A wspA mutation in a clinical isolate of Pseudomonas aeruginosa results in rugose small colonies and enhances biofilm formation

Abstract

ABSTRACT Pseudomonas aeruginosa is a major multidrug-resistant pathogen whose biofilm formation complicates treatment. Rugose small colony variants (RSCVs) exhibit enhanced biofilm production and resistance, posing significant clinical challenges; however, their formation mechanisms in clinical strains remain incompletely understood. Paired clinical isolates AR8023-1 (wild type) and AR8023-2 (RSCV) were chronologically collected from urine samples of a neurosurgery inpatient. Genome comparison via Breseq and plasmid complementation constructed the complemented strain AR8023-2RifR::wspAAR8023-1. All strains were examined for RSCV phenotype, biofilm formation, motility, transcriptomic changes, c-di-GMP quantification, and antimicrobial susceptibility under planktonic and biofilm conditions. Both isolates were identified as ST3420. AR8023-1 exhibited typical smooth morphology, while AR8023-2 displayed an RSCV phenotype with a single SNP—a glutamine deletion at position 289 (ΔQ289) in WspA. Complementation restored wild-type morphology, motility, and biofilm formation. Transcriptomics revealed significant upregulation of c-di-GMP metabolic genes in RSCVs (P < 0.001), suggesting that WspAΔQ289 increases diguanylate cyclase activity and elevates c-di-GMP synthesis. Intracellular c-di-GMP levels were ~3-fold higher in RSCVs than in wild-type and complemented strains (P < 0.001). Biofilm susceptibility testing demonstrated that β-lactam MBICs for RSCVs were 8- to 512-fold higher than those of wild-type and complement isolates. Introduction of the same WspAΔQ289 allele into P. aeruginosa PAO1 did not recapitulate the RSCV phenotype, indicating that strain background contributes to the phenotypic outcome. This study confirms and extends the role of a clinical WspAΔ289Q mutation that elevates c-di-GMP, driving RSCV formation and biofilm-mediated resistance. IMPORTANCE This study further characterizes a specific WspAΔ289Q mutation in clinical Pseudomonas aeruginosa that is associated with activation of the c-di-GMP pathway, driving rugose small colony variant (RSCV) formation and hyper-biofilm phenotypes. The mutation markedly enhances biofilm-mediated β-lactam resistance, which is consistent with treatment failure. Complementation restores wild-type traits, supporting the importance of WspA structural integrity as a key regulator of colony morphology and antibiotic tolerance. These findings provide a molecular biomarker for RSCV surveillance and highlight c-di-GMP signaling as a therapeutic target against biofilm-associated chronic infections, although the phenotype is strain-dependent and the mutation occurs in a previously identified WspA hotspot. This study further characterizes a specific WspAΔ289Q mutation in clinical Pseudomonas aeruginosa that is associated with activation of the c-di-GMP pathway, driving rugose small colony variant (RSCV) formation and hyper-biofilm phenotypes. The mutation markedly enhances biofilm-mediated β-lactam resistance, which is consistent with treatment failure. Complementation restores wild-type traits, supporting the importance of WspA structural integrity as a key regulator of colony morphology and antibiotic tolerance. These findings provide a molecular biomarker for RSCV surveillance and highlight c-di-GMP signaling as a therapeutic target against biofilm-associated chronic infections, although the phenotype is strain-dependent and the mutation occurs in a previously identified WspA hotspot.