wspA mutation mediated Pseudomonas aeruginosa rugose small colony variant and its hyper-biofilm
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Pseudomonas aeruginosa is a major multidrug-resistant pathogen whose biofilm formation complicates treatment. P. aeruginosa r ugose s mall c olony v ariants (RSCVs), characterized by enhanced biofilm production and resistance, pose significant clinical challenges. However, the formation mechanisms remain unclear in clinical strains. Paired clinical P. aeruginosa isolates AR8023-1 (wild-type) and AR8023-2 (RSCV) were collected from urine samples of a neurosurgery inpatient chronologically. The genome comparison was performed using Breseq, and plasmid complementation was performed to construct complemented strain (AR8023-2 RifR :: wspA AR8023-1 ). Then, all the aforementioned strains were examined RSCV phenotype, biofilm formation, and motility, and gene expression differences induced by the wspA mutation were analyzed via transcriptome sequencing, followed by quantification of c-di-GMP. In addition, we assessed antimicrobial susceptibility of the strains under both planktonic and biofilm conditions. The results showed that both AR8023-1 and AR8023-2 were identified as ST3420. AR8023-1 exhibited typical smooth morphology, while AR8023-2 displayed a RSCV phenotype. A single SNP difference was identified between the two strains, characterized by a deletion of glutamine at position 289 (CAG triplet deletion) in the wspA gene of strain AR8023-2. The complement isolates restored wild-type morphology, motility, and biofilm formation. Transcriptomics revealed a significant upregulation of c-di-GMP metabolic genes in RSCVs ( P < 0.001), indicating that the WspAΔ289Q mutation activates diguanylate cyclase (DGC) activity, thereby elevating c-di-GMP synthesis. Intracellular c-di-GMP levels were significantly higher in RSCVs than those in WT and complement isolates, respectively ( P < 0.001). Consequently, biofilm susceptibility testing demonstrated the β-lactams MBIC of RSCVs was ≥8–512-fold higher than those of WT and complement isolates. This study identifies a clinical WspAΔ289Q mutation that elevates c-di-GMP, driving RSCV formation and biofilm-mediated resistance.
IMPORTANCE
P. aeruginosa is a leading multidrug-resistant pathogen whose biofilm formation capability greatly complicates treatment outcomes. This study identifies a novel clinical mutation in the wspA gene (WspAΔ289Q) that drives the emergence of rugose small colony variants (RSCVs), a phenotype linked to heightened biofilm production and antimicrobial resistance. We demonstrate that this mutation upregulates cyclic di-GMP synthesis, leading to increased biofilm formation and markedly reduced susceptibility to β-lactam antibiotics under biofilm conditions. These results provide important insights into the genetic basis of RSCV development in clinical settings and highlight the role of c-di-GMP signaling in biofilm-associated resistance. Our findings underscore the need to target c-di-GMP pathways as a potential strategy for combating persistent P. aeruginosa infections.
