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

描述（由申请人提供）：贾第虫病是全球范围内急性原生动物肠道感染的最常见原因，慢性贾第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，贾第虫病已被指定为世界卫生组织（世卫组织）被忽视的疾病。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贲门附着机制的研究。这里的主要焦点是评估对解释心包附着的两个“经典”假设的支持：“构象变化”模型和“流体动力学”模型。“构象改变”模型认为腹侧椎间盘的构象改变导致了吸吸性附着。我们将使用腹侧椎间盘的高分辨率低温电子断层扫描、活细胞成像和新型附着分析来研究假定的椎间盘构象动力学（目的1）。我们还将研究盘相关膜联蛋白（1-giardins）在盘构象动力学中的作用（目的2），并使用全基因组、高通量随机GFP视觉筛选识别和表征新的盘相关成分（目的3）。贲门附着的“流体动力学模型”假设腹侧鞭毛产生流体动力学电流，使其能够以吸力为基础的附着。为了测试这一选择，我们将分析各种具有运动缺陷的突变体的附着动力学（目的2）。最后，为了分析椎间盘的结构和功能（目的1-3），我们将描述细胞分裂过程中背椎间盘的生物发生和亲代椎间盘的拆卸（目的4）。有丝分裂后，两个背侧子盘组装，亲代腹侧盘和中间体拆卸。利用可光激活GFP标记菌株的实时成像和对微管分解突变体的分析，我们将验证中间体作为背侧子盘的盘成分储存库的假设。公共卫生相关性：肠贾第鞭毛虫是人类的十大寄生虫之一，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，全球六分之一的人口患有贾第虫病。贾第鞭毛虫通过一种未知的机制附着在宿主小肠上。本研究直接探讨了贾第鞭毛虫的附着机制，并将为药物发现提供额外的靶点。我们特别研究了“腹侧椎间盘”的功能，这是一种类似吸盘的结构。

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.