Molecular insights into calcium regulation of Pseudomonas aeruginosa responses to antibiotics and host-related stimuli

The opportunistic human pathogen Pseudomonas aeruginosa (Pa), a leading cause of severe infections, becomes increasingly resistant to antibiotics, including the last resort antibiotic, polymyxin-B (PMB). Previous studies have shown that calcium (Ca²⁺) at the levels encountered during infections increases Pa resistance to PMB. However, the mechanisms of this Ca²⁺ regulation are not known. Here, we identified three novel genes (PA2803, PA3237 and PA5317) that contribute to the Ca²⁺-dependent PMB resistance in Pa. The focus of this work, PA2803 encodes a putative phosphonatase and is a founding member of the PA2803 subfamily that belongs to Haloacid Dehalogenase Superfamily. Since the transcription of this gene is regulated by both Ca²⁺ and inorganic phosphate (Pi), we named it “Pi and Ca²⁺ regulated protein, PcrP”. Congruent with our sequence-based predictions, we showed that PcrP lacks catalytic activity and instead binds protein partners, revealing a novel non-catalytic function for PA2803 subfamily proteins. By using pull-down assays and bacterial two-hybrid system, we identified and validated two protein partners of PcrP: Acp3 and PA3518. We showed that PcrP is involved in oxidative stress response mediated by its interactions with Acp3, which may support its role in PMB resistance. Overall, this study identified a novel protein-binding function for the PA2803 subfamily representative that mediates Pa responses to elevated Ca²⁺ and enhances its PMB resistance.