EF-hand calcium sensor, EfhP, controls transcriptional regulation of iron uptake by calcium in Pseudomonas aeruginosa.

The human pathogen Pseudomonas aeruginosa (Pa) poses a major risk for a range of severe infections, particularly lung infections in patients suffering from cystic fibrosis (CF). As previously reported, the virulent behavior of this pathogen is enhanced by elevated levels of Ca²⁺ that are commonly present in CF nasal and lung fluids. In addition, a Ca²⁺-binding EF-hand protein, EfhP (PA4107), was partially characterized and shown to be critical for the Ca²⁺-regulated virulence in P. aeruginosa. Here, we describe the rapid (10 min, 60 min), and adaptive (12 h) transcriptional responses of PAO1 to elevated Ca²⁺ detected by genome-wide RNA sequencing and show that efhP deletion significantly hindered both rapid and adaptive Ca²⁺ regulation. The most differentially regulated genes included multiple Fe sequestering mechanisms, a large number of extracytoplasmic function sigma factors (ECFσ), and several virulence factors, such as the production of pyocins. The Ca²⁺ regulation of Fe uptake was also observed in CF clinical isolates and appeared to involve the global regulator Fur. In addition, we showed that the efhP transcription is controlled by Ca²⁺ and Fe, and this regulation required a Ca²⁺-dependent two-component regulatory system CarSR. Furthermore, the efhP expression is significantly increased in CF clinical isolates and upon pathogen internalization into epithelial cells. Overall, the results established for the first time that Ca²⁺ controls Fe sequestering mechanisms in P. aeruginosa and that EfhP plays a key role in the regulatory interconnectedness between Ca²⁺ and Fe signaling pathways, the two distinct and important signaling pathways that guide the pathogen's adaptation to the host.IMPORTANCEPseudomonas aeruginosa (Pa) poses a major risk for severe infections, particularly in patients suffering from cystic fibrosis (CF). For the first time, kinetic RNA sequencing analysis identified Pa rapid and adaptive transcriptional responses to Ca²⁺ levels consistent with those present in CF respiratory fluids. The most highly upregulated processes include iron sequestering, iron starvation sigma factors, and self-lysis factors pyocins. An EF-hand Ca²⁺ sensor, EfhP, is required for at least 1/3 of the Ca²⁺ response, including the majority of the iron uptake mechanisms and the production of pyocins. Transcription of efhP itself is regulated by Ca²⁺ and Fe, and increases during interactions with host epithelial cells, suggesting the protein's important role in Pa infections. The findings establish the regulatory interconnectedness between Ca²⁺ and iron signaling pathways that shape Pa transcriptional responses. Therefore, understanding Pa's transcriptional response to Ca²⁺ and associated regulatory mechanisms will serve in the development of future therapeutics targeting Pa's dangerous infections.