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.
(© 2022. The Author(s).)