Microfilaments in Giardia Attachment and Virulence

贾第鞭毛虫附着和毒力中的微丝

• 批准号: 6969605
• 负责人: HEIDI G ELMENDORF
• 金额: $ 28.28万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-15 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6969605
• 关键词: Giardia; actin binding protein; affinity chromatography; biomechanics; cell adhesion; cytoskeletal proteins; fluorescence microscopy; giardiasis; mass spectrometry; microfilaments; molecular /cellular imaging; parasite infection mechanism; proteomics; protozoal genetics; shear stress; virulence; yeast two hybrid system

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.

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，也是一种 B 类生物恐怖制剂，可导致吸收不良性腹泻。贾第鞭毛虫生物学最引人注目但尚未得到充分研究的方面之一是其细胞骨架：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。该 R21 提案将通过启动微丝细胞骨架的新型蛋白质的鉴定并为控制附着机制的生物物理学理解奠定基础来探索这一新的研究领域。将测试以下假设：（1）贾第鞭毛虫中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。新型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进行鉴定，包括亲和层析、沉降、酵母双杂交和质谱分析。 (2) 贾第鞭毛虫对生物和惰性基质的附着将通过质膜背表面下的微丝的整体收缩来介导（以在细胞腹表面下产生负压和吸力）和质膜周围的微丝的整体收缩。腹盘的周长（产生夹紧力）。将首次使用流动池产生流体动力剪切力来量化附着力。通过观察柔性或穿孔基质的动力学和附着力，并使用全干涉反射水荧光 (TIRAF) 显微镜来量化寄生虫腹侧表面和基质之间的距离，确定附着机制。这里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。这项工作将增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。