Cellular and Molecular Physiology of Bloodstream Malaria Parasites

血流疟原虫的细胞和分子生理学

基本信息

批准号：
10272080
负责人：
SANJAY A DESAI
金额：
$ 135.58万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/10272080
关键词：
Animal Model Anions Antimalarials Biology Blood Circulation CRISPR/Cas technology Carrier Proteins Cell physiology Cells Cellular biology Chemicals Chromosome Mapping Chromosomes Collaborations Complex Computational Biology DNA Daughter Destinations Dissociation Dose Energy Transfer Erythrocyte Membrane Erythrocytes Exhibits Fluorescence Future Genes Genetic Genetic Crosses Genetic study Goals Human Image In Vitro Ion Channel Ion Transport Ions Journals Knock-out Link Lipid Biochemistry Location Maintenance Malaria Measurement Membrane Methods Modification Molecular Molecular Chaperones Molecular Genetics Molecular and Cellular Biology Nutrient Organelles Parasites Permeability Pharmaceutical Preparations Pharmacologic Substance Physiological Physiology Plasma Plasmodium Plasmodium falciparum Property Protein Biochemistry Protein Export Pathway Protein Isoforms Proteins Research Role Structure Structure-Activity Relationship Surface Technology Transfection Transgenes Transmembrane Transport Vaccines Vacuole Work biophysical properties burden of illness cost drug development drug discovery experimental study fitness gene cloning genetic linkage analysis genetic manipulation high throughput screening human model human pathogen inhibitor/antagonist knock-down live cell imaging multidisciplinary mutant parasite genome patch clamp pathogen preservation protein protein interaction rhoptry small molecule libraries solute trafficking uptake

项目摘要

In 2020, the Apicomplexan Molecular Physiology Section Malaria examined the role of clag3 genes in formation of the plasmodial surface anion channel (PSAC) at the host membrane of malaria-parasite infected erythrocytes. This conserved channel serves an essential role in parasite nutrient uptake and is a validated antimalarial target. To examine CLAG3 contribution, we first established a central role of clag3 through linkage analysis in the 7G8 x GB4 genetic cross. We then used DNA transfection to produce a P. falciparum line with 90% CLAG3 knockdown. Despite confident genetic mapping of CLAG3s role, this knockdown exhibited remarkably preserved nutrient and solute uptake. Even more surprisingly, transport remained sensitive to a CLAG3 isoform-specific inhibitor despite quantitative knockdown, indicating that low doses of the CLAG3 transgene are sufficient to confer block. Next, we produced a complete CLAG3 knockout line and found it exhibits an incomplete loss of transport activity, in contrast to rhoph2 and rhoph3, two PSAC-associated genes that cannot be disrupted because nutrient uptake is abolished in their absence. Although the CLAG3 knockout did not incur a fitness cost under standard nutrient-rich culture conditions, this parasite could not be propagated in a modified medium that more closely resembles human plasma. These studies suggest that CLAG proteins encoded by other chromosomes can compensate for loss of CLAG3. Our findings also suggest oligomerization of CLAG proteins to form the PSAC pore. They also reveal that CLAG3 is dispensable under standard in vitro conditions but required for propagation under physiological conditions. PLoS Pathogens (2020) 16(2):e1008363. doi: 10.1371/journal.ppat.1008363; PMID: 32069335. In another study, we examined trafficking and assembly of the parasite-encoded RhopH complex, consisting of CLAG3, RhopH2 and RhopH3. We explored protein-protein interactions between RhopH subunits using live-cell imaging and Frster resonance energy transfer (FRET) experiments. Using the GFP derivatives, we generated single- and double-tagged parasite lines and performed live-cell fluorescence measurements. These parasites permitted tracking of the location and association between these proteins within rhoptry organelles shortly after their synthesis. At the end of the intracellular parasite cycle, daughter merozoites egress and reinvade new erythrocytes. Our tagged RhopH subunits were transferred into the new host cells parasitophorous vacuole, from where they were exported and trafficked to the erythrocyte membrane. CLAG3 and RhopH2 remained fully associated at the host membrane. Fluorescence intensity measurements identified stoichiometric increases in exported RhopH protein when erythrocytes are infected with two parasites; whole-cell patch-clamp revealed a concomitant increase in PSAC functional copy number and a dose effect for RhopH contribution to ion and nutrient permeability. This study is the first to demonstrate live-cell FRET imaging in human malaria parasites. Our findings reveal that RhopH subunits traffic to their host membrane destination without dissociation, and suggest quantitative contribution to PSAC formation. mBio (2020), in press.

2020年，Apicomplexan分子生理部门疟疾研究了粘液岩 - 寄生虫感染的红细胞的宿主膜上粘液3基因在形成质粒表面阴离子通道（PSAC）中的作用。该保守的通道在寄生虫营养摄取中起着至关重要的作用，并且是经过验证的抗疟疾靶标。为了检查clag3贡献，我们首先通过7G8 x GB4遗传杂交中的连锁分析确立了clag3的核心作用。然后，我们使用DNA转染产生了一个具有90％CLAG3敲低的恶性疟原虫线。尽管clag3s角色有信心的遗传图，但这种敲低的营养和溶质摄取表现出明显保存的营养。更令人惊讶的是，尽管定量敲低，运输仍然对clag3同工型特异性抑制剂保持敏感，这表明低剂量的clag3转基因足以赋予阻塞。接下来，我们产生了一条完整的clag3敲除线，发现它表现出不完全的运输活性损失，与Rhoph2和Rhoph3相比，这两个与PSAC相关的基因无法破坏，因为在不存在的情况下养分摄取的养分。尽管在标准营养丰富的培养条件下，clag3敲除并未产生适应性成本，但在更类似于人血浆的改良培养基中，这种寄生虫不能传播。这些研究表明，由其他染色体编码的作粘液蛋白可以补偿作粘液的损失。我们的发现还表明，粘液蛋白的低聚以形成PSAC孔。他们还表明，在标准的体外条件下，clag3是可支配的，但在生理条件下传播所必需的。 PLOS病原体（2020）16（2）：E1008363。 doi：10.1371/journal.ppat.1008363; PMID：32069335。在另一项研究中，我们检查了由clag3，Rhoph2和Rhoph3组成的寄生虫编码综合体的运输和组装。我们使用活细胞成像和FRSTER共振能量转移（FRET）实验探索了rhoph亚基之间的蛋白质 - 蛋白质相互作用。使用GFP衍生物，我们生成了单标记的寄生虫线，并进行了活细胞荧光测量。这些寄生虫允许跟踪这些蛋白质在合成后不久这些蛋白质之间的位置和关联。在细胞内寄生虫周期结束时，女儿梅罗洛斯的出口并重新染色了新的红细胞。我们的标记的rhoph亚基被转移到新的宿主细胞寄生虫液泡中，从那里出口并被贩运到红细胞膜。 clag3和Rhoph2在宿主膜上保持完全关联。当红细胞感染了两个寄生虫时，荧光强度测量结果鉴定出出口rhoph蛋白的化学计量增加。全细胞斑块钳显示，PSAC功能拷贝数的增加以及对离子和营养渗透性的贡献的剂量效应。这项研究是第一个证明人类疟疾寄生虫中的活细胞FRET成像的研究。我们的发现表明，偶然的亚基在没有解离的情况下流入其宿主膜目的地，并建议对PSAC形成的定量贡献。 MBIO（2020），印刷中。