Biochemical And Biophysical Parasitology

生物化学和生物物理寄生虫学

• 批准号: 8156811
• 负责人: Fuyuki Tokumasu
• 金额: $ 27.56万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 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/8156811
• 关键词: Biochemical; Parasitology

In this FY, we accomplished and advanced several important projects. They are (1) NO coordination with heme species, (2) Identifications of unique properties of parasite-derived intracellular membranes, (3) study of time-dependent protein trafficking of tetracysteine-tagged parasite KAHRP protein, (4) initiation of new project studying gametocyte metabolism and drug resistance relationship. (1) In this FY, we expanded previous project studying NO production inside parasites. We found that products of two putative parasite genes for NO-producing enzymes are expressed inside parasite. Using resonance Raman spectroscopy, we also found that NO produced in situ interact with heme species and affect equilibrium of crystallization of heme into hemozoin, which directly relates to toxicity of heme against parasites. See the 'research advance' section for more information. This work has been recently published in the Journal of Experimental Parasitology (Early electronic publication). (2) We continued studying parasite-derived membranes with unique biophysical properties. During invasion process, erythrocyte membrane-derived parasitophorous vacuole (PVM) surrounds invading parasite. PVM continuously increase in its size as parasites mature. In addition, new membranes emerge from PVM and form MC. This structure is thought to play crucial roles in trafficking and delivery processes for parasite-derived proteins. However, the source of lipid molecules enough to support the size increase in the new membrane structure is not known. A key to solve this question is cholesterol content as parasite does not have a cholesterol synthesis mechanism. In addition, cholesterol is one of the most important molecules for membrane biophysical properties. The potentially unique membrane properties of PVM and MC could provide important insights for their functions responsible for efficient trafficking and delivery of parasite proteins to host erythrocyte membrane. In previous FY, we identified unique biophysical properties of PVM and MC using Fluorescence Lifetime Microscopy (FLIM) and fluorophores (Di4-ANEPPDHQ) sensitive to cholesterol enriched membrane domains. The data suggested that these membranes have cholesterol level between host erythrocyte membrane and parasite membrane. The data suggested that membrane exchange or transfer occurred during parasite invasion and intraerythrocytic stages. In this FY year, we identified the lipid exchange/transfer between parasite and host erythrocyte membrane. The mixture of Di4-ANEPPDHQ-labelled erythrocytes and non-labeled matured parasites resulted in fluorescently positive PVMs and parasites in the ring stage in the next cycle. This fluorescence expanded throughout the parasite and PVM as it developed to matured stage, but fluorescence intensity at host membrane decreased at later stages, showing that lipid/membrane was transferred from host erythrocyte membrane to parasites during invasion process as well as following intraerythrocytic stages. In contrast, non-labeled erythrocyte mixed with labeled mature parasite did not show fluorescence from host membrane, suggesting the lipid/membrane transfer seems to be directional. On the other hand, non-parasitized erythrocytes labeled with fluorescent cholesterol analogue resulted in labeled new ring and mature parasites but host erythrocytes did not show a significant decrease in fluorescence intensity. These results suggest that lipid transfer mechanism may not be the same between cholesterol and non-sterol lipids such as phospholipids. The manuscript for these data is now under preparation. (3) In previous FY, we established protocol and methods for studying parasite protein trafficking using tetracysteine (TC) tag. we successfully engineered KAHRP-GFP-TC (control) and KAHRP-TC constructs. ReAsH-TC labeling of KAHRP-GFP-TC fusion proteins confirmed expression of TC tag and a comparable fluorescence pattern of protein distribution as GFP fluorescence, showing the capability of TC tag to be used for study of protein trafficking in a live condition. In this year, we selectively labeled old and new proteins with biarsenical fluorophores with two different colors in live condition. We quantified the time required for new protein reach MC from parasite body as well as for the protein to reach host erythrocyte membrane. Our results suggested that 5 hours was required for newly synthesized proteins appear in MC and 9 hours for host erythrocyte membrane appearance. We also found not all proteins were delivered to erythrocyte cytoplasm, but remained inside parasite. These results show that the tetracysteine technology can provide information on a new dynamic aspects of protein trafficking/delivery mechanism which is not otherwise possible with the common GFP-labeling. (4) In this year, we applied our expertise and knowledge on lipid membrane and microscopy technique and initiated a new project studying gametocyte metabolism related to drug resistance. During intraerythrocytic stages of P. falciparum, some parasites start forming gametocyte under the stress such as reductions in energy sources. Gametocytes develop through 5 different stages and eventually become gametes. However, physiological and molecular events for gametocyte metabolism are largely unknown. One physical characteristic is that gametocyte produce a large amount of internal membranes. We also confirmed this using lipid analogue fluorescent dye, Di-4 ANEPPDHQ. Majority of new membranes were produced in erythrocyte cytoplasm as a part of parasitophorous vacuole (PVM) or extension of PVM. However, labeling with Filipin III, a cholesterol-binding fluorophore, did not show any increase in cholesterol in gametocytes, suggesting only the amount of phospholipids increased. We also found in the experiment with reactive oxygen species indicator that gametocytes have significantly lower superoxide level as compare to erythrocytic stages. This was an interesting observation because it suggested that digestions of amino acids from erythrocyte hemoglobin may have decreased and established the new energy source/acquisition pathway. We are currently investigating this possibility by biochemical assay as well as fluorescence indicators for various intracellular species. On the other hand, gametocytes are known to be resistant against many of antimalarial drugs, including chloroquine. Since no known mechanism exist to explain the resistance, we start studying the expression level of PfCRT, a parasite-induced membrane protein whose mutation is responsible for the chloroquine resistance in intraerythrocytic stages. Preliminary result indicated that gametocyte did not show any reactivity against PfCRT antibody in later than stage II. Since wild-type PfCRT is essential for normal parasite growth in asexual stages, no PfCRT expression by IFA suggested that altered food vacuole function in gametocyte. Additional experiments are being performed to answer how PfCRT expression are controlled in gametocyte stages, and to study the general gametocyte energy metabolism. Collaborative Projects (Jichi Med school): We start observing MC structure by scanning electron microscope to study parasite-derived membrane system essential for protein trafficking across the host erythrocyte cytoplasm. By removing the top half membrane of substrate attached erythrocyte, we directly observed inside surface of erythrocyte membrane. Preliminary results showed that MCs were associated with erythrocyte membrane and they are connected physically by tethering membranous structure. This structure was never been reported although it was suggested. This tether sructure may provide important means for protein transport from MC to host erythrocyte membrane.

在此FY中，我们完成并提高了几个重要项目。 They are (1) NO coordination with heme species, (2) Identifications of unique properties of parasite-derived intracellular membranes, (3) study of time-dependent protein trafficking of tetracysteine-tagged parasite KAHRP protein, (4) initiation of new project studying gametocyte metabolism and drug resistance relationship. （1）在此FY中，我们扩大了先前的项目，研究了寄生虫内部没有生产。 我们发现，在寄生虫中表达了两个用于无生产酶的两个推定寄生虫基因的产物。 使用共振拉曼光谱法，我们还发现，没有产生原位与血红素物种相互作用，并影响血红素结晶为血元的平衡，这直接与血红素对寄生虫的毒性有关。 有关更多信息，请参见“研究进步”部分。 这项工作最近发表在《实验寄生虫学杂志》（早期电子出版物）上。 （2）我们继续研究具有独特生物物理特性的寄生虫衍生的膜。 在入侵过程中，红细胞膜衍生的寄生虫液泡（PVM）围绕入侵寄生虫。 随着寄生虫的成熟，PVM的大小不断增加。 此外，新膜从PVM和MC形成。 人们认为这种结构在寄生虫衍生的蛋白质的运输和输送过程中起着至关重要的作用。 然而，尚不清楚脂质分子的来源足以支持新膜结构的尺寸增加。 解决这个问题的关键是胆固醇含量，因为寄生虫没有胆固醇的合成机制。 此外，胆固醇是膜生物物理特性最重要的分子之一。 PVM和MC的潜在独特膜特性可以为其功能提供重要的见解，以使寄生虫蛋白有效地运输和递送寄生膜膜。 在以前的FY中，我们使用荧光寿命显微镜（FLIM）和荧光团（DI4-ANEPPDHQ）鉴定了PVM和MC的独特生物物理特性，对富含胆固醇的膜结构域敏感。 数据表明，这些膜在宿主红细胞膜和寄生虫膜之间具有胆固醇水平。 数据表明，膜交换或转移发生在寄生虫入侵和炎症细胞内阶段。 在这一年中，我们确定了寄生虫和宿主红细胞膜之间的脂质交换/转移。 DI4-AnePPDHQ标记的红细胞和未标记的成熟寄生虫的混合物在下一个循环中导致荧光阳性PVM和寄生虫。 这种荧光在整个寄生虫和PVM中扩展到成熟阶段，但是在后期宿主膜上的荧光强度降低，表明在入侵过程中以及术中术中脂肪/膜从宿主的红细胞膜转移到寄生虫。 相比之下，未标记的红细胞与标记的成熟寄生虫混合在一起没有显示宿主膜的荧光，这表明脂质/膜转移似乎是方向性的。 另一方面，用荧光胆固醇类似物标记的非寄生红细胞导致标记的新环和成熟的寄生虫，但宿主的红细胞并未显示出荧光强度的显着降低。 这些结果表明，脂质转移机制在胆固醇和非脆脂质（例如磷脂）之间可能不相同。 这些数据的手稿现在正在准备中。 （3）在以前的FY中，我们建立了使用四环素皮质（TC）标签研究寄生虫蛋白运输的方案和方法。 我们成功地设计了KAHRP-GFP-TC（对照）和KAHRP-TC构建体。 KAHRP-GFP-TC融合蛋白的REASH-TC标记证实了TC TAG的表达和蛋白质分布的可比荧光模式作为GFP荧光，显示了TC TAG的能力，用于研究活物条件下蛋白质运输的能力。 在今年，我们选择性地将新旧蛋白质标记为具有两种不同颜色的Biarsenical荧光团的新蛋白质。 我们量化了从寄生虫体以及到达宿主红细胞膜的蛋白质到达MC所需的时间。 我们的结果表明，新合成蛋白需要5小时出现在MC中，而9小时才能出现宿主红细胞膜的外观。 我们还发现，并非所有蛋白质都递送到红细胞细胞质中，而是留在寄生虫中。 这些结果表明，四半半胱氨酸技术可以提供有关蛋白质运输/递送机制的新动态方面的信息，而常见的GFP标记不可能。 （4）今年，我们应用了有关脂质膜和显微镜技术的专业知识和知识，并启动了一个研究与耐药性有关的配子细胞代谢的新项目。在恶性疟原虫的肠内肉体内阶段，一些寄生虫开始在应力下形成配子细胞，例如能源减少。 配子细胞通过5个不同的阶段发展，并最终成为配子。 但是，配子细胞代谢的生理和分子事件在很大程度上尚不清楚。 一个物理特征是配子细胞产生大量的内膜。 我们还使用脂质模拟荧光染料DI-4 aneppdHQ证实了这一点。 大多数新膜是在红细胞细胞质中产生的，作为寄生虫液泡（PVM）的一部分或PVM延伸。 然而，用胆固醇结合荧光团菲律宾III标记并未显示配子细胞胆固醇的升高，这仅表明只有磷脂量增加。 我们还在用活性氧指标的实验中发现，与红细胞相比，配子细胞具有明显较低的超氧化物水平。 这是一个有趣的观察结果，因为它表明来自红细胞血红蛋白的氨基酸的消化可能已经减少并确定了新的能源/采集途径。我们目前正在通过生化测定法以及各种细胞内物种的荧光指标研究这种可能性。 另一方面，众所周知，配子细胞对包括氯喹在内的许多抗疟药具有抗性。 由于没有已知的机制来解释抗性，因此我们开始研究PFCRT的表达水平，PFCRT是一种寄生虫诱导的膜蛋白的表达水平，其突变是促炎细胞阶段的氯喹抗性。 初步结果表明，在第二阶段，配子细胞没有对PFCRT抗体的任何反应性。 由于野生型PFCRT对于无性阶段的正常寄生虫生长至关重要，因此IFA的PFCRT表达没有表明在配子细胞中的食物液泡功能改变。 正在进行其他实验，以回答如何在配子细胞阶段控制PFCRT表达，并研究一般的配子型能量代谢。 协作项目（Jichi Med School）：我们通过扫描电子显微镜开始观察MC结构，以研究寄生虫衍生的膜系统对于整个宿主红细胞细胞质的蛋白质运输至关重要。 通过去除附着的红细胞的底物的上半膜，我们直接观察到红细胞膜的表面。 初步结果表明，MC与红细胞膜有关，并且通过束缚膜结构与物理连接。 尽管提出了这种结构，但从未报告过这种结构。该系带可能为从MC到宿主红细胞膜的蛋白质转运提供重要手段。