Cellular and Molecular Physiology of Bloodstream Malaria Parasites

血流疟原虫的细胞和分子生理学

基本信息

批准号：
10927772
负责人：
SANJAY A DESAI
金额：
$ 160.56万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/10927772
关键词：
Acceleration Agonist Anions Antimalarials Biological Assay Biology Blood Circulation CRISPR/Cas technology Carrier Proteins Cation Pumps Cations Cell membrane Cell physiology Cells Cellular biology Cessation of life Chemicals Cholesterol Collaborations Complex Computational Biology DNA Development Dose Egtazic Acid Erythrocyte Membrane Erythrocytes Excision Fluorescent Dyes Future Genes Genetic Genetic study Glucose Goals Homeostasis Human Ion Channel Ion Transport Ionophores Ions Kinetics Knowledge Ligands Link Lipid Biochemistry Malaria Measurement Membrane Metabolic Molecular Molecular Biology Molecular Chaperones Molecular and Cellular Biology Mutation Nutrient Parasite resistance Parasites Parents Permeability Pharmaceutical Preparations Pharmacologic Substance Physiology Plasmids Plasmodium Plasmodium falciparum Process Property Protein Biochemistry Protein Export Pathway Pump Radioisotopes Regulation Research Resistance Structure Structure-Activity Relationship Surface TRPV1 gene Technology Transfection Transmembrane Transport Vaccines Vacuole Vanadates Work biophysical properties burden of illness drug development drug discovery extracellular gene cloning genetic manipulation high throughput screening human model human pathogen improved inhibitor insight model organism multidisciplinary mutant novel parasite genome pathogen small molecule libraries solute uptake vector mosquito

项目摘要

In 2023, the Apicomplexan Molecular Physiology Section Malaria examined Na+ and H+ homeostasis within the intracellular human malaria parasite, Plasmodium falciparum. This parasite maintains this homeostasis through the action of PfATP4, a cation pump localized to the intracellular parasite plasma membrane. PfATP4 is the target of advanced antimalarial leads, which produce many poorly understood metabolic disturbances within infected erythrocytes. To better understand these disturbances, we have now expressed the mammalian ligand-gated TRPV1 ion channel at the parasite plasma membrane to study ion regulation and examine the effects of cation leak. TRPV1 expression was well-tolerated, consistent with negligible ion flux through the nonactivated channel. TRPV1 ligands produced rapid parasite death in the transfectant line at their activating concentrations, but were harmless to the wild-type parent. Activation triggered cholesterol redistribution at the parasite plasma membrane, reproducing effects of PfATP4 inhibitors and directly implicating cation dysregulation in this process. In contrast to predictions, TRPV1 activation in low Na+ media accentuated parasite killing but a PfATP4 inhibitor had unchanged efficacy. Selection of a ligand-resistant mutant revealed a previously uncharacterized G683V mutation in TRPV1 that occludes the lower channel gate, implicating reduced permeability as a mechanism for parasite resistance to antimalarials targeting ion homeostasis. Our findings provide key insights into malaria parasite ion regulation and will guide mechanism-of-action studies for advanced antimalarial leads that act at the host-pathogen interface. PLoS One 18:e0283776 (2023). PMID: 37014920 In another study, we examined Ca++ uptake and efflux at the erythrocyte membrane. Ca++ is required for numerous cellular developmental activities and is required by intracellular malaria parasites. Despite its requirement, Ca++ is maintained at very low concentrations in human erythrocytes by an efficient PMCA Ca++ extrusion pump. Although much of our knowledge about this Ca++ extrusion pump derives from studies with human erythrocytes, kinetic studies of Ca++ transport for these cells are limited to radioisotope flux measurements. Here, we developed a robust, microplate-based assay for erythrocyte Ca++ efflux using extracellular fluorescent Ca++ indicators. We optimized Ca++ loading with the A23187 ionophore, established conditions for removal of the ionophore, and adjusted fluorescent dye sensitivity by addition of extracellular EGTA to allow continuous tracking of Ca++ efflux. Efflux kinetics were accelerated by glucose and inhibited in a dose-dependent manner by the nonspecific inhibitor vanadate, revealing that Ca++ pump activity can be tracked in a 384-well microplate format. These studies enable radioisotope-free kinetic measurements of the Ca++ pump and should facilitate screens for specific inhibitors of this essential transport activity. Eur. Biophys. J. 52:101-110 (2023). PMID: 36512028

2023年，Apicomplexan分子生理学部分疟疾检查了细胞内人类疟疾寄生虫疟原虫恶性疟原虫中的Na+和H+稳态。该寄生虫通过PFATP4的作用（一种阳离子泵）固定在细胞内寄生虫质膜。 PFATP4是晚期抗疟疾铅的靶标，它在感染的红细胞中产生了许多知识的代谢障碍。为了更好地理解这些干扰，我们现在已经表达了寄生虫质膜的哺乳动物配体门控的TRPV1离子通道，以研究离子调节并检查阳离子泄漏的影响。 TRPV1表达良好，与通过未激活通道微不足道的离子通量一致。 TRPV1配体以其激活浓度在转染线中产生快速的寄生虫死亡，但对野生型父母无害。激活触发寄生虫质膜的胆固醇再分布，pfatp4抑制剂的再现作用，并在此过程中直接牵涉到阳离子失调。与预测相反，低Na+培养基中的TRPV1激活加剧了寄生虫的杀戮，但PFATP4抑制剂具有不变的功效。选择配体的耐药突变体揭示了TRPV1中先前未表征的G683V突变，该突变会阻塞下通道栅极，这暗示了降低的渗透性是靶向离子稳态的寄生虫抗性的机制。我们的发现提供了对疟疾寄生虫离子调节的关键见解，并将指导在宿主 - 病原体界面作用的晚期抗疟疾铅的作用机理研究。 PLOS ONE 18：E0283776（2023）。 PMID：37014920 在另一项研究中，我们检查了红细胞膜上的Ca ++摄取和外排。 Ca ++是许多细胞发育活性所必需的，并且需要细胞内疟疾寄生虫。尽管需要它，但通过有效的PMCA CA ++挤出泵，Ca ++在人类红细胞中仍保持非常低的浓度。尽管我们对这种CA ++挤出泵的许多了解来自对人类红细胞的研究，但这些细胞的Ca ++转运的动力学研究仅限于放射性同位素通量测量。在这里，我们使用细胞外荧光Ca ++指示器开发了一种强大的基于微板的测定法，用于红细胞Ca ++外排。我们通过添加细胞外EGTA来优化用A23187离子载体，确定的离子载体和调整后的荧光染料敏感性的CA ++负载，以允许连续跟踪Ca ++流出。外排动力学通过葡萄糖加速，并通过非特异性抑制剂钒酸盐以剂量依赖性方式抑制，这表明Ca ++泵活性可以以384孔微孔板格式跟踪。这些研究实现了Ca ++泵的无放射性动力学测量，并应促进筛选该基本运输活性的特定抑制剂。欧元。生物。 J. 52：101-110（2023）。 PMID：36512028

项目成果

期刊论文数量（41）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Malaria parasites tolerate a broad range of ionic environments and do not require host cation remodelling.

DOI：
10.1111/mmi.12159
发表时间：
2013-04
期刊：
Molecular microbiology
影响因子：
3.6
作者：
Pillai AD;Addo R;Sharma P;Nguitragool W;Srinivasan P;Desai SA
通讯作者：
Desai SA

NK cells inhibit Plasmodium falciparum growth in red blood cells via antibody-dependent cellular cytotoxicity.

DOI：
10.7554/elife.36806
发表时间：
2018-06-26
期刊：
eLife
影响因子：
7.7
作者：
Arora G;Hart GT;Manzella-Lapeira J;Doritchamou JY;Narum DL;Thomas LM;Brzostowski J;Rajagopalan S;Doumbo OK;Traore B;Miller LH;Pierce SK;Duffy PE;Crompton PD;Desai SA;Long EO
通讯作者：
Long EO

Conditional permeabilization of the P. falciparum plasma membrane in infected cells links cation influx to reduced membrane integrity.

受感染细胞中恶性疟原虫质膜的条件透化将阳离子流入与膜完整性降低联系起来。