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 当前资助的研究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的信号传导。这些研究包括建立 通过监测P中Ca2+水平的变化，Ca2+稳态的信号传导模式。 在与宿主细胞相互作用的过程中，铜绿菌，包括侵袭，细胞内存活和 逃脱。为了实现这些目标，请仔细检查宿主和病原体CA2+稳态 一侧及其对另一侧对空间和临时CA2+改变的响应是 必需的。为了监视PA与宿主细胞之间空间关系的变化，我们计划 结合荧光标签（GFP，RFP，YFP）和荧光（DAPI，Alexa Fluor） 腓林蛋白）或免疫荧光染色。监视微空间和临时易位 PA与宿主单元相互作用的亚细胞隔室内的自由Ca2+ CA2+敏感荧光遗传编码的钙指标（GECIS）设计为 位于面向细胞外空间的外膜附近，在periplasm中，附近 细胞质膜面向细胞质和细胞质中。所有这些方法是 基于荧光分子的应用。因此，我们要求支持 购买倒荧光相的对比显微镜Leica DMI8，高级级 模块化研究显微镜。这将是我们实验室中的第一个荧光显微镜。这 请求的仪器将放置在Pi的实验室中，并将用于所有使用 PI实验室的学生和Lutter博士的合作实验室。所有参加的学生将 经过培训，可以由公司代表使用该仪器和参与软件。 预期的研究进展将显着增强我们研究钙信号传导的能力 在铜绿假单胞菌中与其真核宿主相互作用。获得的新知识将有所改善 我们对这一重要人类病原体的理解，并为发展效率提高 用于控制或防止其致命感染的手段。