Our studies initially focused on the pathogenesis of established CF airways infection and, taking clues from these studies, explored whether these variables could uniquely contribute to the early pathogenesis of P. aeruginosa infection in CF airways.
Morphometric analyses of freshly excised lungs by three techniques demonstrated that P. aeruginosa
grows as macrocolonies in the airway intraluminal rather than the epithelial surface compartment (Figure , a–c). These findings contradict recent hypotheses emanating from in vitro model systems that focus on high-salt/defensin inactivation (26
) or luminal epithelial cell binding (4
), which predict bacterial infection of CF airway epithelial cells themselves (5
). However, our data are consistent with those from animal models that have demonstrated the adherence of P. aeruginosa
to respiratory mucus (27
), and three previous qualitative studies of CF postmortem lungs that identified P. aeruginosa
in airway lumens rather than on airway epithelial cells (30
). Furthermore, they are also consistent with our studies of NSEs that revealed P. aeruginosa
preferentially bound to mucus rather than epithelial cell surfaces (Figure , d–f). A key extension of the in vivo characterization of CF airways infection is that P. aeruginosa
occupies an intraluminal niche that is markedly hypoxic (Figure , a–b).
If the CF airways disease reflects infection of mucus, how is this process initiated and perpetuated? A sequence consistent with several aspects of the “low volume/reduced mucus clearance” hypothesis (10
) for CF pathogenesis is outlined in Figure . First, as compared with NL airway epithelial function (compare Figure a), data have been reported that CF airway epithelia excessively absorb Na+
(and water) from the lumen, deplete the periciliary liquid layer (PCL), and slow/abolish mucus clearance (Figure b) (10
). Accelerated Na+
absorption, which reflects the absence of CFTR’s normal inhibitory activity on ENaC (36
), is fueled by an increased turnover rate of ATP-consuming Na+
-ATPase pumps (37
) leading to two- to threefold increases in CF airway epithelial O2
Figure 5 Schematic model of the pathogenic events hypothesized to lead to chronic P. aeruginosa infection in airways of CF patients. (a) On normal airway epithelia, a thin mucus layer (light green) resides atop the PCL (clear). The presence of the low-viscosity (more ...)
Second, persistent mucin secretion into stationary mucus generates plaques/plugs (16
) (Figure c). The combination of thickened mucus and raised O2
consumption by CF epithelia generated steep O2
gradients within adherent mucus (Figure c). Importantly, the steep pO2
gradient in ASL/mucus was specific for CF epithelia because it was not reproduced in cultures from another genetic airways disease with an infectious phenotype, PCD (Figure f).
Third, bacteria deposited on thickened mucus can penetrate into hypoxic zones (Figure d). When the normal rotational mucus transport ceased due to excessive volume absorption, the vertical “currents” within transported mucus were abolished, but motile P. aeruginosa still penetrated thickened mucus (Figure , c and d). Note that environmental P. aeruginosa strains such as those that characterize early infection are motile and would likely penetrate mucus readily.
Fourth, P. aeruginosa
can grow in hypoxic/anaerobic CF mucus (Figure a). In part, growth under anaerobic conditions may be supported by the terminal electron acceptor, nitrate (~20 μM), contained in ASL. Furthermore, we show that increased alginate production was a characteristic feature of PAO1 strains in response to hypoxia, particularly with growth in low concentrations of nitrate that mimic ASL (Figure , b–d), and this characteristic is also a feature of environmental P. aeruginosa
strains. We speculate that the increased alginate formation may represent a stress response to hypoxia that is part of the process that forms biofilmlike macrocolonies, the predominant phenotype of P. aeruginosa
in CF airways (3
). Interestingly, Staphylococcus aureus
also responds to the hypoxic environment of CF mucus with a switch from nonmucoid to a mucoid phenotype (40
Finally, the capacity of P. aeruginosa
to proliferate in hypoxic mucus will generate fully hypoxic (anaerobic) conditions in patients with persistent CF airways infection (Figure , Figure , e and f, and Figure e). Hassett et al. reported that P. aeruginosa
alginate production was maintained by anaerobic conditions (21
). The reduced O2
tension in the mucopurulent intraluminal contents of CF airways may, therefore, be one variable contributing to the persistence of P. aeruginosa
macrocolonies in CF airways. The consequences of the macrocolony growth state have been explored in detail and include resistance to antibiotics (42
) and host phagocyte killing (Figure f) and (42
), all of which contribute to the persistence of P. aeruginosa
infection and the chronic destructive airways disease characteristics of CF.
In summary, our data demonstrate that the P. aeruginosa
infection of CF airways occurs within the luminal (mucus) rather than the epithelial cell surface compartment. Thus, we speculate that mucus clearance is a key feature of innate lung defense (44
), and a fundamental defect leading to chronic CF lung infections is the failure to effectively clear mucus that contains bound bacteria from the lung (10
). Hypoxic gradients exist within poorly cleared/adherent mucus, consequent to CF-specific increases in epithelial O2
consumption, and inhaled P. aeruginosa
respond to hypoxic mucus with alginate production and macrocolony formation, which allows them to evade host defenses and produce a chronic destructive lung disease. These data lead us to conclude that therapeutic strategies to treat CF lung disease should include novel drugs designed to clear the lung of retained mucus plaques/plugs, which initiate and perpetuate CF lung disease, and antibiotics that effectively treat P. aeruginosa
growing under hypoxic/anaerobic conditions.