EF-hand protein, EfhP, specifically binds Ca2+ and mediates Ca2+ regulation of virulence in a human pathogen Pseudomonas aeruginosa

Abstract

Abstract 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.