YfiBNR Mediates Cyclic di-GMP Dependent Small Colony Variant Formation and Persistence in Pseudomonas aeruginosa

Abstract

During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important player in P. aeruginosa persistence, and implicates a central role for c-di-GMP, and by extension the SCV phenotype in chronic infections.

Author Summary

During long-term chronic infections of cystic fibrosis patients, Pseudomonas aeruginosa adapts to the lung environment, generating various different morphotypes including small colony variants (SCVs), small, strongly adherent colonies whose appearance correlates with persistence of infection. The SCV morphology is strongly associated with increased levels of the signaling molecule cyclic di-GMP. In this study we investigated the connection between cyclic di-GMP, SCV and persistence of infection. Following a genetic screen for mutants that displayed SCV morphologies, we identified and characterized the YfiBNR system. YfiN is a membrane-bound cyclic di-GMP producing enzyme, whose activity is tightly controlled by YfiR and YfiB. Cyclic di-GMP produced by YfiN boosts exopolysaccharide synthesis, generating an SCV morphotype upon YfiR-mediated release of YfiN repression. The resulting YfiN-mediated SCV morphotype is highly resistant to macrophage phagocytosis in vitro, suggesting a role for the SCV phenotype in immune system evasion. Consistent with this, YfiN de-repression increased the persistence of P. aeruginosa in long-term infections in a mouse model. The observation that the addition of antibiotics decreased the number of suppressors, and the relative fitness disadvantage of the YfiN-mediated SCV morphotype in liquid culture, suggested that SCV-mediated persistence might be favored during antimicrobial chemotherapy.