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，顶复门分子生理学组疟疾研究组检查了 clag3 基因在疟原虫感染红细胞宿主膜上疟原虫表面阴离子通道 (PSAC) 形成中的作用。 这种保守的通道在寄生虫营养吸收中发挥着重要作用，并且是经过验证的抗疟靶点。 为了检查 CLAG3 的贡献，我们首先通过 7G8 x GB4 遗传杂交中的连锁分析确定了 clag3 的核心作用。 然后，我们使用 DNA 转染来产生 90% CLAG3 敲低的恶性疟原虫品系。 尽管对 CLAG3 的作用有信心的基因图谱，这种敲低却表现出显着保留的营养和溶质吸收。更令人惊讶的是，尽管存在定量敲低，转运仍然对 CLAG3 同种型特异性抑制剂敏感，这表明低剂量的 CLAG3 转基因足以产生阻断。 接下来，我们生产了一个完整的 CLAG3 敲除系，发现它表现出运输活性的不完全丧失，与 rhoph2 和 rhoph3 相比，这两个 PSAC 相关基因不能被破坏，因为它们缺失时营养吸收会被消除。尽管 CLAG3 敲除在标准营养丰富的培养条件下不会产生适应性成本，但这种寄生虫无法在更类似于人类血浆的改良培养基中繁殖。这些研究表明其他染色体编码的 CLAG 蛋白可以补偿 CLAG3 的损失。 我们的研究结果还表明 CLAG 蛋白寡聚形成 PSAC 孔。 他们还揭示，CLAG3 在标准体外条件下是可有可无的，但在生理条件下繁殖是必需的。 PLoS 病原体 (2020) 16(2):e1008363。 doi：10.1371/journal.ppat.1008363；电话号码：32069335。 在另一项研究中，我们检查了寄生虫编码的 RhopH 复合物的运输和组装，该复合物由 CLAG3、RhopH2 和 RhopH3 组成。 我们利用活细胞成像和 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，我们生成了单标记和双标记寄生虫系并进行了活细胞荧光测量。 这些寄生虫允许在合成后不久追踪这些蛋白质在棒状细胞器内的位置和关联。 在细胞内寄生虫周期结束时，子代裂殖子离开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寄生液泡中，从那里它们被输出并运输到红细胞膜上。 CLAG3 和 RhopH2 在宿主膜上保持完全结合。 荧光强度测量发现，当红细胞感染两种寄生虫时，输出的 RhopH 蛋白的化学计量增加；全细胞膜片钳揭示了 PSAC 功能拷贝数的伴随增加以及 RhopH 对离子和营养物渗透性贡献的剂量效应。 这项研究首次展示了人类疟疾寄生虫的活细胞 FRET 成像。 我们的研究结果表明，RhopH 亚基在不解离的情况下运输至其宿主膜目的地，并表明对 PSAC 形成的定量贡献。 mBio (2020)，正在出版。