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

Abstract

Calcium (Ca²⁺) is well known as a second messenger in eukaryotes, where Ca²⁺ signaling controls life-sustaining cellular processes. Although bacteria produce the components required for Ca²⁺ signaling, little is known about the mechanisms of bacterial Ca²⁺ signaling. Previously, we have identified a putative Ca²⁺-binding protein EfhP (PA4107) with two canonical EF-hand motifs and reported that EfhP mediates Ca²⁺ regulation of virulence factors production and infectivity in Pseudomonas aeruginosa, a human pathogen causing life-threatening infections. Here, we show that EfhP selectively binds Ca²⁺ with 13.7 µM affinity, and that mutations at the +X and -Z positions within each or both EF-hand motifs abolished Ca²⁺ binding. We also show that the hydrophobicity of EfhP increased in a Ca²⁺-dependent manner, however no such response was detected in the mutated proteins. ¹⁵ N-NMR showed Ca²⁺-dependent chemical shifts in EfhP confirming Ca²⁺-binding triggered structural rearrangements in the protein. Deletion of efhP impaired P. aeruginosa survival in macrophages and virulence in vivo. Disabling EfhP Ca²⁺ binding abolished Ca²⁺ induction of pyocyanin production in vitro. These data confirm that EfhP selectively binds Ca²⁺, which triggers its structural changes required for the Ca²⁺ regulation of P. aeruginosa virulence, thus establishing the role of EfhP as a Ca²⁺ sensor.