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.
Highlights
Many bacterial pathogens are able to establish long-term chronic infections within their respective hosts
The yfiBNR operon is an small colony variants (SCVs)-related locus in Pseudomonas aeruginosa
To begin our analysis of the SCV phenotype, we sought to identify those loci in P. aeruginosa PA01 whose disruption led to a characteristic SCV morphology and behavior
Summary
Many bacterial pathogens are able to establish long-term chronic infections within their respective hosts. The strategies and mechanisms employed by pathogens to persist against attacks by the host immune system and the action of antimicrobial substances are still relatively poorly understood An appreciation of these processes is needed to develop strategies that help to avoid complications with antibiotic resistance development and infection relapses associated with prolonged colonization of host tissue. Burkholderia cepacia SCVs are associated with increased serum resistance and fatal systemic infections post lung transplantation [8]. These studies suggest that during the course of chronic lung infections, SCVs are selected for due to a fitness advantage in this unique environment, and that they might play an important role in the pathogenesis of P. aeruginosa lung infections
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