EF-hand protein, EfhP, specifically binds Ca(2+) and mediates Ca(2+) regulation of virulence in a human pathogen Pseudomonas aeruginosa

Calcium (Ca(2+)) is well known as a second messenger in eukaryotes, where Ca(2+) signaling controls life-sustaining cellular processes. Although bacteria produce the components required for Ca(2+) signaling, little is known about the mechanisms of bacterial Ca(2+) signaling. Previously, we have identified a putative Ca(2+)-binding protein EfhP (PA4107) with two canonical EF-hand motifs and reported that EfhP mediates Ca(2+) regulation of virulence factors production and infectivity in Pseudomonas aeruginosa, a human pathogen causing life-threatening infections. Here, we show that EfhP selectively binds Ca(2+) with 13.7 µM affinity, and that mutations at the +X and −Z positions within each or both EF-hand motifs abolished Ca(2+) binding. We also show that the hydrophobicity of EfhP increased in a Ca(2+)-dependent manner, however no such response was detected in the mutated proteins. (15) N-NMR showed Ca(2+)-dependent chemical shifts in EfhP confirming Ca(2+)-binding triggered structural rearrangements in the protein. Deletion of efhP impaired P. aeruginosa survival in macrophages and virulence in vivo. Disabling EfhP Ca(2+) binding abolished Ca(2+) induction of pyocyanin production in vitro. These data confirm that EfhP selectively binds Ca(2+), which triggers its structural changes required for the Ca(2+) regulation of P. aeruginosa virulence, thus establishing the role of EfhP as a Ca(2+) sensor.