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.

EF-Hand Calcium Sensor, EfhP, Controls Transcriptional Regulation of Iron Uptake by Calcium inPseudomonas aeruginosa

“…2+ . Previously, we have identified and partially characterized Pa Ca 2+ sensor, EfhP (25,49). Here we aimed to characterize the role of this protein in PAO1 transcriptional response to Ca 2+ .…”

“…Previous studies have identified Ca 2+ regulation of numerous Pa virulence factors (33,34,79) contributing to increased mortality in in vivo infection models (35,49,80).…”

“…Previous studies have identified Ca 2+ regulation of numerous Pa virulence factors ( 33, 34, 79 ) contributing to increased mortality in in vivo infection models ( 35, 49, 80 ). The EF hand Ca 2+ sensor, EfhP, was shown to control Pa Ca 2+ responses including the induction of pyoverdine ( 25 ) and pyocyanin production ( 49 ) and elevated virulence in lettuce leaf and G. mellonella infection models ( 49, 50 ). In this study, we utilized RNA sequencing analysis to characterize the Pa rapid and adaptive transcriptional responses to Ca 2+ which potentially drive the observed increases in virulence.…”

“…These data support a hypothesis that EfhP plays a key role in orchestrating the Ca 2+ control over Pa gene expression. Furthermore, efhP is highly conserved in Pseudomonas genus with even greater sequence conservation among Pa CF clinical isolates vs non-CF clinical and environmental isolates ( 49 ), which provides additional evidence of its role in human infections. The predicted periplasmic localization of EfhP ( 49 ) may equip Pa with a mechanism to sense and respond to extracellular concentrations of Ca 2+ .…”

“…Furthermore, efhP is highly conserved in Pseudomonas genus with even greater sequence conservation among Pa CF clinical isolates vs non-CF clinical and environmental isolates ( 49 ), which provides additional evidence of its role in human infections. The predicted periplasmic localization of EfhP ( 49 ) may equip Pa with a mechanism to sense and respond to extracellular concentrations of Ca 2+ . Since Pa faces a rapid transition to millimolar levels of Ca 2+ upon invading human airways, which are further heightened during CF, it is important to characterize the development of Ca 2+ transcriptional responses over time.…”