Role of calcium sensor EfhP in regulation of virulence in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe chronic infections in the lungs of Cystic Fibrosis (CF) patients, where elevated levels of calcium (Ca2+) are commonly detected. Our group has discovered that elevated Ca2+ increases antibiotic resistance, enhances biofilm formation, and induces the production of several virulence factors in P. aeruginosa. We also identified several components of Ca2+ regulatory network, which included a novel putative Ca2+ sensor, which we designated EfhP based on the encoded Ca2+-binding EF hand domains. We hypothesized that EfhP mediates Ca2+ regulation of the pathogen ability to cause disease in an animal host as we earlier showed in plants. To test this hypothesis and define the role of EfhP in Ca2+-dependent virulence of this pathogen, we used Galleria mellonella wax worm as an animal model. We injected the wax worms with the wild type and efhP deletion mutant that were grown either in the presence or absence of 5 mM Ca2+. The results showed that growth in the presence of Ca2+ enhanced the ability of P. aeruginosa to kill in the animal model. We also showed that efhP plays in role in virulence, in that upon removal of the gene less worms were killed compared to the wildtype. In addition, we assessed the transcriptional regulation of efhP in response to Ca2+ and Fe2+ using efhP promoter construct. We determined that there is potential regulatory connection between Ca2+ and Fe2+ in the transcription of efhP. Additionally, we identified several proteins that have a role in regulating efhP transcription, specifically CarP, CarR and BfmS. These findings pertaining to transcriptional regulation of efhP will allow for a better understanding on how to inhibit EfhP functions. This knowledge is essential for future development of strategies and approaches for preventing or controlling P. aeruginosa infections