Cystic fibrosis is a widespread genetic disease that leads to progressive disability and early death. The principal cause of mortality and morbidity in CF patients is a progressive deterioration of the respiratory system caused by a chronic infection of the patients’ lungs, mainly by the opportunistic bacterial pathogen Pseudomonas aeruginosa. CF lung infections can be treated with antibiotics, however full clearance is not possible due to the protective environment of the CF lung and the adaptation of infective species to a persistent lifestyle. This presents serious challenges for the long-term chemotherapy of CF patients.
New research reveals that Small Colony Variants (SCVs) of P. aeruginosa are be a hallmark of chronic infection in cystic fibrosis (CF) patients. These new results suggest that SCV-mediated persistence might be a good target for antimicrobial chemotherapy. Adaptive P. aeruginosa morphotypes include SCVs, slow-growing and strongly adherent variants that frequently arise in chronic lung infections. Because the appearance of SCVs correlates with poor lung function and antibiotic resistance, they have long been suspected of mediating the P. aeruginosa persistence phenotype in CF infections. In this study, the researchers characterized a signaling system in P. aeruginosa called YfiBNR, mutations in which lead to the generation of SCV variants. Activation of YfiBNR resulted in increased levels of the signaling molecule c-di-GMP, which in turn triggered massive production of exopolysaccharides and drastically reduced growth rates, two hallmarks of SCV behavior. YfiN-mediated SCVs were shown to be highly resistant to macrophage phagocytosis, suggesting a role for the SCV phenotype in immune system evasion. Consistent with this, activation of YfiN significantly increased the persistence of P. aeruginosa in long-term infections in a mouse model, establishing a firm causal link between SCV and persistence in chronic P. aeruginosa infections. The authors conclude that c-di-GMP has long been a key suspect in chronic behavior of bacterial pathogens. The finding that the c-di-GMP-mediated SCV phenotype confers a persistent advantage in mice provides the first direct evidence in favor of such a model. This study opens up new avenues to specifically counteract persistent infections in CF.
YfiBNR Mediates Cyclic di-GMP Dependent Small Colony Variant Formation and Persistence in Pseudomonas aeruginosa. PLoS Pathog 6(3): e1000804. doi:10.1371/journal.ppat.1000804
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 SCVrelated 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 subinhibitory 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.