부산대학교 도서관

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.sup.2+) are elevated. Previously, we showed that P. aeruginosa responds to externally added (Ca.sup.2+) through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular (Ca.sup.2+) level, followed by its removal to the basal submicromolar level. However, the molecular mechanisms responsible for regulating (Ca.sup.2+) induced virulence factor production and (Ca.sup.2+) homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa to elevated [(Ca.sup.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 (CaCl.sub.2) 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.sup.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 beta-propeller protein, PA0327 (here called carP). Mutations in both carO and carP affected (Ca.sup.2+) homeostasis, reducing the ability of P. aeruginosa to export excess (Ca.sup.2+) . In addition, a mutation in carP had a pleotropic effect in a (Ca.sup.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.sup.2+) and responding through its regulatory targets that modulate (Ca.sup.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.sup.2+) concentrations, yet the cells maintain intracellular (Ca.sup.2+) at levels that are orders of magnitude less than that of the external environment. In addition, (Ca.sup.2+) signals P. aeruginosa to induce the production of several virulence factors. Compared to eukaryotes, little is known about how bacteria maintain (Ca.sup.2+) homeostasis or how (Ca.sup.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.sup.2+) levels. CarRS modulates (Ca.sup.2+) signaling and (Ca.sup.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.sup.2+) and relays that information for (Ca.sup.2+) dependent cellular processes.