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

Abstract

ABSTRACTThe human pathogenPseudomonas aeruginosaposes 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 inP. 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 thatefhPdeletion 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 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 theefhPtranscription is controlled by Ca2+and Fe, and this regulation required Ca2+-dependent two-component regulatory system CarSR. Furthermore, theefhPexpression 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 inP. aeruginosaand 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 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 identifiedParapid 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 all the iron uptake mechanisms and production of pyocins. Transcription ofefhPitself is regulated by Ca2+, Fe, and increases during interactions with host epithelial cells, suggesting the protein’s important role inPainfections. The findings establish the regulatory interconnectedness between Ca2+and iron signaling pathways that shapePatranscriptional responses. Therefore, understanding Pa’s transcriptional response to Ca2+and associated regulatory mechanisms will serve the development of future therapeutics targetingPadangerous infections.