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，顶复体分子生理组为理解疟原虫感染后红细胞通透性增加的分子基础做出了基础性贡献。宿主细胞通透性的改变在几十年前就已为人所知，不同的研究小组记录了有机和无机溶质吸收增加的范围。在过去的十年里，我们小组和其他人的膜片钳研究确定，增加的摄取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否来自宿主或寄生虫一直存在激烈的争论。我们现在通过使用高通量抑制剂筛选找到一种分离特异性PSAC拮抗剂（ISPA-28）来解决这些问题，该拮抗剂可以特异性地阻断山梨醇摄取到感染Dd2寄生虫系的红细胞；感染HB3寄生虫系的细胞的通道被阻断的效率降低了800倍。ISPA-28的转运研究揭示了在阻断糖、氨基酸、有机阳离子和阴离子的摄取能力方面的平行差异，表明一个共享的离子通道介导了这些不同溶质的摄取。膜片钳研究暗示PSAC是这个共享离子通道。