Calcium-Regulated Protein CarP Responds to Multiple Host Signals and Mediates Regulation of Pseudomonas aeruginosa Virulence by Calcium.

Pseudomonas aeruginosa is an opportunistic pathogen causing life-threatening infections. Previously, we showed that elevated calcium (Ca2+) levels increase the production of virulence factors in P. aeruginosa In an effort to characterize the Ca2+ regulatory network, we identified a Ca 2+-regulated β-propeller protein, CarP, and showed that expression of the encoding gene is controlled by the Ca2+-regulated two-component system CarSR. Here, by using a Galleria melonella model, we showed that CarP plays a role in regulating P. aeruginosa virulence. By using transcriptome sequencing (RNA-Seq), reverse transcription (RT)-PCR, quantitative RT-PCR (RT-qPCR), and promoter fusions, we determined that carP is transcribed into at least two transcripts and regulated by several bacterial and host factors. The transcription of carP is elevated in response to Ca2+ in P. aeruginosa cystic fibrosis isolates and PAO1 laboratory strain. Elevated Fe2+ also induces carP The simultaneous addition of Ca2+ and Fe2+ increased the carP promoter activity synergistically, which requires the presence of CarR. In silico analysis of the intergenic sequence upstream of carP predicted recognition sites of RhlR/LasR, OxyR, and LexA, suggesting regulation by quorum sensing (QS) and oxidative stress. In agreement, the carP promoter was activated in response to stationary-phase PAO1 supernatant and required the presence of elevated Ca2+ and CarR but remained silent in the triple mutant lacking rhlI, lasI, and pqsA synthases. We also showed that carP transcription is regulated by oxidative stress and that CarP contributes to P. aeruginosa Ca2+-dependent H2O2 tolerance. The multifactorial regulation of carP suggests that CarP plays an important role in P. aeruginosa adaptations to host environments.IMPORTANCE P. aeruginosa is a human pathogen causing life-threatening infections. It is particularly notorious for its ability to adapt to diverse environments within the host. Understanding the signals and the signaling pathways enabling P. aeruginosa adaptation is imperative for developing effective therapies to treat infections caused by this organism. One host signal of particular importance is calcium. Previously, we identified a component of the P. aeruginosa calcium-signaling network, CarP, whose expression is induced by elevated levels of calcium. Here, we show that carP plays an important role in P. aeruginosa virulence and is upregulated in P. aeruginosa strains isolated from sputa of patients with cystic fibrosis. We also identified several bacterial and host factors that regulate the transcription of carP Such multifactorial regulation highlights the interconnectedness between regulatory circuits and, together with the pleotropic effect of CarP on virulence, suggests the importance of this protein in P. aeruginosa adaptations to the host.