A novel non-catalytic function of PA2803-encoded PcrP contributes to polymyxin B resistance in P. aeruginosa and redefines the functional role of the PA2803 subfamily.

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 (Ca ²⁺ ) at the levels encountered during infections increases Pa resistance to PMB. However, the mechanisms of this Ca ²⁺ regulation are not known. Here, we identified three novel genes (PA2803, PA3237 and PA5317) that contribute to the Ca ²⁺ -dependent PMB resistance in Pa. PA2803, the focus of this work, encodes a putative phosphonatase and is a founding member of the PA2803 subfamily from the Haloacid Dehalogenase Superfamily. Since the transcription of this gene is regulated by both Ca ²⁺ and inorganic phosphate (P _i ), we named it “P _i and Ca ²⁺ regulated protein, PcrP”. Congruent with 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 show that PcrP is involved in oxidative stress responses in Pa, which are likely mediated by its interactions with Acp3, and may support its role in PMB resistance. In addition, PcrP imparts a Ca ²⁺ -dependent growth advantage to Pa during P _i starvation and plays a role in polyphosphate accumulation in a Ca ²⁺ -dependent manner. Overall, this study identified a novel protein-binding function for the PA2803 subfamily representative that mediates Pa responses to elevated Ca ²⁺ and P _i starvation and enhances PMB resistance.