The Plasmodial Surface Anion Channel And Malaria Parasite Nutrient Acquisition

2013年，顶复门分子生理学科继续研究疟疾寄生虫感染后红细胞通透性增加的分子基础和生理作用。 我们的研究暗示了疟原虫感染后宿主红细胞膜上一种不寻常的离子通道。 这个通道，疟原虫表面阴离子通道（PSAC），是由clag 3基因，这是保守的，并限于疟疾寄生虫。 PSAC的确切组成仍然存在争议，因为clag 3基因与其他生物体中已知的离子通道没有明显的同源性。 我们正在努力通过生物化学、分子和遗传学研究来解决这种不确定性。 在一系列研究中，我们获得了clag 3基因和clag 2基因的独立证据，clag 2基因是寄生虫2号染色体上的寄生虫。 在这里，我们使用了两个杀稻瘟菌素S-抗性寄生虫线改变PSAC活性，以检查分子基础。 全基因组测序没有发现任何突变体的DNA水平的变化。 相反，表达谱显示clag 3和clag 2经历了这些突变体的显著沉默（相对于野生型亲本系，表达降低18至140倍）。 全基因组表达微阵列实验排除了寄生虫基因表达的全局变化。 clag基因的沉默是由于相关组蛋白上的特定表观遗传标记。 生物化学研究表明，沉默中止生产的通道蛋白。 通过在组成型启动子下表达clag 3基因来防止沉默的DNA转染阻止了杀稻瘟菌素S抗性的获得，揭示了这种抗药性机制需要基因沉默。 本研究通过定量分析clag基因沉默对PSAC活性的影响，有助于我们了解PSAC的结构和功能。 它还揭示了一种新的表观遗传机制的抗疟药物耐药性相关的不同寄生虫的目标。 我们还探讨了感染后红细胞通透性增加的确切作用。 来自许多小组的研究表明，PSAC可能1）在寄生虫营养获取中起作用，2）用于重塑红细胞阳离子浓度以使寄生虫受益，或3）对细胞内寄生虫没有重要作用。 在一项研究中，我们探讨营养收购作为一个可能的PSAC的作用，通过检查寄生虫杀死PSAC抑制剂。 我们发现，当外部营养浓度降低到生理水平时，来自不同类别的PSAC抑制剂表现出显著改善的功效;不阻断PSAC的抗疟药的类似研究显示功效没有变化，排除我们工程培养基的非特异性效应。 连锁分析，DNA转染实验，并产生一个特定的异位同源重组的选择都牵连clag 3基因的寄生虫生长抑制PSAC抑制剂。 这些发现表明PSAC在细胞内寄生虫营养获取中起作用。 他们应该刺激针对这种寄生虫通道的药物发现工作。 在一项进一步研究PSAC作用的独立研究中，我们通过在新型蔗糖基培养基中建立连续培养物，挑战了普遍接受的关于寄生虫离子需求的假设。 以蔗糖作为主要的杀虫剂，K+和Cl-作为主要的胞外离子，我们获得了寄生虫的生长和繁殖率与生理介质中的那些没有区别。 这些条件消除了长期已知的细胞内Na+通过PSAC的增加，不包括红细胞阳离子重塑的要求。 我们还解剖了Na+，K+和Cl-的需求，发现意想不到的低浓度的每种离子满足寄生虫的需求。 令人惊讶的是，生长没有受到不利影响，高达148 mM的K+，这表明低细胞外K+不是一个必要的触发红细胞入侵。 与此同时，裂殖子的出口和入侵需要一个阈值离子强度，这表明在这些阶段的大分子之间的关键静电相互作用。 这些发现提供了深入了解疟疾的跨膜信号传导，并揭示了宿主和寄生虫离子需求之间的根本差异。

In 2013, the Apicomplexan Molecular Physiology Section continued studies into the molecular basis and physiological role of increased erythrocyte permeability after infection with malaria parasites. Our studies implicate an unusual ion channel at the host erythrocyte membrane after infection with malaria parasites. This channel, the plasmodial surface anion channel (PSAC), is determined by clag3 genes, which are conserved in and restricted to malaria parasites. The precise composition of PSAC remains debated because the clag3 genes do not have obvious homology to known ion channels in other organisms. We are working to address this uncertainty with biochemical, molecular and genetic studies. In one line of studies, we obtained independent evidence for clag3 genes and additionally the clag2 gene, a paralog on parasite chromosome 2. Here, we used two blasticidin S-resistant parasite lines with altered PSAC activity to examine molecular basis. Whole-genome sequencing did not reveal DNA level changes in either mutant. Expression profiling instead revealed that clag3 and clag2 underwent marked silencing these mutants (18- to 140-fold reduced expression relative to the wild-type parental lines). Whole-genome expression microarray experiments excluded global changes in parasite gene expression. Silencing of the clag genes was due to specific epigenetic marks on associated histones. Biochemical studies revealed that silencing aborted production of the channel protein. DNA transfection to prevent silencing by expression of the clag3 gene under a constitutive promoter prevented acquisition of blasticidin S resistance, revealing that gene silencing is required by this drug resistance mechanism. This study contributes to our understanding of PSAC structure and function by quantifying the effects of clag gene silencing on PSAC activity. It also reveals a novel epigenetic mechanism of antimalarial drug resistance relevant to diverse parasite targets. We also explore the precise roles served by increased erythrocyte permeability after infection. Studies from numerous groups have suggested that PSAC may 1) function in parasite nutrient acquisition, 2) serve to remodel erythrocyte cation concentrations for parasite benefit, or 3) serve no essential role for the intracellular parasite. In one study, we explore nutrient acquisition as a possible PSAC role by examining parasite killing by PSAC inhibitors. We found that PSAC inhibitors from various classes exhibited markedly improved efficacy when external nutrient concentrations are reduced to physiological levels; similar studies with antimalarials that do not block PSAC showed no change in efficacy, excluding nonspecific effects of our engineered media. Linkage analysis, DNA transfection experiments, and selections yielding a specific ectopic homologous recombination all implicated clag3 genes in parasite growth inhibition by PSAC inhibitors. These findings indicate that PSAC functions in intracellular parasite nutrient acquisition. They should stimulate drug discovery efforts targeting this parasite channel. In an independent study that examined PSACs role further, we challenged generally accepted assumptions about the parasites ionic requirements by establishing continuous culture in novel sucrose-based media. With sucrose as the primary osmoticant and K+ and Cl- as the main extracellular ions, we obtained parasite growth and propagation at rates indistinguishable from those in physiological media. These conditions abolish long-known increases in intracellular Na+ via PSAC, excluding a requirement for erythrocyte cation remodeling. We also dissected Na+, K+, and Cl- requirements and found that unexpectedly low concentrations of each ion meet the parasites demands. Surprisingly, growth was not adversely affected by up to 148 mM K+, suggesting that low extracellular K+ is not an essential trigger for erythrocyte invasion. At the same time, merozoite egress and invasion required a threshold ionic strength, suggesting critical electrostatic interactions between macromolecules at these stages. These findings provide insights into transmembrane signaling in malaria and reveal fundamental differences between host and parasite ionic requirements.