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 (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. 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 Ca 2+ -dependent growth advantage 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, which mediates Pa responses to elevated Ca 2+ and P i starvation and enhances PMB resistance. IMPORTANCE Pseudomonas 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 (Ca 2+ ) 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 Ca 2+ -dependent PMB resistance in Pa . One of them, PA2803 , regulated by Ca 2+ and phosphate, was named phosphate and Ca 2+ -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.