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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.

SUBMITTER: Kayastha BB

PROVIDER: S-EPMC9132961 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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

Kayastha Biraj B BB Kubo Aya A Burch-Konda Jacob J Dohmen Rosalie L RL McCoy Jacee L JL Rogers Rendi R RR Mares Sergio S Bevere Justin J Huckaby Annalisa A Witt William W Peng Shuxia S Chaudhary Bharat B Mohanty Smita S Barbier Mariette M Cook Gabriel G Deng Junpeng J Patrauchan Marianna A MA

Scientific reports 20220525 1

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 viru ...[more]

PMID: 35614085

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Project description:Pseudomonas aeruginosa is a facultative human pathogen, and a major cause of nosocomial infections and severe chronic infections in endocarditis and in cystic fibrosis (CF) patients. Calcium (Ca2+) accumulates in pulmonary fluids of CF patients, and plays a role in the hyperinflammatory response to bacterial infection. Earlier we showed that P. aeruginosa responds to increased Ca2+ levels, primarily through the increased production of secreted virulence factors. Here we describe the role of putative Ca2+-binding protein, with an EF-hand domain, PA4107 (EfhP), in this response. Deletion mutations of efhP were generated in P. aeruginosa strain PAO1 and CF pulmonary isolate, strain FRD1. The lack of EfhP abolished the ability of P. aeruginosa PAO1 to maintain intracellular Ca2+ homeostasis. Quantitative high-resolution 2D-PAGE showed that the efhP deletion also affected the proteomes of both strains during growth with added Ca2+. The greatest proteome effects occurred when the pulmonary isolate was cultured in biofilms. Among the proteins that were significantly less abundant or absent in the mutant strains were proteins involved in iron acquisition, biosynthesis of pyocyanin, proteases, and stress response proteins. In support, the phenotypic responses of FRD1 ΔefhP showed that the mutant strain lost its ability to produce pyocyanin, developed less biofilm, and had decreased resistance to oxidative stress (H2O2) when cultured at high [Ca2+]. Furthermore, the mutant strain was unable to produce alginate when grown at high [Ca2+] and no iron. The effect of the ΔefhP mutations on virulence was determined in a lettuce model of infection. Growth of wild-type P. aeruginosa strains at high [Ca2+] causes an increased area of disease. In contrast, the lack of efhP prevented this Ca2+-induced increase in the diseased zone. The results indicate that EfhP is important for Ca2+ homeostasis and virulence of P. aeruginosa when it encounters host environments with high [Ca2+].

Project description:High binding affinity and selectivity of metal ions are essential to the function of metalloproteins. Thus, understanding the factors that determine these binding characteristics is of major interest for both fundamental mechanistic investigations and guiding of the design of novel metalloproteins. In this work, we perform QM cluster model calculations and quantum mechanics/molecular mechanics (QM/MM) free energy simulations to understand the binding selectivity of Ca2+ and Mg2+ in the wild-type carp parvalbumin and its mutant. While a nonpolarizable MM model (CHARMM36) does not lead to the correct experimental trend, treatment of the metal binding site with the DFTB3 model in a QM/MM framework leads to relative binding free energies (ΔΔGbind) comparable with experimental data. For the wild-type (WT) protein, the calculated ΔΔGbind is ∼6.6 kcal/mol in comparison with the experimental value of 5.6 kcal/mol. The good agreement highlights the value of a QM description of the metal binding site and supports the role of electronic polarization and charge transfer to metal binding selectivity. For the D51A/E101D/F102W mutant, different binding site models lead to considerable variations in computed binding affinities. With a coordination number of seven for Ca2+, which is shown by QM/MM metadynamics simulations to be the dominant coordination number for the mutant, the calculated relative binding affinity is ∼4.8 kcal/mol, in fair agreement with the experimental value of 1.6 kcal/mol. The WT protein is observed to feature a flexible binding site that accommodates a range of coordination numbers for Ca2+, which is essential to the high binding selectivity for Ca2+ over Mg2+. In the mutant, the E101D mutation reduces the flexibility of the binding site and limits the dominant coordination number of Ca2+ to be seven, thereby leading to reduced binding selectivity against Mg2+. Our results highlight that the binding selectivity of metal ions depends on both the structural and dynamical properties of the protein binding site.

Dataset Information

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

Publications

EF-hand protein, EfhP, specifically binds Ca<sup>2+</sup> and mediates Ca<sup>2+</sup> regulation of virulence in a human pathogen Pseudomonas aeruginosa.

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