Biofilm Maturation in Carbapenem-resistant Pseudomonas aeruginosa Is Regulated by the sRNA PA213 and Its Corresponding Encoded Small Protein.

Objectives: Biofilm formation is a key factor contributing to the persistence and resistance of carbapenem-resistant Pseudomonas aeruginosa (CRPA). This study aims to elucidate the regulatory role of PA213, a previously uncharacterized and hypoxia-inducible small RNA, in promoting CRPA biofilm maturation.

Methods: We collected 113 clinical isolates for species identification and antimicrobial profiling. Comparative transcriptomic analyses were conducted between carbapenem-susceptible and resistant strains to identify functional sRNA candidates. The expression and structure of PA213 were characterized using qRT-PCR, RACE, and secondary structure prediction. A Flag-tagged PA213 fusion protein was confirmed by Western blot. PA213's phenotypic effects were assessed through assays of biofilm formation, motility, pyocyanin production, and extracellular matrix regulation. Biofilm architecture was visualized by SEM, and viability was analyzed via CFU counts and PI staining. Protein partners were identified via GST-pulldown and LC-MS, and interaction with OprI was confirmed through immunofluorescence and biochemical validation. The impact of PA213 on quorum sensing (QS) gene expression was examined by RT-qPCR based on RNA-seq datasets.

Results: PA213 is a novel, uncharacterized sRNA that is highly expressed in clinical CRPA isolates. This sRNA featured a typical stem-loop structure and a 279-nucleotide open reading frame (ORF), which encoded a low-molecular-weight polypeptide. PA213 was significantly upregulated in clinical CRPA under hypoxia and encoded a small peptide with high conservation. Functionally, PA213 acted as a multifunctional regulator, enhancing biofilm formation, inhibiting pyocyanin synthesis, promoting swimming motility, and reducing virulence and pathogenicity. It enhanced biofilm formation during microcolony (12-48 h) and maturation (48-72 h) phases, without increasing EPS production or relying on the las/rhl QS pathway. It ensured sufficient functional bacterial density and maintained the three-dimensional structure of the biofilm by modulating viable bacterial proliferation. Mechanistically, the protein encoded by PA213 directly interacted with OprI, mediating spatial stabilization of mature biofilms.

Conclusion: PA213 functions as a bifunctional sRNA-peptide regulator that promotes CRPA biofilm maturation via non-coding and protein-mediated mechanisms, offering new insights into hypoxia-responsive biofilm regulation and antimicrobial resistance evolution.