The PA2803-encoded PcrP exhibits a novel non-catalytic function and contributes to polymyxin B resistance in P. aeruginosa.

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. 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 Ca2+ and inorganic phosphate (Pi), we named it "Pi and Ca2+-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. By using pull-down assays and bacterial two-hybrid systems, we identified and validated two protein partners of PcrP: Acp3 and PA3518. We showed 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 Ca2+-dependent growth advantage during Pi starvation and plays a role in polyphosphate accumulation in a Ca2+-dependent manner. Overall, this study identified a novel protein-binding function for the PA2803 subfamily representative, which mediates Pa responses to elevated Ca2+ and Pi starvation and enhances PMB resistance.IMPORTANCEPseudomonas aeruginosa (Pa) is a critical human pathogen that presents significant clinical challenges, underscoring the urgent need for understanding its resistance mechanisms. Previous studies have shown that calcium (Ca2+) at the levels detected during infections increases Pa resistance to the last resort antibiotic polymyxin B (PMB). For the first time, we identified three novel genes, whose products are required for the Ca2+-dependent PMB resistance in Pa. One of them, PA2803, regulated by Ca2+ and phosphate, was named phosphate and Ca2+-regulated protein, PcrP. This study discovered a novel protein-binding function of PcrP and identified two protein partners. Given the high level of sequence conservation within the PA2803 subfamily, the protein-binding function may be shared by other members of the PA2803 subfamily.