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Investigation of the molecular and mechanistic basis for attachment by Giardia lamblia.

机译:贾第鞭毛虫附着的分子和机理基础的研究。

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摘要

Giardia lamblia is a protozoan parasite responsible for widespread diarrheal disease in humans and animals. Attachment to the host intestinal mucosa is necessary for establishing infection, but how Giardia performs this attachment is poorly understood. This work illuminates two aspects of Giardia attachment: a molecular analysis to identify microfilament-associated proteins and a mechanistic analysis to examine the dynamics of parasite attachment and detachment.The microfilament system plays an important role in attachment, yet no known microfilament protein except actin in Giardia has been identified. We developed an actin co-sedimentation/mass spectrometry assay using human beta-actin to identify possible microfilament-associated proteins in Giardia. We found that only Giardia actin and two proteins in the alpha-giardin family (alpha-1 giardin and alpha-7.3 giardin) were specifically enriched in F-actin pellets (P0.2). The specific co-existing of Giardia actin and F-actin suggests that the parasite and human actins can co-polymerize, an interesting finding for such divergent species. The two alpha giardins identified in this study belong to annexin family and localized to the plasma membrane, where actin also localizes, supporting an in vivo role for the observed in vitro interactions.The dynamic changes of the relative topology of substrate and parasite have never been observed and would provide important evidence to understand the attachment mechanism. We used total internal reflection fluorescence (TIRF) microscopy to observe parasites surface-labeled with Alexa-488 conjugated wheat germ agglutinin attach to a glass substrate. We found that the bare zone (a cytoplasmic protrusion through the structurally-unsupported center of the ventral disk), the lateral crest at the periphery of the ventral disk, and the lateral shields at the posterior end of the ventral disk are in closest apposition to the substrate, with the bare zone showing most the dramatic changes during attachment and detachment. In addition, observations of the dynamics of fluorescent microspheres indicated the presence of fluid flow under the surface of the ventral disk of attached parasites. These data provide support for a negative pressure model of attachment.Together these studies advance our understanding of both the machinery of attachment and the biomechanical properties of attachment and point to new directions for chemotherapeutic research.
机译:贾第鞭毛虫是一种原生动物寄生虫,可导致人类和动物广泛的腹泻病。对宿主肠道粘膜的附着对于建立感染是必要的,但是贾第鞭毛虫如何进行这种附着却知之甚少。这项工作阐明了贾第鞭毛虫附着的两个方面:分子分析以鉴定微丝相关蛋白和机制分析以检查寄生虫的附着和脱离的动力学。微丝系统在附着中起着重要作用,但除肌动蛋白外其他未知的微丝蛋白贾第虫已被鉴定。我们开发了一种肌动蛋白共沉淀/质谱测定法,使用人β-肌动蛋白来鉴定贾第鞭毛虫中可能的微丝相关蛋白。我们发现只有贾第鞭毛虫肌动蛋白和α-贾第蛋白家族中的两个蛋白质(α-1贾第蛋白和α-7.3贾第蛋白)在F-肌动蛋白沉淀中特异性富集(P <0.2)。贾第鞭毛虫肌动蛋白和F-肌动蛋白的特定共存表明,寄生虫和人肌动蛋白可以共聚,这对于这种不同的物种是一个有趣的发现。在这项研究中鉴定出的两个αgiardins属于膜联蛋白家族并位于质膜上,肌动蛋白也位于该膜上,支持观察到的体外相互作用的体内作用。从未发现底物和寄生虫相对拓扑的动态变化观察并为理解依恋机制提供重要证据。我们使用全内反射荧光(TIRF)显微镜观察了用Alexa-488共轭小麦胚芽凝集素表面标记的寄生虫,该寄生虫附着在玻璃基板上。我们发现裸露区域(穿过腹盘结构上不受支撑的中心的胞质突起),腹盘外围的侧面波峰和腹盘后端的侧面护盾与之最接近在基材上,裸露区域在附着和分离过程中显示出最明显的变化。另外,对荧光微球动力学的观察表明,在附着的寄生虫的腹盘表面下方存在流体流动。这些数据为附着的负压模型提供了支持。这些研究共同促进了我们对附着机制和附着生物力学特性的理解,并为化学治疗研究指明了新的方向。

著录项

  • 作者

    Luo, Haibei.;

  • 作者单位

    Georgetown University.;

  • 授予单位 Georgetown University.;
  • 学科 Biology Microbiology.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 159 p.
  • 总页数 159
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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