Abstract
Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life-threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca(2+)) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca(2+) through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca(2+) level, followed by its removal to the basal submicromolar level. However, the molecular mechanisms responsible for regulating Ca(2+)-induced virulence factor production and Ca(2+) homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa to elevated [Ca(2+)] in both planktonic cultures and biofilms. Among the genes induced by CaCl2 in strain PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR), while the closely related two-component regulators phoPQ and pmrAB were repressed by CaCl2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR and performed transcriptome analysis of the mutant strain at low and high [Ca(2+)]. Among the genes regulated by CarSR in response to CaCl2 are the predicted periplasmic OB-fold protein, PA0320 (here called carO), and the inner membrane-anchored five-bladed β-propeller protein, PA0327 (here called carP). Mutations in both carO and carP affected Ca(2+) homeostasis, reducing the ability of P. aeruginosa to export excess Ca(2+). In addition, a mutation in carP had a pleotropic effect in a Ca(2+)-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca(2+) and responding through its regulatory targets that modulate Ca(2+) homeostasis, surface-associated motility, and the production of the virulence factor pyocyanin. Importance: During infectious disease, Pseudomonas aeruginosa encounters environments with high calcium (Ca(2+)) concentrations, yet the cells maintain intracellular Ca(2+) at levels that are orders of magnitude less than that of the external environment. In addition, Ca(2+) signals P. aeruginosa to induce the production of several virulence factors. Compared to eukaryotes, little is known about how bacteria maintain Ca(2+) homeostasis or how Ca(2+) acts as a signal. In this study, we identified a two-component regulatory system in P. aeruginosa PAO1, termed CarRS, that is induced at elevated Ca(2+) levels. CarRS modulates Ca(2+) signaling and Ca(2+) homeostasis through its regulatory targets, CarO and CarP. The results demonstrate that P. aeruginosa uses a two-component regulatory system to sense external Ca(2+) and relays that information for Ca(2+)-dependent cellular processes.