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）：贾迪亚兰布利亚是一种无处不在的人类病原体，是导致麦芽性腹泻的B类生物恐怖剂。贾第鞭毛虫生物学的最引人注目但最令人难以理解的方面之一是其细胞骨架：微丝是贾第鞭毛虫机械地附着在肠上皮细胞上的能力，因此允许寄生虫建立感染。该R21提案将通过识别微丝细胞骨架的新蛋白质并确定对附着的机制的生物物理理解的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明显不同，因此将其确定为重要的药物靶标。新型的肌动蛋白结合蛋白将通过生化，遗传和蛋白质组学手段，包括亲和色谱，沉积，酵母两杂种和质谱法鉴定。 （2）将贾第鞭毛虫附着在生物学和惰性底物上的附着将通过在质膜背侧表面的积分收缩（以产生细胞腹表面下的负压和吸收力）和在腹膜外膜周围（产生腹侧磁盘上产生胸膜）（产生clifter a clifter a Clifter a Clifter a Clifters a Climimeter）的集成收缩。使用流细胞创建流体动力剪切力，将首次对附着力进行定量。附着的机制将通过观察柔性或穿孔底物的依恋的动力学和力来确定，并使用总干扰反射反射水荧光（TIRAF）显微镜来量化寄生虫腹表面和底物之间的距离。此处提出的研究将为贾第鞭毛虫使用微丝产生依恋力的机制进行更详细的研究奠定基础。这项工作将促进我们对寄生虫发病机理的理解，并发展我们对微丝作为药物设计目标的了解。