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，Apicomexan分子生理学研究组疟疾研究了clag3基因在疟疾寄生虫感染的红细胞膜上形成疟原虫表面阴离子通道(PSAC)中的作用。这个保守的通道在寄生虫的营养吸收中起着至关重要的作用，是一个有效的抗疟疾靶标。为了检验CLAG3的贡献，我们首先通过连锁分析确定了CLAG3在7G8×GB4遗传杂交中的核心作用。然后，我们用DNA转染法获得了一株CLAG3基因敲除率为90%的恶性疟原虫。尽管对CLAG3s的作用有信心的遗传图谱，但这种敲除显示出显著保存的营养和溶质吸收。更令人惊讶的是，尽管CLAG3基因被定量敲除，但转运仍然对CLAG3亚型特异性抑制剂敏感，这表明低剂量的CLAG3转基因足以阻断。接下来，我们制作了一个完整的CLAG3基因敲除系，发现它表现出不完全的运输活性丧失，而不是Rhph2和Rhph3，这两个与PSAC相关的基因不能被干扰，因为它们的营养吸收在没有它们的情况下被取消。尽管在标准的营养丰富的培养条件下，CLAG3基因敲除不会产生健康成本，但这种寄生虫不能在更接近于人类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蛋白可以弥补CLAG3的丢失。我们的发现还表明，CLAG蛋白的寡聚作用形成了PSAC孔。它们还揭示了CLAG3在标准的体外条件下是必不可少的，但在生理条件下是繁殖所必需的。《公共科学图书馆·病原体》(2020)16(2)：e1008363。DOI：10.1371/Joural.ppat.1008363；PMID：32069335。 在另一项研究中，我们研究了寄生虫编码的Rhop H复合体的贩运和组装，该复合体由CLAG3、Rhop H2和Rhop H3组成。我们使用活细胞成像和Frster共振能量转移(FRET)实验研究了Rhop H亚基之间的蛋白质-蛋白质相互作用。使用GFP衍生物，我们产生了单标记和双标记寄生虫系，并进行了活细胞荧光测量。这些寄生虫在它们合成后不久就可以跟踪这些蛋白质在杆状细胞器中的位置和联系。在细胞内寄生虫周期结束时，子裂殖子离开并重新侵入新的红细胞。我们标记的RHopH亚基被转移到新的寄主细胞的寄生液泡中，从那里它们被输出并运输到红细胞膜。CLAG3和Rhop H2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Rhop H蛋白的化学计量比增加；全细胞膜片钳显示PSAC功能拷贝数随之增加，并且Rhop H对离子和营养通透性的贡献存在剂量效应。这项研究首次展示了人类疟疾寄生虫的活细胞FRET成像。我们的发现表明，Rhop H亚单位没有解离地运输到它们的宿主膜目的地，这表明它对PSAC的形成有定量的贡献。MBio(2020)，正在印刷中。