Microfilaments in Giardia Attachment and Virulence

描述(由申请人提供)：蓝氏贾第鞭毛虫是一种普遍存在的人类病原体，是一种B类生物恐怖分子，可导致吸收不良的腹泻。贾第鞭毛虫生物学中最引人注目但研究不足的方面之一是它的细胞骨架：微丝负责贾第鞭毛虫机械附着到肠道上皮细胞的能力，从而允许寄生虫建立感染。这项R21提案将通过启动对微丝细胞骨架新蛋白的鉴定来探索这一新的研究领域，并为生物物理理解依附控制机制奠定基础。将检验以下假设：(1)贾第虫体内的微丝细胞骨架将比以前认识到的更加多样化，从而将其确立为重要的药物靶点。新的肌动蛋白结合蛋白将通过生化、遗传学和蛋白质组学手段进行鉴定，包括亲和层析、沉淀、酵母双杂交和质谱学。(2)贾第鞭毛虫在生物和惰性底物上的附着将通过质膜背面下的微丝的整体收缩(在细胞腹面下产生负压和吸力)和腹盘周围的微丝的整合收缩(产生抓紧力)来调节。附着力将首次使用流动池来创建流体动力剪切力来量化。附着机制将通过观察附着在柔性或穿孔底物上的动力学和作用力，并通过使用全干涉反射水荧光(TIRAF)显微镜来量化寄生虫腹面和底物之间的距离来确定。这里提出的研究将为更详细地研究贾第虫利用微丝产生附着力的机制奠定基础。这项工作将促进我们对寄生虫发病机制的理解，并发展我们对微丝作为药物设计目标的认识。

DESCRIPTION (provided by the applicant): Giardia lamblia is a ubiquitous human pathogen and a Category B bioterrorism agent that causes mal-absorptive diarrhea. One of the most compelling yet understudied aspects of Giardia biology is its cytoskeleton: microfilaments are responsible for the ability of Giardia to mechanically attach to intestinal epithelial cells thus permitting the parasite to establish infections. This R21 proposal will explore this new area of research by initiating identification of novel proteins of the microfilament cytoskeleton and establishing the foundations of biophysical understanding of the mechanism by which attachment is controlled. The following hypotheses will be tested: (1) The microfilament cytoskeleton in Giardia will be significantly more divergent than previously recognized thus establishing it as an important drug target. Novel actin-binding proteins will be identified through biochemical, genetic and proteomics means, including affinity chromatography, sedimentation, yeast two-hybrid, and mass spectrometry. (2) Attachment of Giardia to biological and inert substrates will be mediated by integrated contraction of the microfilaments under the dorsal surface of the plasma membrane (to generate negative pressure and a suction force under the ventral surface of the cell) and of microfilaments around the perimeter of the ventral disk (to generate a clutching force). The force of attachment will be quantified for the first time using flow cells to create hydrodynamic shear forces. The mechanism of attachment will be determined by observing the dynamics and force of attachment to flexible or perforated substrates and by using total interference reflection aqueous-fluorescence (TIRAF) microscopy to quantify the distance between the parasite ventral surface and substrate. The studies proposed here will lay the groundwork for a more detailed study of the mechanism by which Giardia uses microfilaments to generate attachment force. This work will advance our understanding of parasite pathogenesis and develop our knowledge of microfilaments as a target for drug design.