A physical linkage between cystic fibrosis airway surface dehydration and Pseudomonas aeruginosa biofilms

Abstract

A vexing problem in cystic fibrosis (CF) pathogenesis has been to explain the high prevalence of Pseudomonas aeruginosa biofilms in CF airways. We speculated that airway surface liquid (ASL) hyperabsorption generates a concentrated airway mucus that interacts with P. aeruginosa to promote biofilms. To model CF vs. normal airway infections, normal (2.5% solids) and CF-like concentrated (8% solids) mucus were prepared, placed in flat chambers, and infected with an Ϸ5 ؋ 10 3 strain PAO1 P. aeruginosa. Although bacteria grew to 10 10 cfu/ml in both mucus concentrations, macrocolony formation was detected only in the CF-like (8% solids) mucus. Biophysical and functional measurements revealed that concentrated mucus exhibited properties that restrict bacterial motility and small molecule diffusion, resulting in high local bacterial densities with high autoinducer concentrations. These properties also rendered secondary forms of antimicrobial defense, e.g., lactoferrin, ineffective in preventing biofilm formation in a CF-like mucus environment. These data link airway surface liquid hyperabsorption to the high incidence of P. aeruginosa biofilms in CF via changes in the hydration-dependent physical-chemical properties of mucus and suggest that the thickened mucus gel model will be useful to develop therapies of P. aeruginosa biofilms in CF airways.mucus ͉ rheology C ystic fibrosis (CF) lung disease reflects the chronic bacterial infection of intrapulmonary airways with Pseudomonas aeruginosa biofilms (1, 2). P. aeruginosa biofilms have mostly been studied in flow chambers (3-7) that are good models for the biofilms that form on venous or urethral catheters under conditions of high flow rates and relatively high oxygen tensions. However, biofilms in CF airways form in thickened (concentrated) mucus gels that are relatively hypoxic and adherent to airway surfaces (see Fig. 1A) (8, 9). Recently, Sriramulu et al. (10) investigated the role autoregulators and amino acids play in the tightness of biofilm formation in a mucus stimulant of constant hydration. For the present study, we designed a culture system to investigate whether dehydration of the CF mucus environment may predispose to P. aeruginosa biofilm formation.A key aspect of the model was the selection of mucus concentrations that mimic normal and CF mucus. Although measurements of mucus concentration, i.e., the percentage of solids content, from CF subjects before infection have not been reported, estimates from cell cultures (11) and sputum (12-18) suggest that CF mucus is at least three or four times more concentrated than normal. Therefore, mucus was obtained from well differentiated human airway cultures and was isotonically concentrated (1) to produce mucus of normal (2.5% solids wt/wt) and CF-like (8%) concentrations (Fig. 1B). Aliquots of each were deposited in (i) an open chamber to model a mucus plaque adherent to CF airway surfaces with a residual lumen allowing airflow and (ii) a closed chamber to mimic a mucus plug occluding a CF airway (Fig. 1 A). P....

Proteases and cystic fibrosis

“…Both bacterial exoproducts and neutrophil mediators upregulate production of mucin glycoproteins, the major macromolecular constituent of mucus (Rose and Voynow, 2006), and increase mucus secretion into the airway resulting in a favorable milieu for bacterial proliferation. Due to aberrant ion and water flux in the airway, mucus plaques become viscous which favors the development of biofilms (Matsui et al, 2006) and impedes neutrophil phagocytosis of bacteria (Matsui et al, 2005). Over time, airway secretory remodeling takes place with epithelial loss and heterogeneous zones of proliferation , hypertrophy of goblet cells, and hyperplasia of submucosal gland cells (Hays and Fahy, 2006).…”

Low oxygen induces the type III secretion system in Pseudomonas aeruginosa via modulation of the small RNAs rsmZ and rsmY

“…In CF patients, mutation(s) in the cystic fibrosis transmembrane conductance regulator (CFTR) results in thickened mucus formation in the respiratory airway and leads to reduced oxygen availability deep within the lungs (AlvarezOrtega & Harwood, 2007;Boucher, 2004;Matsui et al, 2006;Worlitzsch et al, 2002;Yoon et al, 2002). Long-term persistence of P. aeruginosa in the CF lung is aided by its ability to reside in the dense hyper-mucoid lumen as hypoxic biofilm-like masses, leading to chronic infections refractory to the host immune response and antibiotic treatment (AlvarezOrtega & Harwood, 2007;Matsui et al, 2006;Stewart & Franklin, 2008;Worlitzsch et al, 2002).…”

Inhibitors of pendrin anion exchange identified in a small molecule screen increase airway surface liquid volume in cystic fibrosis

“…In addition to forming a permeability barrier, tethered mucins in the PCL generate an osmotic pressure to regulate PCL hydration (52). The osmotic potential exerted by CF mucus (.8% solids), but not healthy mucus (;2% solids), was demonstrated to be sufficient to dehydrate the PCL and collapse cilia (52), and to restrict mobility of bacteria and foster formation of bacterial biofilm precursor macrocolonies (53). Restoration of CFTR expression in CF primary epithelial cells by viral transduction significantly increases ASL depth, ciliary beat frequency, and mucus transport, supporting the idea that loss of CFTR function mediates ASL dehydration (54).…”