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 enable the accurate measurements of the temporal and spatial changes of Ca2+ concentration within subcellular spaces of P. aeruginosa cells, we request to replace the currently obsolete plate reader Biotek with the updated version of the instrument, Cytation 5. The new instrument offers enhanced capabilities and accuracy of measurements. It allows low-noise reading of monochromator-based absorbance, tunable fluorescence, and luminescence. Importantly, it will also include imaging using 20X – 60X objectives, high contrast brightfield and four different imaging cubes for fluorescence. It allows tight temperature control with a 4-zone temperature control system and prevents condensation on plate lids. It offers dual reagent injectors able for multimode detection. 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.

NARRATIVE Calcium is an essential intracellular messenger in eukaryotes, regulating major cellular functions. Calcium signaling in bacteria also controls key processes including their ability to infect human hosts. However, the molecular mechanisms of calcium signaling in bacteria are not well known, and its role in the interactions between bacterial pathogens and host cells has not been recognized. The proposed research will characterize the molecular events involved in the transition of the human pathogen of high clinical importance into host cells. This new knowledge is necessary to understand the behavior of human pathogens during invasion and will ultimately enable developing means to control devastating infections.