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

描述（由申请方提供）：贾第虫病是全球急性原虫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）忽视的疾病。贾第鞭毛虫是一种寄生性原生生物，其致病性依赖于通过腹盘（一种新的细胞骨架结构）附着在肠道微绒毛上。缺乏生物物理学支持和明显缺乏分子证据阻碍了对所提出的心包附着机制的研究。这里的主要重点是评估支持两个“经典”的假设，解释心内膜附着：“构象变化”模型和“流体动力学”模型。“构象变化”模型提出腹侧盘的构象变化导致基于吸力的附着。我们将使用腹侧椎间盘的高分辨率冷冻电子断层扫描、活细胞成像和新型附着试验（目的1）来研究假定的椎间盘构象动力学。我们还将研究椎间盘相关膜联蛋白（1-giardins）在椎间盘构象动力学中的作用（Aim 2），并使用全基因组，高通量随机GFP视觉筛选（Aim 3）识别和表征新的椎间盘相关成分。心包附着的“流体动力学模型”假设腹侧鞭毛产生流体动力学电流，从而实现基于抽吸的附着。为了测试这种替代方案，我们将在具有运动缺陷的各种突变体中测定附着动力学（Aim 2）。最后，为了告知我们对椎间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椎间盘的生物发生和亲本椎间盘的解体（目标4）。有丝分裂后，两个背侧子盘组装，父母的腹侧盘和中间体拆卸。使用可光活化的GFP标记的菌株的实时成像和微管分解突变体的分析，我们将测试中间体作为背侧子盘的盘组件的储库的假设。公共卫生相关性：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虫病。贾第鞭毛虫通过一种不确定的机制附着在宿主小肠上。本文提出的研究直接研究了贾第虫的附着机制，并将为药物发现提供额外的靶点。具体来说，我们调查的功能“腹盘”的吸盘状结构。

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.