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分子生理科为了解感染疟疾寄生虫后红细胞通透性增加的分子基础做出了重要贡献。几十年来，宿主细胞渗透性的改变已经为人所知，不同的小组记录了有机和无机溶质的范围，并增加了吸收。在过去的十年里，我们小组和其他人进行的膜片钳研究确定，摄取增加是一个或多个离子通道作用的结果。然而，关于不同离子通道的数量以及它们是来自宿主还是来自寄生虫，一直存在激烈的争论。我们现在已经解决了这些问题，方法是使用高通量的抑制剂筛选，找到一种分离株特异性的PSAC拮抗剂(ISPA-28)，它能特异性地阻止受DD2寄生虫系感染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要低800倍。用ISPA-28进行的转运研究表明，在阻止糖、氨基酸、有机阳离子和阴离子的吸收方面存在平行的差异，表明单一的共享离子通道介导了这些不同溶质的吸收。膜片钳研究表明PSAC是这种共享的离子通道。 然后，我们使用Thomas Wellems博士创造的DD2×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，大多数后代无性系表达的通道与两个亲本之一相同。然后，我们利用已知的微卫星遗传模式进行QTL分析，将ISPA-28区块定位到寄生虫3号染色体上的一个单一基因座上。从该基因座选择15个基因进行DNA导入疟疾寄生虫的实验，以探索可能对PSAC活性的功能贡献。该基因涉及两个相关基因，称为clag3基因，以前被认为在细胞黏附或侵袭中起作用。我们发现这些基因经历了表达切换，并使用体外选择来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ISPA-28在PSAC形成中的作用，并确定ISPA-28结合在通道蛋白C末端附近的可变区。根据PSAC功能研究的需要，我们还提出了特异性抗体，并利用蛋白酶敏感性将该蛋白定位到宿主膜上。最后，最近产生的PSAC突变体被发现在clag3产物的预测跨膜区内有点突变，进一步支持了clag3基因在PSAC形成中的中心作用。 与PSAC活性的严格保守一致，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。功能研究表明，这种离子通道似乎对细胞内寄生虫起着至关重要的作用。对致病基因的识别为疟疾的治疗干预提供了一个明确的目标。