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Pseudomonas aeruginosa  is a facultative human pathogen, and a major cause of nosocomial infections and severe chronic infections in endocarditis and in cystic fibrosis (CF) patients. Calcium (Ca 2+ ) accumulates in pulmonary fluids of CF patients, and plays a role in the hyperinflamatory response to bacterial infection. Earlier we showed that  P. aeruginosa  responds to increased Ca 2+  levels, primarily through the increased production of secreted virulence factors. Here we describe the role of putative Ca 2+ -binding protein, with an EF-hand domain, PA4107 (EfhP), in this response. Deletion mutations of  efhP  were generated in  P. aeruginosa  strain PAO1 and CF pulmonary isolate, strain FRD1. The lack of EfhP abolished the ability of  P. aeruginosa  PAO1 to maintain intracellular Ca 2+  homeostasis. Quantitative high-resolution 2D-PAGE showed that the  efhP  deletion also affected the proteomes of both strains during growth with added Ca 2+ . The greatest proteome effects occurred when the pulmonary isolate was cultured in biofilms. Among the proteins that were significantly less abundant or absent in the mutant strains were proteins involved in iron acquisition, biosynthesis of pyocyanin, proteases, and stress response proteins. In support, the phenotypic responses of FRD1 Δ efhP  showed that the mutant strain lost its ability to produce pyocyanin, developed less biofilm, and had decreased resistance to oxidative stress (H 2 O 2 ) when cultured at high [Ca 2+ ]. Furthermore, the mutant strain was unable to produce alginate when grown at high [Ca 2+ ] and no iron. The effect of the Δ efhP  mutations on virulence was determined in a lettuce model of infection. Growth of wild-type  P. aeruginosa  strains at high [Ca 2+ ] causes an increased area of disease. In contrast, the lack of  efhP  prevented this Ca 2+ -induced increase in the diseased zone. The results indicate that EfhP is important for Ca 2+  homeostasis and virulence of  P. aeruginosa  when it encounters host environments with high [Ca 2+ ].

A Pseudomonas aeruginosa EF-Hand Protein, EfhP (PA4107), Modulates Stress Responses and Virulence at High Calcium Concentration