Microfilaments in Giardia Attachment and Virulence

描述（由申请方提供）：贾第鞭毛虫是一种普遍存在的人类病原体，是一种导致吸收不良性腹泻的B类生物恐怖因子。贾第虫生物学最引人注目但研究不足的方面之一是其细胞骨架：微丝负责贾第虫机械附着肠上皮细胞的能力，从而允许寄生虫建立感染。这个R21的建议将探索这一新的研究领域，通过启动识别新的蛋白质的微丝细胞骨架和建立生物物理理解的基础上的机制，其中附件是控制。以下假设将被检验：（1）贾第虫的微丝骨架将比以前认识到的更加明显的发散，从而确定它作为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，遗传学和蛋白质组学手段，包括亲和层析，沉淀，酵母双杂交和质谱鉴定。(2)贾第虫与生物和惰性基质的附着将通过质膜背表面下的微丝（以在细胞腹表面下产生负压和吸力）和腹盘周边周围的微丝（以产生抓持力）的整体收缩来介导。将首次使用流动池量化附着力，以产生流体动力学剪切力。通过观察附着到柔性或穿孔基底的动力学和力，并通过使用全干涉反射水性荧光（TIRAF）显微镜来量化寄生虫腹面和基底之间的距离，确定附着机制。本文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。这项工作将推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知识。

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.