets for drug discovery. Specifically we investigate the function of the "ventral disc" a suction cup-like structure.