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

The opportunistic human pathogen <i>Pseudomonas aeruginosa</i> (<i>Pa</i>), 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 <i>Pa</i> resistance to PMB. However, the mechanisms of this Ca²⁺ regulation are not known. Here, we identified three novel genes (<i>PA2803, PA3237</i>, and <i>PA5317</i>) that contribute to the Ca²⁺-dependent PMB resistance in <i>Pa. PA2803</i>, 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 (P_i), we named it "P_i 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 <i>Pa</i>, 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 P_i 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 <i>Pa</i> responses to elevated Ca²⁺ and P_i starvation and enhances PMB resistance.IMPORTANCE<i>Pseudomonas aeruginosa</i> (<i>Pa</i>) 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 <i>Pa</i> 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 <i>Pa</i>. One of them, <i>PA2803</i>, 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.