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，顶复门分子生理学部分为了解疟疾寄生虫感染后红细胞通透性增加的分子基础做出了根本性贡献。 几十年来，宿主细胞渗透性的改变已为人所知，各个研究小组都记录了吸收增加的有机和无机溶质的范围。 在过去的十年中，我们小组和其他人的膜片钳研究确定，吸收的增加是由一个或多个离子通道的作用引起的。 然而，不同离子通道的数量以及它们是宿主还是寄生虫来源一直存在激烈争论。 现在，我们通过使用高通量抑制剂筛选来解决这些问题，以找到一种分离物特异性PSAC拮抗剂（ISPA-28），该拮抗剂可以特异性阻止感染Dd2寄生虫系的红细胞摄取山梨醇；感染 HB3 寄生虫系的细胞的通道被阻断的效率低 800 倍。 ISPA-28 的转运研究揭示了阻断糖、氨基酸、有机阳离子和阴离子吸收能力的平行差异，表明单一共享离子通道介导这些不同溶质的吸收。 膜片钳研究表明PSAC 是这种共享离子通道。 然后，我们使用 Thomas Wellems 博士生成的 Dd2 x HB3 遗传杂交来追踪 ISPA-28 亲和力的遗传。 我们的研究表明遗传相对简单，大多数后代克隆表达的通道与两个亲本系之一相同。 然后，我们使用已知微卫星遗传模式的 QTL 分析将 ISPA-28 块映射到寄生虫 3 号染色体上的单个基因座。选择来自该基因座的 15 个基因进行疟疾寄生虫的 DNA 转染实验，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涉及该位点的两个相关基因；这些基因被称为 clag3 基因，以前被认为在细胞粘附或侵袭中发挥作用。 我们发现这些基因经历表达转换并使用体外选择来识别表达一种或另一种等位基因的寄生虫。 这些选择证实了在 PSAC 形成中的作用，并确定 ISPA-28 结合在通道蛋白 C 末端附近的可变结构域。 我们还按照PSAC 功能研究的要求，制备了特异性抗体并使用蛋白酶敏感性将蛋白质定位到宿主膜上。 最后，发现最近产生的PSAC突变体在clag3产物的预测跨膜结构域内具有点突变，进一步支持clag3基因在PSAC形成中的核心作用。 与 PSAC 活性的严格保守性一致，clag 基因家族在迄今为止研究的所有啮齿动物、鸟类和灵长类疟疾物种中都是保守的。 正如功能研究表明的那样，该离子通道似乎对细胞内寄生虫发挥着重要作用。 责任基因的鉴定为疟疾的治疗干预提供了明确的目标。