Cellular and Molecular Physiology of Bloodstream Malaria Parasites

血流疟原虫的细胞和分子生理学

• 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
• 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

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 Molecular Physiology Section Malaria研究了clag 3基因在疟疾寄生虫感染的红细胞宿主膜上形成疟原虫表面阴离子通道（PSAC）中的作用。 这种保守的通道在寄生虫的营养吸收中起着重要作用，是一种经过验证的抗疟靶点。 为了检查CLAG 3贡献，我们首先通过连锁分析在7 G8 X GB 4遗传杂交中确立了clag 3的中心作用。 然后，我们使用DNA转染来产生具有90%CLAG3敲低的恶性疟原虫系。 尽管有信心的CLAG 3的作用的遗传图谱，这种敲低表现出显着保存营养和溶质的吸收。甚至更令人惊讶的是，尽管定量敲低，转运仍然对CLAG 3亚型特异性抑制剂敏感，表明低剂量的CLAG 3转基因足以产生阻断。 接下来，我们产生了一个完整的CLAG 3敲除系，并发现它表现出运输活性的不完全丧失，与rhoph 2和rhoph 3相反，这两个PSAC相关基因不能被破坏，因为它们缺失时营养吸收被消除。虽然CLAG 3敲除在标准营养丰富的培养条件下不会产生适应性成本，但这种寄生虫不能在更类似于人血浆的改良培养基中繁殖。这些研究表明，由其他染色体编码的CLAG蛋白可以补偿CLAG 3的丢失。 我们的研究结果还表明CLAG蛋白的寡聚化形成PSAC孔。 它们还揭示了CLAG 3在标准体外条件下是可降解的，但在生理条件下是繁殖所需的。PLoS Pathogens（2020）16（2）：e1008363. doi：10.1371/journal.ppat.1008363; PMID：32069335. 在另一项研究中，我们研究了寄生虫编码的RhopH复合物的运输和组装，该复合物由CLAG 3，RhopH 2和RhopH 3组成。 我们探讨了RhopH亚基之间的蛋白质-蛋白质相互作用，使用活细胞成像和Frster共振能量转移（FRET）实验。 使用GFP衍生物，我们产生了单和双标记的寄生虫系，并进行了活细胞荧光测量。 这些寄生虫允许跟踪的位置和这些蛋白质之间的关联在棒状体细胞器合成后不久。 在细胞内寄生虫周期结束时，子裂殖子排出并重新侵入新的红细胞。 我们标记的RhopH亚基被转移到新的宿主细胞的寄生空泡中，从那里它们被输出并运输到红细胞膜。 CLAG 3和RhopH 2在宿主细胞膜上保持完全结合。 荧光强度测量确定出口RhopH蛋白质的化学计量增加时，红细胞感染两种寄生虫;全细胞膜片钳技术揭示了PSAC功能拷贝数的同时增加和RhopH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活细胞FRET成像在人类疟疾寄生虫。 我们的研究结果表明，RhopH亚基交通到其宿主膜的目的地没有解离，并建议定量贡献PSAC的形成。 mBio（2020），in press.