Microfilaments in Giardia Attachment and Virulence

描述（由申请人提供）：贾第鞭毛虫是一种普遍存在的人类病原体和B类生物恐怖制剂，导致吸收不良腹泻。贾第鞭毛虫生物学中最引人注目但尚未得到充分研究的一个方面是它的细胞骨架：微丝负责贾第鞭毛虫机械附着在肠上皮细胞上的能力，从而允许寄生虫建立感染。本R21提案将通过开始鉴定微丝细胞骨架的新蛋白质和建立对附着控制机制的生物物理理解的基础来探索这一新的研究领域。以下假设将得到验证：(1)贾第鞭毛虫的微丝细胞骨架将比以前认识到的更加分化，从而确定它是一个重要的药物靶点。新的肌动蛋白结合蛋白将通过生物化学、遗传和蛋白质组学手段鉴定，包括亲和层析、沉淀、酵母双杂交和质谱。(2)贾第鞭毛虫附着在生物和惰性基质上，将通过质膜背表面下的微丝（产生负压和细胞腹面下的吸力）和腹盘周围的微丝（产生抓握力）的整体收缩来介导。附着力将首次量化使用流动细胞来产生流体动力剪切力。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力来确定，并通过使用全干涉反射水荧光（TIRAF）显微镜来量化寄生虫腹部表面与底物之间的距离。本文提出的研究将为更详细地研究贾第鞭毛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靶点的认识。

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.