Host Ca2+, actin, and ATP production in rickettsia-endothelial cell dysfunction

立克次体内皮细胞功能障碍中宿主 Ca2、肌动蛋白和 ATP 的产生

• 批准号: 10659249
• 负责人: JOHN STEPHEN Dumler
• 金额: $ 18.98万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-05 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10659249
• 关键词: ATP phosphohydrolase; Actin-Binding Protein; Actins; Actomyosin; Adenine Nucleotide Translocase; Affect; Bacteria; Blood Vessels; Brain; Brazil; Buffers; Ca(2+)-Transporting ATPase; Calcium; Calcium Channel; Calcium Channel Blockers; Calmodulin; Case Fatality Rates; Cation Pumps; Cell Death; Cell Line; Cell physiology; Cells; Cellular Structures; Cessation of life; Chelating Agents; Colombia; Communicable Diseases; Consumption; Cytoskeleton; Cytosol; Data; Disease; Endoplasmic Reticulum; Endothelial Cells; Endothelium; Energy-Generating Resources; Event; Fatal Outcome; Functional disorder; Genetic Transcription; Genome; Germ Cells; Goals; Growth; Human; Hypotension; Impairment; In Vitro; Infection; Intervention; Invaded; Investigation; Link; Lung; Lyme Disease; Measures; Membrane; Metabolic; Mexico; Microscopy; Molecular; Monitor; Multiple Organ Failure; Myosin Light Chain Kinase; Nutrient; Nutritional; Organ; Organism; PIK3CG gene; PMCA1 protein; Parasites; Pathogenicity; Pathologic; Permeability; Phospholipase; Phosphorylation; Phosphotransferases; Preventive therapy; Process; Production; Proliferating; Property; Proteins; Pump; Regulation; Research; Resolution; Resources; Rickettsia; Rickettsia Infections; Rickettsia parkeri; Rickettsia rickettsii; Rocky Mountain Spotted Fever; Role; Sepsis; Shock; Signal Pathway; Signaling Molecule; Small Interfering RNA; System; Tick-Borne Infections; Time; Transcriptional Regulation; Vascular Endothelial Cell; Vascular Permeabilities; Virulence; Visualization; Work; adverse outcome; antagonist; blood perfusion; cadherin 5; calcium-dependent protein kinase; cell motility; chelation; design; electric impedance; fitness; hypoperfusion; improved; inhibitor; ischemic injury; microbial; monolayer; parasitism; pathogen; pharmacologic; prevent; receptor; recruit; release of sequestered calcium ion into cytoplasm; response; spotted fever; temporal measurement; voltage

Project Summary/Abstract Spotted fever group rickettsioses (SFGR) are in aggregate the second most common tick-borne infections in the U.S. and account for considerable severe disease and death, with case fatality rates over 10% in many regions of the world. Rocky Mountain spotted fever is the prototypical disease in this group for which the major pathophysiologic adverse consequence is increased vascular permeability, leading to hypotension, hypoperfusion, and ischemic injury to the lungs, brain and other organs. SFG rickettsiae infect endothelial cells and parasitize host ATP for growth and spread. Our preliminary studies showed that regulation of calcium flux in infected endothelial cell barriers using calcium chelators or specific calcium channel blockers abrogates vascular permeability. We propose that spotted fever rickettsiae utilize a nutritional virulence strategy to subvert host calcium handling – through an acquired “channelopathy” - that drives increases in access to cellular metabolic substrates, but that in turn damages key functions of endothelial cells. Our hypothesis is that increasing competition for cellular ATP by rickettsiae impairs ATP-dependent Ca2+ pumps, which in turn leads to increased intracellular calcium concentrations, activation of Ca2+ channels, Ca2+-dependent kinases, signaling pathways and transcriptional regulation that increase cellular content of key nutrients for rickettsial growth. The consequences of improved rickettsial fitness ultimately leads to reorganization of cellular cytoskeleton, disassembly of inter-endothelial junctions, and inevitably, increased vascular permeability. The proposal will measure intracellular calcium and ATP concentrations in the presence or absence of calcium channel-blocking agent, while monitoring for endothelial barrier dysfunction in both early and late stages of Rickettsia parkeri infection. Evidence of a role for calcium flux in rickettsial vascular permeability includes localized calcium “puffs” with transient focal cytosolic ATP depletion and local cytoskeletal restructuring as early post-invasion events. This is predicted to be followed by global increases in intracellular calcium, ATP depletion, cytoskeleton restructuring, and inter-endothelial junction disassembly with massive rickettsial proliferation, sensitive to buffering cytosolic calcium concentrations through chelation or blocking or silencing of key calcium channels. Rickettsial load will also be determined to understand the effect of these manipulations on microbial fitness. This work will identify key mechanisms that permit changes in vascular permeability with spotted fever rickettsia infection, perhaps driven by rickettsial effector proteins, and potential targets for host-based pharmacologic intervention to prevent severe and fatal outcomes of SFGR and potentially other infectious diseases.

项目总结/摘要 斑点热群立克次体病（SFGR）是美国第二大常见的蜱传感染， 造成相当严重的疾病和死亡，世界许多地区的病死率超过10%。落基 山地斑疹热是这一人群的典型疾病，其主要病理生理不良后果是 血管通透性增加，导致低血压、灌注不足和肺、脑和其他组织的缺血性损伤。 机关SFG立克次体感染内皮细胞并寄生宿主ATP以进行生长和扩散。我们的初步研究 显示，使用钙螯合剂或特异性钙离子调节感染的内皮细胞屏障中的钙通量， 通道阻断剂消除血管通透性。我们认为斑点热立克次体利用营养毒力 通过获得性“通道病”破坏宿主钙处理的策略， 代谢底物，但这反过来又损害了内皮细胞的关键功能。我们的假设是 立克次氏体对细胞ATP的竞争损害ATP依赖性Ca 2+泵，这反过来又导致增加 细胞内钙浓度、Ca 2+通道激活、Ca 2+依赖性激酶、信号传导途径和 转录调节增加立克次体生长的关键营养物质的细胞含量。改善的后果 立克次体适应性最终导致细胞骨架的重组，内皮间连接的解体， 不可避免地增加了血管通透性。该提案将测量细胞内钙和ATP浓度， 存在或不存在钙通道阻滞剂，同时监测两种早期 和晚期立克次体感染。钙流在立克次体血管通透性中作用的证据 包括局部钙“泡沫”与短暂的局灶性细胞溶质ATP耗竭和局部细胞骨架重建， 入侵后的事件。据预测，这之后是细胞内钙的整体增加，ATP耗竭， 细胞骨架重组，内皮间连接解体，伴有大量立克次体增殖，对 通过螯合或阻断或沉默关键钙通道来缓冲胞质钙浓度。立克次 还将测定负荷以了解这些操作对微生物适合度的影响。这项工作将确定 斑点热立克次体感染导致血管通透性改变的关键机制，可能是由 立克次体效应蛋白，以及基于宿主的药物干预以预防严重和致命的 SFGR和其他潜在传染病的结果。