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

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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.