Molecular mechanisms of attachment by the ventral disc in Giardia

贾第鞭毛虫腹盘附着的分子机制

• 批准号: 10335204
• 负责人: SCOTT C DAWSON
• 金额: $ 52.14万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10335204
• 关键词: 3-Dimensional; Acute Diarrhea; Affect; Animal Model; Animals; Architecture; Biological; Biological Assay; Biological Testing; Biophysics; CRISPR interference; Cells; Chronic diarrhea; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cyst; Cytoplasm; Data; Defect; Development; Dorsal; Electron Microscopy; Elements; Flagella; Foundations; Genetic; Gerbils; Giardia; Image; In Vitro; Infection; Intestinal parasite; Intestines; Knock-out; Lateral; Lead; Life Cycle Stages; Light; Link; Mediating; Microtubule Stabilization; Microtubules; Modeling; Molecular; Molecular Conformation; Movement; Organelles; Parasites; Peristalsis; Persons; Phenotype; Proteins; Research; Resistance; Resolution; Role; Severities; Small Intestines; Spectrometry, Mass, Secondary Ion; Structural defect; Structure; Suction; Surface; Testing; Theoretical model; Time; Work; Zoonoses; base; bioluminescence imaging; cell motility; diarrheal disease; evidence base; flexibility; gut colonization; host colonization; hydrodynamic model; imaging study; in vivo; insight; knock-down; link protein; mutant; novel; prevent; protein complex; seal; targeted treatment; tool

Giardia is a widespread zoonotic intestinal parasite that causes acute and chronic diarrheal disease in more than 280 million people each year. Motile trophozoites colonize and attach to the small intestine with the ventral disc, a complex microtubule organelle. Attachment is required for infection as it allows Giardia to resist peristalsis. Theoretical models of attachment must be grounded in accurate biological data. For 50 years, hydrodynamic suction has been extensively modeled as the leading mechanism for Giardia attachment, yet this model of attachment lacks empirical support. Our pioneering work on disc architecture and composition, combined with our development of CRISPR-mediated knockdowns and knockouts and bioluminescent imaging of infection dynamics in animals, enable us to genetically test the structural and/or functional roles of DAPs required for disc conformational dynamics in attachment. During early stages of attachment, we discovered that regions of the disc undergo specific conformational changes. These changes, along with the presence of a curved disc, likely create a “seal” that enables attachment to the surface and resistance to shear forces. We have also recently identified and localized 87 disc-associated proteins (DAPs) to the specific structural and functional regions of the disc involved in maintaining and modulating disc conformations. Here we use our new CRISPR-based genetic tools to create specific classes of DAP mutants associated with key regions of the ventral disc that are likely required for its domed structure, as well as DAP mutants in key regions associated with flexible movements. We evaluate structural and attachment defects in CRISPR-interference (CRISPRi) DAP knockdowns (10 total) using high-resolution live imaging, electron microscopy, and biophysical assays. In Aim 1, we interrogate the role of the conspicuous microribbon and crossbridge complexes of the disc in mediating disc curvature. In Aim 2, we define the structural and functional roles of overlap zone DAPs that may structurally link the upper and lower portions of the disc, enabling proper domed conformation. In Aim 3, we interrogate the molecular mechanisms of DAPs associated with disc margin and ventral groove movements that contribute to formation of the lateral crest seal needed to resist shear forces. Lastly, we evaluate aberrant infection dynamics of two disc structural mutants using in vivo bioluminescent imaging (BLI) in an animal model of infection. Therapies that target parasite attachment would limit host colonization and limit the dissemination of infectious cysts.

贾第虫是一种广泛分布的人畜共患肠道寄生虫，可引起急性和慢性腹泻 每年有超过2.8亿人患病。活动的滋养体定殖并附着于 小肠腹盘，一个复杂的微管细胞器。附件是 因为它可以让贾第虫抵抗寄生虫病。依恋的理论模型 必须基于准确的生物学数据50年来，流体动力吸力一直是 被广泛地建模为贾第虫附着的主要机制，然而这种模型 依恋缺乏实证支持。我们在光盘结构和组成方面的开创性工作， 结合我们开发的CRISPR介导的敲除和基因敲除， 动物感染动力学的生物发光成像，使我们能够从基因上测试 DAPs在椎间盘构象动力学中的结构和/或功能作用 附件.在附着的早期阶段，我们发现椎间盘的某些区域 特定的构象变化。这些变化，沿着椎间盘弯曲， 可能产生一种“密封”，能够附着到表面上并抵抗剪切力。我们 最近还鉴定并定位了87种椎间盘相关蛋白（DAP）， 参与维持和调节椎间盘的椎间盘结构和功能区域 构象在这里，我们使用我们新的基于CRISPR的遗传工具来创建特定类别的 DAP突变体与腹侧椎间盘的关键区域相关，这些区域可能是其生长所需的。 圆顶结构，以及与灵活运动相关的关键区域的DAP突变体。 我们评估了CRISPR干扰（CRISPRi）DAP中的结构和附着缺陷， 使用高分辨率实时成像、电子显微镜和生物物理学进行击倒（共10次） 测定。在目标1中，我们询问了明显的微通道和交叉桥的作用， 椎间盘复合体介导椎间盘弯曲。在目标2中，我们定义了结构和 重叠区DAP的功能作用，这些DAP可以在结构上连接 盘，使适当的圆顶构造。在目标3中，我们询问分子 与椎间盘边缘和腹侧沟运动相关的DAP机制， 有助于形成抵抗剪切力所需的侧向牙顶密封。最后，我们评估 使用体内生物发光成像的两种盘结构突变体的异常感染动力学 (BLI)在动物感染模型中。针对寄生虫附着的治疗将限制宿主 定植和限制传染性囊肿的传播。