EF-Hand Calcium Sensor, EfhP, Controls Transcriptional Regulation of Iron Uptake by Calcium in Pseudomonas aeruginosa

Abstract

The human pathogen Pseudomonas aeruginosa 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 Ca ²⁺ that are commonly present in CF nasal and lung fluids. In addition, a Ca ²⁺ -binding EF-hand protein, EfhP (PA4107), was partially characterized and shown to be critical for the Ca ²⁺ -regulated virulence in P. aeruginosa . Here we describe the rapid (10 min, 60 min), and adaptive (12 h) transcriptional responses of PAO1 to elevated Ca ²⁺ detected by genome-wide RNA sequencing and show that efhP deletion significantly hindered both rapid and adaptive Ca ²⁺ 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 production of pyocins. The Ca ²⁺ 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 Ca ²⁺ and Fe, and this regulation required Ca ²⁺ -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 Ca ²⁺ controls Fe sequestering mechanisms in P. aeruginosa and that EfhP plays a key role in the regulatory interconnectedness between Ca ²⁺ and Fe signaling pathways, the two distinct and important signaling pathways that guide the pathogen's adaptation to host.

Importance: Pseudomonas 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 Ca ²⁺ 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 Ca ²⁺ sensor, EfhP, is required for at least 1/3 of the Ca ²⁺ response, including all the iron uptake mechanisms and production of pyocins. Transcription of efhP itself is regulated by Ca ²⁺ , 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 Ca ²⁺ and iron signaling pathways that shape Pa transcriptional responses. Therefore, understanding Pa's transcriptional response to Ca ²⁺ and associated regulatory mechanisms will serve the development of future therapeutics targeting Pa dangerous infections.