Cellular and Molecular Physiology of Bloodstream Malaria Parasites

2020年，apiccomplexes an Molecular Physiology Section Malaria研究了clag3基因在疟原虫感染红细胞宿主膜上形成疟原虫表面阴离子通道（plasmodial surface阴离子通道，PSAC）中的作用。这一保守通道在寄生虫营养摄取中起着重要作用，是一种经过验证的抗疟疾靶点。为了检验CLAG3的贡献，我们首先通过7G8 x GB4遗传杂交的连锁分析确定了CLAG3的中心作用。然后，我们使用DNA转染产生了CLAG3敲除90%的恶性疟原虫。尽管对CLAG3s的作用有信心，但这种基因敲除表现出显著保存的营养和溶质摄取。更令人惊讶的是，尽管定量敲除，转运对CLAG3亚型特异性抑制剂仍然敏感，这表明低剂量的CLAG3转基因足以产生阻断作用。接下来，我们制作了一个完整的CLAG3敲除系，并发现它表现出运输活性的不完全丧失，与rhoph2和rhoph3相反，这两个psac相关基因不能被破坏，因为营养吸收在它们缺失时被取消。虽然CLAG3敲除在标准富营养培养条件下不会造成适应性损失，但这种寄生虫不能在更接近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的缺失。我们的发现还表明CLAG蛋白的寡聚化形成了PSAC孔。他们还揭示了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。科学通报，2020,16(2):e1008363。doi: 10.1371 / journal.ppat.1008363;PMID: 32069335。

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.