Calcium signaling controls Pseudomonas aeruginosa invasion and adaptation to the host intracellular environment

钙信号控制铜绿假单胞菌入侵和适应宿主细胞内环境

• 批准号: 10851424
• 负责人: Marianna Patrauchan
• 金额: $ 8.81万
• 依托单位: OKLAHOMA STATE UNIVERSITY STILLWATER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10851424
• 关键词: Calcium Signaling; Cell membrane; Cells; Collaborations; Computer software; Cytoplasm; Development; Engineering; Environment; Extracellular Space; Fluorescence; Funding; Genetic; Goals; Homeostasis; Infection; Invaded; Knowledge; Membrane; Microscope; Monitor; Pattern; Phase; Pseudomonas aeruginosa; Research; Role; Side; Signal Transduction; Stains; Students; Training; calcium indicator; fluorescence microscope; human pathogen; improved; instrument; pathogen; periplasm; prevent; response; spatial relationship; student participation

ABSTRACT The currently funded research 2R15GM124670-02 is focused on discovering the role of Ca2+ signaling in the interactions of P. aeruginosa with host cells. These studies include establishing the signaling patterns of Ca2+ homeostasis by monitoring the changes of Ca2+ levels in P. aeruginosa during interactions with host cells, including invasion, intracellular survival, and escape. To achieve these goals, a careful examination of host and pathogen Ca2+ homeostasis on one side and their cellular responses to spatial and temporal Ca2+ alterations on the other is required. To monitor the changes in spatial relationship between Pa and host cells, we plan to use a combination of fluorescent tagging (GFP, RFP, YFP) and fluorescent (DAPI, Alexa Fluor Phalloidin) or immunofluorescent staining. To monitor micro-spatial and temporal translocations of free Ca2+ within subcellular compartments where Pa interacts with host cells, we plan to use the Ca2+-sensitive fluorescent Genetic Encoded Calcium Indicators (GECIs) engineered to localize near the outer membrane facing the extracellular space, in the periplasm, near the cytoplasmic membrane facing the cytoplasm, and in the cytoplasm. All these approaches are based on the application of fluorescent molecules. Therefore, we are requesting to support a purchase of Inverted Fluorescence Phase Contrast microscope, Leica DMi8, the premium-class modular research microscope. This will be our first fluorescence microscope in the lab. The requested instrument will be placed in the PI’s lab and will be available for the use by all the students in the PI’s lab and the collaborating lab of Dr. Lutter. All the participating students will be trained to use the instrument and the accompanying software by the company representative. The anticipated research advances will significantly enhance our ability to study calcium signaling in P. aeruginosa interacting with its eukaryotic host. The obtained new knowledge will improve our understanding of this important human pathogen and inform the development of more efficient means for controlling or preventing its deadly infections.

抽象的 目前资助的研究2R15GM124670-02专注于发现Ca2+的作用 铜绿假单胞菌与宿主细胞相互作用中的信号传导。这些研究包括建立 通过监测 P.Ca2+ 水平的变化来了解 Ca2+ 稳态的信号模式。 铜绿假单胞菌与宿主细胞相互作用，包括侵袭、细胞内存活和 逃脱。为了实现这些目标，需要仔细检查宿主和病原体的 Ca2+ 稳态 一方面是它们的细胞对空间和时间 Ca2+ 变化的反应，另一方面是 必需的。为了监测Pa和宿主细胞之间空间关系的变化，我们计划 结合使用荧光标记（GFP、RFP、YFP）和荧光标记（DAPI、Alexa Fluor 鬼笔环肽）或免疫荧光染色。监测微空间和时间易位 亚细胞区室中的游离 Ca2+ 在 Pa 与宿主细胞相互作用的地方，我们计划使用 Ca2+ 敏感荧光基因编码钙指示剂 (GECI) 设计用于 定位在面向细胞外空间的外膜附近，在周质中，靠近 细胞质膜面向细胞质，并位于细胞质中。所有这些方法都是 基于荧光分子的应用。因此，我们请求支持 购买顶级倒置荧光相差显微镜 Leica DMi8 模块化研究显微镜。这将是我们实验室中的第一台荧光显微镜。这 所请求的仪器将放置在 PI 的实验室中，可供所有人员使用 PI实验室和Lutter博士合作实验室的学生。所有参赛学生将 由公司代表培训如何使用仪器和随附软件。 预期的研究进展将显着增强我们研究钙信号传导的能力 铜绿假单胞菌与其真核宿主相互作用。获得的新知识将会提高 我们对这种重要的人类病原体的了解并为开发更有效的方法提供信息 控制或预防其致命感染的方法。