Cystic Fibrosis: A background
Cystic fibrosis: Cystic fibrosis (CF) is an autosomal recessive disorder that is the most common lethal genetic disease among Caucasians, with an incidence of about 1 in 3200 newborns in the U.S. CF is caused by mutation of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR gene encodes an epithelial cell chloride channel that also regulates sodium (and possibly other) channels in the apical plasma membrane. Thus, deficiency of CFTR function results in abnormal regulation of electrolyte and fluid balance in specific tissues. While CF affects multiple organ systems, including the reproductive tract and sweat glands, morbidity and mortality are primarily related to disease of the gastrointestinal and respiratory tracts.
Individuals with CF in 2006 have a median predicted survival age of 37 years, an increase of about 10 years that has occurred over the past two decades. This increase in median survival is attributable to advances in the treatment of gastrointestinal and lung disease. Despite improved medical therapy and the advent of lung transplantation, pulmonary disease remains the principal cause of morbidity and mortality in CF. Some individuals with CF still die from lung disease during their teenage years, and for those who survive into adulthood, the severity of lung disease is a major determinant of quality of life.
CF lung disease begins in infancy or early childhood. It is characterized by chronic bacterial infection and severe inflammation that leads to progressive deterioration in lung function. The airways, rather than the lung parenchyma, are the sites of inflammation and infection. CF lung disease is characterized by secretion of copious amounts of thick mucus and migration of a predominantly neutrophilic infiltrate into the airways. This “acute” infiltrate is not replaced by a “chronic” mononuclear cell infiltrate, as normally occurs when physiologic acute inflammation resolves, but instead persists. Although Staphylococcus aureus is the most common initial pathogen in the first few years of life, epidemiological studies have demonstrated the opportunistic pathogen P. aeruginosa to be the most important pathogen in progressive, severe CF lung disease.
P. aeruginosa is the most important pathogen in the CF airway. P. aeruginosa is acquired from environmental reservoirs and can cause both acute and chronic infections, depending on the clinical context. Acute P. aeruginosa infections can be rapidly fatal. In contrast, in specific clinical conditions such as CF, diffuse panbronchiolitis, primary ciliary dyskinesia, endotracheal intubation, and idiopathic bronchiectasis, P. aeruginosa can cause chronic airway infections that persist over the course of months or years. Despite this persistent and exuberant inflammatory response, P. aeruginosa infection persists in the CF airway, in part due to a series of adaptive bacterial changes that are not yet well defined.
Adaptation of P. aeruginosa to the airway is important for cystic fibrosis lung disease. In the setting of longstanding infection, P. aeruginosa isolates are phenotypically distinct from acute infection isolates. These characteristics include loss of flagellar-dependent and pilin-dependent motility, lipopolysaccharide (LPS) changes including loss of O-antigen and alteration in lipid A structure, increased auxotrophy, decreased secretion of virulence factors, inability to produce pyocyanin and phage, antibiotic resistance, and mucoidy. The most thorough analysis of P. aeruginosa early adaptation, utilizing longitudinal, clonally related collections of isolates from CF patients, was performed by our group. This study identified previously undescribed adaptations that confer changes in intercellular signaling, biofilm formation, basic metabolism, antibiotic susceptibility and in the production of virulence factors associated with acute infections.
Genomics and the natural history of P. aeruginosa in the CF airway: Mutations in lasR occur commonly during chronic CF infections. Whole-genome sequencing of clinical and environmental isolates of P. aeruginosa, performed at the UW, demonstrated previously that over 80% of the genome of strain PAO1 is shared (with only 0.5% divergence at the nucleotide level) among three P. aeruginosa isolates, two from CF patients and one from the environment. Despite such conservation, substantial mutational adaptation occurs in P. aeruginosa during the eight years of CF infections. In this study, the whole-genome sequences were defined for a pair of CF clinical isolates of clonally-related P. aeruginosa from a single patient: One isolated at patient age 3 months, and the other 7.5 years later. In addition, a selection of genes mutated in this patient’s isolates was sequenced among isolates from 29 additional CF patients. This analysis identified several mutations even more common to the late (adapted) isolates that had not been previously identified. For example, lasR, encoding a transcriptional regulator of quorum sensing that impacts virulence and biofilm formation, was mutated in the late isolates from 18 of 29 children with CF, suggesting that lasR mutation is among the most common adaptive changes that occur in P. aeruginosa childhood CF infections. This observation was subsequently confirmed in an exploratory study of isolates from our local CF clinic population, in which lasR mutants were found in 31% of 166 isolates, a prevalence similar to the frequency of mucoidy in the same population. A limited number of lasR mutants have undergone further characterization of the natural history of P. aeruginosa adaptation within CF airways using the combined results from DNA microarrays, proteomics, and phenotypic analysis. This analysis has shown that lasR mutation leads to multiple phenotypic consequences that likely adversely impact clinical course, including the ability to grow on nutrients commonly found in the CF airway and antibiotic resistance.
P. aeruginosa lasR mutants exhibit characteristics predicted to impact clinical course. lasR encodes a transcriptional regulator that sits atop a hierarchical signaling system known as quorum sensing. Quorum sensing signaling has been shown to occur in CF patient airways, and P. aeruginosa with quorum sensing mutations are attenuated for virulence in rat models of acute infection.. Nevertheless, it is generally accepted that acute infection models do not accurately reflect the chronic nature of CF airway infections. To further confuse matters, publications examining the importance of quorum sensing in bacterial evolution have examined the growth characteristics of lasR mutant P. aeruginosa under experimental conditions that require intact quorum sensing functions to grow (i.e., with protein as the sole carbon source, which requires the expression of a lasR-regulated protease). These studies found that, under these specific experimental conditions, lasR mutants benefited from the presence of strains with intact lasR to “complement” quorum sensing functions. Thus, under these conditions, lasR mutants were referred to as “cheaters”. Yet these conditions are not reflective of the CF airway, which includes many nutrient sources (including amino acids) that do not require the intact lasR gene product to grow. This abundance of nutrients may explain why lasR mutants were isolated frequently from patients without wild-type co-isolates. We have discovered several phenotypes due to LasR inactivation that would be predicted to confer a growth advantage in the CF airway, and to adversely impact clinical course. For example, we found that lasR inactivating mutation decreases susceptibility to all three of the most commonly used antibiotics used in CF care: b-lactams, aminoglycosides, and fluoroquinolones. We have also demonstrated that two of these antibiotics, tobramycin and ciprofloxacin, can select for loss of lasR, further providing a rationale for the frequency of these mutants within CF airways.