The impact of pseudomonas aeruginosa genetic adaptation to the cystic fibrosis airway on inflammation and bacterial persistence

By adulthood, the lungs of most Cystic Fibrosis (CF) patients become chronically infected by the bacterium Pseudomonas aeruginosa. Once chronic, these infections are virtually ineradicable and often fatal, and are characterized by severe neutrophilic inflammation and a persistent bacterial biofilm infection. In the early stages of infection, P. aeruginosa strains found in CF airways are indistinguishable from environmental strains, but as P. aeruginosa adapts to the CF lung, it undergoes extensive genetic adaptation and microevolution. Phenotypic changes commonly occurring in CF-adapted P. aeruginosa strains include mucoidy, the loss of motility, loss of acute virulence factors like secreted proteases, and loss of LasR quorum sensing. Despite the loss of functions used to invade and injure the host, the emergence of CF-adapted P. aeruginosa strains in CF airways is paradoxically associated with more severe lung disease and worst patient outcomes. We hypothesized that CF-adapted P. aeruginosa isolates alter host pulmonary immune responses in a manner that increases neutrophil-mediated inflammation and impedes bacterial clearance, thereby contributing to lung disease progression. Using in vitro human airway epithelial cell (AEC) culture models and a murine model of chronic P. aeruginosa airway infection, we determined that P. aeruginosa isolates defective in LasR quorum sensing, or in the production of the LasR-regulated secreted protease LasB, were unable to degrade pro-inflammatory cytokines secreted by AEC, unlike wild-type P. aeruginosa strains. Genetically engineered ΔlasR and ΔlasB mutant strains were associated with hyper-inflammatory cytokine responses and increased neutrophil recruitment in vitro and in vivo. Additionally, infection with the ΔlasR and ΔlasB mutants led to greater lung immunopathology and, in the case of the ΔlasB strain, greater morbidity. In CF patients, we found that the frequency of lasR mutants in the lungs was positively correlated with plasma IL-8 levels, a marker of neutrophil inflammation. Next, we compared the in vivo virulence and immunogenicity of two clonally-related P. aeruginosa isolates recovered from a single patient 7.5 years apart: the Late isolate, which had numerous CF-adapted phenotypic changes including a loss of LasR quorum sensing and secreted LasB activity, and a non-adapted Early isolate. We found that the Late isolate caused less early mortality and morbidity in a murine model of chronic airway infection compared to the Early isolate, but displayed enhanced persistence by day 10 post infection (p.i.). We demonstrated that the kinetics of cytokine responses in the lungs of Early and Late-infected mice differed; the Early isolate stimulated a robust early pro-inflammatory and pro-neutrophilic response that was quickly shut down, and the Late isolate stimulated less vigorous pro-inflammatory response but a more prolonged neutrophil chemokine response. The Late isolate elicited greater neutrophil recruitment into the lungs by days 4 and 10 p.i. compared to the Early isolate, demonstrating that this CF-adapted isolate was able to persist despite causing a robust neutrophil response. Together, our studies suggest that bacterial adaptive changes may worsen pulmonary inflammation and contribute directly to the pathogenesis and progression of chronic lung disease in CF patients. Understanding how phenotypic changes occurring in CF-adapted P. aeruginosa isolates modulate host inflammatory responses and promote bacteria persistence in vivo can provide us insights into the pathogenesis of chronic infections, a paradigm distinct from acute virulence.

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