Pseudomonas aeruginosa (Pa) is an opportunistic pathogen that causes severe infections in immunocompromised individuals, including those with cystic fibrosis (CF). Pa forms antibiotic-resistant biofilms, making infections difficult to treat and often lethal. In the CF lung, elevated Ca2+ concentrations are encountered by Pa, which our group has shown to induce virulence factor production. To understand the molecular mechanisms behind this regulation, we identified the novel inner membrane protein CarP, which plays a crucial role in Ca2+-induced pyocyanin production, oxidative stress tolerance, Ca2+ homeostasis, and host immune interactions. Although CarP’s molecular function remains unknown, we hypothesize it interacts with other proteins. To test this, we utilized a FLAG tag co-immunoprecipitation approach, generating two expression constructs: membrane-bound and periplasmic CarP, each with a 3xFLAG tag fused to the C-terminus. Pull-down assays will be performed with each construct, and periplasmic and cytoplasmic samples will be analyzed by mass spectrometry to identify CarP’s protein-binding partners. This research will provide important insights into CarP’s molecular function and the mechanisms of Ca2+-induced virulence in Pa.
