The Plasmodial Surface Anion Channel And Malaria Parasite Nutrient Acquisition

疟原虫表面阴离子通道与疟原虫营养获取

• 批准号: 8336147
• 负责人: SANJAY A DESAI
• 金额: $ 77.06万
• 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/8336147
• 关键词: Address; Affinity; Alleles; Amino Acids; Anions; Antibodies; Antimalarials; Basic Science; Binding; Biology; Birds; Cations; Cell physiology; Cells; Cellular biology; Chemicals; Chromosomes, Human, Pair 3; DNA; Erythrocytes; Future; Gene Family; Genes; Genetic; Genetic Crosses; Goals; Growth; Human; In Vitro; Infection; Informatics; Inheritance Patterns; Ion Channel; Ion Transport; Lipid Biochemistry; Malaria; Maps; Mediating; Membrane; Microsatellite Repeats; Molecular; Molecular Genetics; Molecular and Cellular Biology; Nutrient; Parasites; Peptide Hydrolases; Permeability; Pharmacology; Physiology; Plasma; Plasmodium falciparum; Play; Point Mutation; Predisposition; Primates; Property; Protein Biochemistry; Proteins; Quantitative Trait Loci; Rodent; Role; Sorbitol; Structure; Structure-Activity Relationship; Surface; Therapeutic Intervention; Transfection; Transmembrane Domain; Transmembrane Transport; Work; base; drug development; drug discovery; gene cloning; high throughput screening; inhibitor/antagonist; mutant; patch clamp; research study; small molecule libraries; solute; sugar; uptake

In 2011, the Apicomplexan Molecular Physiology Section made a fundamental contribution to understanding the molecular basis of increased erythrocyte permeability after infection with malaria parasites. Altered host cell permeability has been known for several decades, with various groups documenting the range of organic and inorganic solutes with increased uptake. Over the past ten years, patch-clamp studies from our group and from others determined that increased uptake results from the action of one or more ion channels. However, both the number of distinct ion channels and whether they are of host or parasite origin have been intensely debated. We have now addresses these questions by using a high-throughput inhibitor screen to find an isolate-specific PSAC antagonist (ISPA-28) that specifically blocks sorbitol uptake into red cells infected with the Dd2 parasite line; cells infected with the HB3 parasite line have channels that are blocked 800-fold less effectively. Transport studies with ISPA-28 revealed parallel differences in the ability to block uptake of sugars, amino acids, organic cations, and anions, indicating that a single shared ion channel mediates the uptake of these diverse solutes. Patch-clamp studies implicated PSAC as this shared ion channel. We then used a Dd2 x HB3 genetic cross generated by Dr. Thomas Wellems to track inheritance of ISPA-28 affinity. Our studies indicated relatively simple inheritance with most progeny clones expressing channels identical to one of the two parental lines. We then used QTL analysis with known microsatellite inheritance patterns to map ISPA-28 block to a single locus on the parasites chromosome 3. 15 genes from this locus were selected for DNA transfection experiments into malaria parasites to explore possible functional contributions to PSAC activity. piggyBac-mediated complementation and allelic exchange transfections implicated two related genes from the locus; these genes, known as clag3 genes, were previously thought to play roles in cytoadherence or invasion. We found that these genes undergo expression switching and used in vitro selections to identify parasites that express one or the other allele. These selections confirmed a role in PSAC formation and determined that ISPA-28 binds at a variable domain near the C-terminus of the channel protein. We also raised specific antibodies and used protease susceptibility to localize the protein to the host membrane, as required by functional studies of PSAC. Finally, a recently generated PSAC mutant was found to have a point mutation within a predicted transmembrane domain of the clag3 product, further supporting a central role of clag3 genes in formation of PSAC. Consistent with strict conservation of PSAC activity, the clag gene family is conserved in all rodent, avian and primate malaria species studied to date. As suggested by functional studies, this ion channel appears to serve an essential role for the intracellular parasite. Identification of the responsible genes provides a definitive target for therapeutic intervention against malaria.

2011年，APICOMPLEXAN分子生理部门为理解疟原虫感染后的红细胞渗透性增加的分子基础做出了基本贡献。 宿主细胞渗透率改变了数十年，各组都记录了摄取增加的有机和无机溶质范围。 在过去的十年中，来自我们小组的斑块钳研究确定摄取增加是由于一个或多个离子通道的作用而导致的。 但是，两种不同的离子通道的数量以及它们是否是宿主还是寄生虫来源都进行了激烈的争论。 现在，我们通过使用高通量抑制剂筛选来找到这些问题，以找到分离株特异性的PSAC拮抗剂（ISPA-28），该拮抗剂（ISPA-28）专门阻止了山梨糖醇摄取到感染DD2寄生虫线的红细胞中；感染HB3寄生虫线的细胞的通道障碍的有效较小800倍。 使用ISPA-28的运输研究表明，阻止糖，氨基酸，有机阳离子和阴离子的摄取能力平行差异，表明单个共享离子通道介导了这些多样的溶质的吸收。 斑块钳研究将PSAC视为该共享离子通道。 然后，我们使用了Thomas Wellems博士生成的DD2 X HB3遗传交叉来跟踪ISPA-28亲和力的遗传。 我们的研究表明，大多数后代克隆表达与两个亲本线相同的通道的继承相对简单。 然后，我们使用具有已知微卫星遗传模式的QTL分析将ISPA-28块映射到寄生虫3。3。15基因的单个基因座，从该基因座选择15个基因以将DNA转染实验转染到疟原虫中，以探索对PSAC活性的可能功能贡献。 Piggybac介导的互补和等位基因交换转染暗示了两个相关基因。这些基因被称为clag3基因，以前被认为在细胞疗法或侵袭中起着作用。 我们发现这些基因经历表达开关并在体外选择中使用以识别表达一个或另一个等位基因的寄生虫。 这些选择证实了在PSAC形成中的作用，并确定ISPA-28在通道蛋白的C末端附近的可变结构域结合。 我们还提出了特定的抗体，并使用蛋白酶的敏感性，根据PSAC的功能研究要求将蛋白质定位于宿主膜。 最后，发现最近生成的PSAC突变体在clag3产物的预测跨膜结构域中具有点突变，进一步支持了clag3基因在PSAC形成中的中心作用。 与严格的PSAC活性保存一致，在迄今为止研究的所有啮齿动物，鸟类和灵长类动物疟疾物种中，粘液基因家族均保守。 正如功能研究所建议的那样，该离子通道似乎对细胞内寄生虫起着至关重要的作用。 负责基因的识别为针对疟疾的治疗干预提供了明确的靶标。