Molecular mechanisms of attachment by the ventral disc in Giardia

贾第虫是一种广泛传播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2.8亿人罹患这种疾病。可移动的滋养体定植并附着于 小肠有腹盘，有一个复杂的微管细胞器。附件是 感染所需，因为它允许贾第鞭毛虫抵抗蠕动。依恋的理论模型 必须以准确的生物数据为基础。50年来，流体动力吸力一直是 被广泛建模为贾第虫附着的主导机制，但这个模型 依恋缺乏实证支持。我们在光盘架构和合成方面的开创性工作， 结合我们开发的CRISPR介导的击倒和击倒和 动物感染动态的生物发光成像，使我们能够从基因上测试 盘状构象动力学所需的DAP的结构和/或功能作用 依恋。在附着的早期阶段，我们发现椎间盘的某些区域经历了 特定的构象变化。这些变化，以及弯曲圆盘的存在， 可能会产生一种“密封”，使其能够附着在表面并抵抗剪切力。我们 最近，我还发现并定位了87个光盘相关蛋白(DAP)的特异性 维护和调制光盘所涉及的光盘的结构和功能区域 构象。在这里，我们使用基于CRISPR的新遗传工具来创建特定类别的 与腹侧盘关键区域相关的DAP突变体，可能是其 穹顶结构，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击倒(共10个) 化验。在目标1中，我们询问了显著的微带和交叉桥的作用 间盘复合体在调节间盘曲率中的作用。在目标2中，我们定义了结构和 重叠区DAP的功能作用，可以在结构上连接上和下部分 圆盘，实现正确的圆顶结构。在目标3中，我们询问分子 DAP与间盘边缘和腹沟运动相关的机制 有助于形成抵抗剪切力所需的侧冠密封。最后，我们评估了 活体生物发光成像研究两种盘状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方法将限制宿主 并限制传染性包囊的传播。

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.