DYNAMICS OF CRYPTOSPORIDIUM PARVUM HOST-CELL INTERACTION

小隐孢子虫宿主细胞相互作用的动力学

• 批准号: 5205561
• 负责人: Jeffrey K Griffiths
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5205561
• 关键词: Cryptosporidium; active transport; apical membrane; brush border membrane; cell cell interaction; cell death; confocal scanning microscopy; cytoplasm; cytoskeleton; fluorescent dye /probe; genetic regulation; genetic transcription; histochemistry /cytochemistry; host organism interaction; intracellular transport; life cycle; membrane permeability; microinjections; organelles; polymerase chain reaction; spores; tissue /cell culture; Cryptosporidium; active transport; apical membrane; brush border membrane; cell cell interaction; cell death; confocal scanning microscopy; cytoplasm; cytoskeleton; fluorescent dye /probe; genetic regulation; genetic transcription; histochemistry /cytochemistry; host organism interaction; intracellular transport; life cycle; membrane permeability; microinjections; organelles; polymerase chain reaction; spores; tissue /cell culture

The goal of this continuing collaborative project is to a assess key subcellular and molecular events that occur in the Cryptosporidium infected host cell that may provide rational targets for drug therapy. We have shown that C, parvum induces a set of profound changes in Caco-2 cell monolayers, such as cell and monolayer increase permeability, cell death, and cytoskeletal changes. Building on these results, we have 2 major specific aims and 3 minor specific aims; 1) Characterize the host-parasite interface. The feeder organelle is a unique interface between the cytoplasms of the parasite and the host. The function of the feeder organelle is unknown. It is the likely route for nutrient utilized by the para site. It is also the likely route that any therapeutic drug must take to enter the parasite. Using a variety of techniques including microinjection, selective permeabilization, and passive permeabilization to load the host cell confocal microscopy as they transit from the host cytosol into the parasite. 2) Identify the cytoskeletal components that form the electron dense bands beneath the feeder organelle, and characterize host cytoskeletal regulation induced by the parasite. Beneath the unique feeder organelle lie electron structural building blocks, the kinetic of appearance, and the regulation f actin and other key host cytoskeletal elements in relation to these changes, using IFA techniques and PCR. 3) Determine how infection alters the apical membranes of the host, as reflected in the presence of sucrase-isomaltase, an apical brush border enzyme. The parasite causes extensive remodelling of the apical cell surface. Little is known about the functional significance of this remodelling, including that of the parasitophorous membrane. We will characterize these changes using a marker for the apical membrane surface, sucrase-isomaltase. 4) Genetically alter Caco-2 cells to prevent or retard parasite induced death. No continuous in vitro culture method exists, forcing propagation of the parasite in animals. We will introduce the bcl-2 gene into Caco-2 cells in the hopes of preventing programmed cell death, and if successful characterize the differences between parental and transformed cell lines. Early cell death in vitro may be responsible for poor cell culture propagation of C. parvum. 5) Confirm observations that the oocyst has a delivery an targeting function. We have preliminary evidence that the oocyst delivers and targets the sporozoite to the epithelial cell surface, protecting it from attack in the lumen of the gut. If true this could have major ramifications ont he design of luminally active therapeutic drugs.

这个持续的协作项目的目标是评估密钥 感染隐孢子虫中发生的亚细胞和分子事件 可能为药物治疗提供合理靶标的宿主细胞。 我们有 表明C，Parvum诱导Caco-2细胞的一系列深刻变化 单层，例如细胞和单层增加渗透率，细胞死亡， 和细胞骨架变化。 在这些结果的基础上，我们有2个专业 具体目的和3个次要特定目标； 1）表征主寄生虫界面。 馈线细胞器是 寄生虫和宿主的细胞肿块之间的独特接口。 这 馈线细胞器的功能尚不清楚。 这可能是 Para网站使用的营养素。这也是任何可能的路线 必须服用治疗药物才能进入寄生虫。 使用各种 包括显微注射，选择性通透性和 被动透化以加载宿主细胞共聚焦显微镜 从宿主胞质溶胶转移到寄生虫。 2）确定形成电子致密带的细胞骨架成分 在进料器细胞器的下方，并表征宿主细胞骨架调节 由寄生虫诱导。 在独特的进料器细胞器谎言电子之下 结构构建块，外观动力学和调节 f肌动蛋白和其他关键宿主与这些有关 更改，使用IFA技术和PCR。 3）确定感染如何改变宿主的顶膜，为 反映在Sucrase-Isomaltase的存在（顶端刷子边框）的存在下 酶。 寄生虫引起顶端细胞的大量重塑 表面。 对此的功能意义知之甚少 重塑，包括寄生膜的重塑。 我们将 使用顶膜表面的标记来表征这些变化， Sucrase-Isomaltase。 4）基因改变CACO-2细胞以预防或延迟寄生虫诱导 死亡。 不存在连续的体外培养方法，迫使繁殖 动物中的寄生虫。 我们将将Bcl-2基因引入CACO-2 为了防止编程的细胞死亡，如果成功的话，细胞 表征父母和转化的细胞系之间的差异。 体外早期细胞死亡可能导致细胞培养不良 C. parvum的传播。 5）确认观察到卵囊具有靶向 功能。 我们有初步证据表明卵囊提供了和 将孢子岩靶向上皮细胞表面，保护它免受 在肠道内攻击。 如果是真的，这可能会有重大 分析的阵容活性治疗药物的设计。