EF-hand calcium sensor, EfhP, controls transcriptional regulation of iron uptake by calcium in <i>Pseudomonas aeruginosa</i>

The human pathogen <i>Pseudomonas aeruginosa</i> (<i>Pa</i>) 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 <i>P. aeruginosa</i>. 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 <i>efhP</i> 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 <i>efhP</i> transcription is controlled by Ca²⁺ and Fe, and this regulation required a Ca²⁺-dependent two-component regulatory system CarSR. Furthermore, the <i>efhP</i> 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 <i>P. aeruginosa</i> 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.IMPORTANCE<i>Pseudomonas aeruginosa</i> (<i>Pa</i>) poses a major risk for severe infections, particularly in patients suffering from cystic fibrosis (CF). For the first time, kinetic RNA sequencing analysis identified <i>Pa</i> 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 <i>efhP</i> itself is regulated by Ca²⁺ and Fe, and increases during interactions with host epithelial cells, suggesting the protein's important role in <i>Pa</i> infections. The findings establish the regulatory interconnectedness between Ca²⁺ and iron signaling pathways that shape <i>Pa</i> transcriptional responses. Therefore, understanding Pa's transcriptional response to Ca²⁺ and associated regulatory mechanisms will serve in the development of future therapeutics targeting <i>Pa</i>'s dangerous infections.