CYTOSTOME-FOOD VACUOLE INTERACTIONS IN PLASMODIUM FALCIPARUM

恶性疟原虫中细胞造口术-食物泡的相互作用

• 批准号: 8172288
• 负责人: Theodore F Taraschi
• 金额: $ 1.08万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-07 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8172288
• 关键词: Antimalarials; Atlases; Azithromycin; Biological Preservation; Case Study; Cell membrane; Cells; Cessation of life; Child; Chloroquine; Computer Retrieval of Information on Scientific Projects Database; Cytosol; Data Collection; Development; Disease; Drug resistance; Dynamin; Electrons; Erythrocytes; Event; Figs - dietary; Food; Freezing; Funding; Grant; Guanosine Triphosphate Phosphohydrolases; Hemoglobin; Human; Image; Ingestion; Inner mitochondrial membrane; Institution; Knowledge; Lead; Learning; Liver; Malaria; Membrane; Membrane Fusion; Microtomy; Modeling; Morphology; Organelles; Pamphlets; Parasitemia; Parasites; Pathway interactions; Phase; Plasmodium falciparum; Pregnancy; Pregnant Women; Preventive; Procedures; Process; Proteins; Rattus; Research; Research Personnel; Resolution; Resources; Route; Source; Specimen; Staining method; Stains; Structure; Surface; Techniques; Three-Dimensional Image; Three-Dimensional Imaging; Three-dimensional analysis; Tissues; Tomogram; Transport Process; Triad Acrylic Resin; Tube; Tubular formation; United States National Institutes of Health; Vaccines; Vacuole; Vesicle; World Health Organization; abstracting; comparative; crista membrane; cryogenics; electron tomography; falls; feeding; global health; improved; inhibitor/antagonist; mortality; pressure; prevent; programs; reconstruction; small molecule; tomography; transmission process; Antimalarials; Atlases; Azithromycin; Biological Preservation; Case Study; Cell membrane; Cells; Cessation of life; Child; Chloroquine; Computer Retrieval of Information on Scientific Projects Database; Cytosol; Data Collection; Development; Disease; Drug resistance; Dynamin; Electrons; Erythrocytes; Event; Figs - dietary; Food; Freezing; Funding; Grant; Guanosine Triphosphate Phosphohydrolases; Hemoglobin; Human; Image; Ingestion; Inner mitochondrial membrane; Institution; Knowledge; Lead; Learning; Liver; Malaria; Membrane; Membrane Fusion; Microtomy; Modeling; Morphology; Organelles; Pamphlets; Parasitemia; Parasites; Pathway interactions; Phase; Plasmodium falciparum; Pregnancy; Pregnant Women; Preventive; Procedures; Process; Proteins; Rattus; Research; Research Personnel; Resolution; Resources; Route; Source; Specimen; Staining method; Stains; Structure; Surface; Techniques; Three-Dimensional Image; Three-Dimensional Imaging; Three-dimensional analysis; Tissues; Tomogram; Transport Process; Triad Acrylic Resin; Tube; Tubular formation; United States National Institutes of Health; Vaccines; Vacuole; Vesicle; World Health Organization; abstracting; comparative; crista membrane; cryogenics; electron tomography; falls; feeding; global health; improved; inhibitor/antagonist; mortality; pressure; prevent; programs; reconstruction; small molecule; tomography; transmission process

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: Malaria is a devastating illness that each year infects over 500 million people, resulting in the death of over 1 million people the majority of which are pregnant women and children (WHO, 2008). There are currently no vaccines against Plasmodium falciparum, the parasite responsible for the mortality associated with this disease and drug resistance to current therapies is rapidly spreading (Chico et al., 2008). In Lazarus et al., 2008, we proposed a new model for the transport of hemoglobin from the host erythrocyte cytosol to the digestive vacuole (DV) in intracellular malaria parasites (Fig. 1). This appears to involve a double-membrane, tubular structure formed from the vacuolar membrane that surrounds the intra-erythrocytic parasite (PVM) and the parasite plasma membrane (PPM). This structure is referred to as a cytostome. We have collaborated with the RVBC at the Wadsworth Center to produce a tomogram of an infected erythrocyte that supports a key feature of our new model (Fig. 2). We have many examples from thin section transmission EM of steps 2 and 3, but previously had only captured one image that could represent steps 4 and 5. We suspect that there is a fusion event between the double membrane tubular cytostome and the parasite DV, which is required for hemoglobin transfer. The morphological characterization of the cytostomal pathway is vital to understanding key events in the hemoglobin transport process. We've demonstrated that the delivery of host cell hemoglobin to the DV of intra-erythrocytic P. falciparum parasites is an obligate parasite process, as inhibition of this route arrested parasite development and lead to the elimination of parasitemia from culture (Lazarus, Schneider, and Taraschi, 2008). Treatment with a small molecule inhibitor which targets the GTPase activity of dynamin, a protein believed to be associated with cytostome, caused the disruption of cytostome morphology and prevented hemoglobin transport to the DV, resulting in the arrest of parasite development. These results validated the cytostome feeding mechanism as a new target for antimalarial therapy. Experimental Plan: We suspect that the membranes of the cytostome and the DV fuse, but we've been unable to visualize this event. Likewise the electron dense collar that is observed around the cytosome membrane probably is involved in its formation. Its preservation in conventionally stained and embedded sections is poor, preventing us from learning anything useful about its subunit structure (possibly dynamin rings). The current tomographic reconstruction is promising but we need to move to cryogenic specimen preparative procedures to maximize the information obtained from 3D analysis. Parasitized erythrocytes could be rapidly frozen using the RVBC's BalTec high-pressure freezer, then either freeze-substituted and conventionally sectioned, or kept in the frozen hydrated state and thinned for tomographic data collection by FIB-milling (TRD1). Comparative results from the RVBC on conventionally prepared and frozen-hydrated tissue and isolated organelles give us confidence that our ability to detect evidence of membrane fusion events and substructures involved in membrane remodeling or interactions will be greatly improved with frozen-hydrated specimens (Hsieh et al., 2006; Mannella et al., 2006a; Renken et al., 2009). Full 360¿ tilt range tomography (TRD1) would be a major advantage for determining the structure of the cylindrical cytosomal collar in sections cut from the freeze-substituted material. Likewise, use of phase plates to enhance contrast (TRD2) should yield better resolution with the beam-sensitive frozen-hydrated specimens than is currently possible with extreme defocus phase contrast. If the collars are identical or fall into definable classes, it will be possible to apply averaging techniques (Renken et al., 2009) which would greatly improve the resolution of the collar structure. We expect that the unique facilities and expertise available at the Wadsworth Center will help us generate 3D images of the intracellular parasite that will greatly advance our knowledge of the critical hemoglobin transport pathway. If we are successful, this information will be of great benefit to our program to identify small molecules that inhibit this process and could help guide the development of new antimalarials. References Chico, RM, R Pittrof, B Greenwood and D Chandramohan. 2008. Azithromycin-chloroquine and the intermittent preventive treatment of malaria in pregnancy Malar.J 7:255. Hsieh CE, A Leith, CA Mannella, J Frank and M Marko. 2006. Towards high-resolution three-dimensional imaging of native mammalian tissue: electron tomography of frozen-hydrated rat liver sections. J.Struct. Biol., 153:1-13. Lazarus MD, TG Schneider and TF Taraschi. 2008. A new model for hemoglobin ingestion and transport by the human malaria parasite Plasmodium falciparum J Cell Sci. 121:1937-1949. Mannella CA. 2006. Structure and dynamics of mitochondrial inner membrane cristae Biochim. Biophys. Acta 1763:542-548. WHO. World Health Organization, Global Health Atlas. (2008--pamphlet), Renken C, C Hsieh, M Marko, B Rath, A Leith, T Wagenknecht, J Frank and CA Mannella, 2009. Structure of frozen-hydrated triad junctions: a case study in motif searching inside tomograms. J. Struct. Biol. 165:53-63. (cover picture) Fig. 1: Key Events in Hemoglobin Transport to the Parasite DV. 1) Cytostome nucleation  indentation of PVM / PPM and formation of electron dense collar. 2) Cytostome elongation and formation of a hemoglobin filled cytostome. 3) Cytostomal tube elongation  a hemoglobin-filled tube extends to the DV. 4) PVM fusion/fission  PVM fuses and membrane fission creates a hemoglobin containing vesicle. 5) PPM fusion/fission  PPM fuses at the parasite surface and with the DV and delivers a single-membrane hemoglobin containing vesicle to the DV. Steps 4 and 5 are thought to be very rapid events.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 抽象的： 疟疾是一种毁灭性的疾病，每年都会感染超过5亿的人，导致超过100万人死亡，其中大多数是孕妇和儿童（Who，2008年）。目前没有针对恶性疟原虫的疫苗，寄生虫是导致与这种疾病相关的死亡率和对当前疗法耐药性的抗死亡率（Chico等，2008）。在Lazarus等人，2008年，我们提出了一种新的模型，用于在细胞内疟疾寄生虫中从宿主红细胞胞质醇转运到消化效果（DV）的新模型（图1）。这似乎涉及由围绕着肉眼内寄生虫（PVM）和寄生虫质膜（PPM）形成的双膜管状结构。该结构称为细胞击败。我们已经与Wadsworth Center的RVBC合作，制作了一个感染的红细胞的整整图，该图层支持我们新模型的关键特征（图2）。我们有许多来自步骤2和3的薄截面传输EM的示例，但以前只捕获了一个可以代表步骤4和5的图像。我们怀疑双膜管状细胞固醇与寄生虫DV之间存在融合事件，这是血红蛋白转移所必需的。途径对于理解血红蛋白传输过程中的关键事件至关重要。我们已经证明，将宿主细胞血红蛋白传递到肉毒p. p. p. p. parciparum寄生虫的DV是一个强大的寄生虫过程，因为抑制这种途径阻碍了寄生虫的发展并导致消除寄生虫从培养中消除（Lazarus，Schneider，Schneider和Taraschi，2008年）。用小分子抑制剂治疗Dynamin的GTPase活性，该蛋白质（一种被认为与细胞抑制剂相关的蛋白质）导致细胞抑制剂形态的破坏并阻止血红蛋白转运到DV，从而导致寄生虫发育的停滞。这些结果验证了胞质剂喂养机制，作为抗疟疾治疗的新靶标。 实验计划：我们怀疑细胞击和DV保险丝的机制，但我们无法可视化这一事件。同样，在胞体膜周围观察到的电子致密领可能与其形成有关。它在常规染色和嵌入的部分中的保存很差，使我们无法学习任何有关其亚基结构（可能是dynamin环）有用的东西。当前的断层扫描重建是有希望的，但我们需要采取急速标本准备的程序，以最大程度地从3D分析获得的信息。使用RVBC的Baltec高压冰柜可以快速冷冻寄生的红细胞，然后是冻结取代的和 常规切片或保持冷冻水合状态，并通过纤维填充（TRD1）进行层析成像数据。 RVBC的比较结果对常规制备和冷冻水合的组织和分离的细胞器使我们有信心，我们有信心检测膜融合事件的证据和涉及膜重塑或相互作用的子结构的能力将与Frozen-Hydrated样品相比，将大大改进（HSIEH等，2006; Mannella et and; ren an; ren。完整的360—倾斜范围断层扫描（TRD1）将是确定从冻结叠加材料切割的截面中圆柱细胞球衣领结构的主要优势。同样，使用相板来增强对比度（TRD2）应与横梁敏感的冷冻水合样品相比，与当前极为散焦相对的对比相比。如果项圈相同或属于可确定的类别，则可以应用平均技术（Renken等，2009），这将大大改善项圈结构的分辨率。我们预计，沃兹沃思中心可用的独特设施和专业知识将帮助我们生成细胞内寄生虫的3D图像，这将大大提高我们对关键血红蛋白传输途径的了解。如果我们成功，这些信息将对我们的计划有很大的好处，以识别抑制此过程的小分子，并可以帮助指导新的抗疟药的发展。 参考 Chico，RM，R Pittrof，B Greenwood和D Chandramohan。 2008。阿奇霉素 - 氯喹和妊娠疟疾的间歇性预防治疗疟疾。J7：255。 Hsieh CE，Leith，CA Mannella，J Frank和M Marko。 2006。迈向天然哺乳动物组织的高分辨率三维成像：冷冻水合大鼠肝切片的电子断层扫描。 J. -Struct。 Biol。，153：1-13。 Lazarus MD，TG Schneider和TF Taraschi。 2008年。人疟疾寄生虫疟原虫J细胞Sci摄入血红蛋白的新模型和运输。 121：1937-1949。 Mannella CA。 2006。线粒体内膜Cristae Biochim的结构和动力学。生物。 ACTA 1763：542-548。 WHO。世界卫生组织，全球健康地图集。 （2008-邮编）， Renken C，C Hsieh，M Marko，B Rath，A Leith，T Wagenknecht，J Frank和Ca Mannella，2009年。冷冻水合的三合会结构的结构：用于搜索内部图像中的主题的案例研究。 J. struct。生物。 165：53-63。 （封面图片） 图1：血红蛋白转运到寄生虫DV的关键事件。 1）PVM / PPM的胞核成核缩进和电子致密领的形成。 2）血红蛋白填充的细胞抑制剂的细胞抑制和形成。 3）细胞抑制管的伸长量延伸到DV。 4）PVM融合/裂变PVM融合和膜裂变会产生含有囊泡的血红蛋白。 5）ppm融合/裂变ppm在寄生虫表面和DV处融合，并提供一个包含含量的单个膜血红蛋白到DV。步骤4和5被认为是非常迅速的事件。