The lysogenic filamentous Pseudomonas bacteriophage phage Pf slows mucociliary transport

Abstract

Pseudomonas aeruginosa is a major pulmonary pathogen causing chronic pulmonary infections in people with cystic fibrosis (CF). The P. aeruginosa filamentous and lysogenic bacteriophage, Pf phage, is abundant in the airways of many people with CF and has been associated with poor outcomes in a cross-sectional cohort study. Previous studies have identified roles for Pf phage in biofilm formation, specifically forming higher-order birefringent, liquid crystals when in contact with other biopolymers in biofilms. Liquid crystalline biofilms are more adherent and viscous than those without liquid crystals. A key feature of biofilms is to enhance bacterial adherence and resist physical clearance. The effect of Pf phage on mucociliary transport is unknown. We found that primary CF and non-CF nasal epithelial cells cultured at air-liquid interface treated with Pf phage exhibit liquid crystalline structures in the overlying mucus. On these cell cultures, Pf phage entangles cilia but does not affect ciliary beat frequency. In both these in vitro cell cultures and in an ex vivo porcine trachea model, introduction of Pf phage decreases mucociliary transport velocity. Pf phage also blocks the rescue of mucociliary transport by CF transmembrane conductance regulator modulators in CF cultures. Thus, Pf phage may contribute to the pathogenesis of P. aeruginosa-associated CF lung disease via induction of liquid crystalline characteristics to airway secretions, leading to impaired mucociliary transport. Targeting Pf phage may be useful in treatment CF as well as other settings of chronic P. aeruginosa infections.

Keywords: bacteriophage; cystic fibrosis; mucociliary transport.

Fig. 1.

Pf forms aggregates that entangle the cilia. SEM imaging (12,000×) of WT human nasal epithelial culture (HNEC) at ALI treated with apical PBS (A) or Pf (B) and of CF HNEC at ALI similarly treated with apical PBS (C) or Pf (D).

Fig. 2.

Ciliary beat frequency of human nasal epithelial cell cultures at ALI in the presence of pf phage. Ciliary beat frequency measured by video microscopy on A) WT and CF primary cells treated with either Pf phage or of control of PBS, and B) F508del homozygous donor cells were pretreated with ETI or PBS daily for 48 hours and then treated with Pf phage or PBS. C) Percent active area per imaged field of ALI culture insert for each condition. Analysis by one-way ANOVA with Tukey's multiple comparison (A to C). Unmarked comparisons are not significant. P-value = * < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001. E/T/I, elexacaftor/tezacaftor/ivacaftor; WT, wild type.

Fig. 3.

Mucus transport velocity of human nasal epithelial cell cultures at ALI in the presence of pf phage. MCV was measured by video monitoring of beads placed on top of epithelial cells at ALI culture. A) CF and WT nasal epithelial cells grown at ALI were treated with Pf phage or PBS depicted as mean bead velocity as a measure of MCV. B) CF cells with PBS, Pf phage, and/or E/T/I (given as pretreatment 48 hours prior to phage treatment). C) WT cells treated with PBS, Pf phage, control phage Fd, or control phage DMSvir3. Analysis by Kruskal–Wallis with Dunn's multiple comparison tests for individual comparisons (A and B) and one-way ANOVA with Tukey's multiple comparison (A to C). P-value * < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001. Multiple particle tracking performed on WT cultures represented by D) ensemble effective diffusivity and E) ensemble mean square distance, over a range of time scales from 10 to 500 ms demonstrate a substantial decrease in the active transport of beads across the epithelial surface reflecting an impairment of the mucociliary active transport mechanism. The shaded area represents 95% CIs. Blue = PBS treated and red = Pf phage treated. Videos of particle tracking are included in Figs. S2 and S3. Deff, ensemble effective diffusivity; DMS, DMS3vir phage; E/T/I, elexacaftor/tezacaftor/ivacaftor; Fd, Fd phage; MSD, ensemble mean squared distance; PBS, phosphate-buffered saline; WT, wild type.

Fig. 4.

MCV in ex vivo newborn piglet trachea treated with Pf phage. MCV measurement performed by tracking of ink toner particles and sequential imaging over 30 minutes. The setup of the ex vivo trachea is depicted in (A). Pf phage added at A concentration of 10¹⁰/mL. Control treated with comparable volume of PBS. Both conditions treated with 0.3 µM carbachol and 10 µM formoterol for maximal mucociliary transport.(39) Tracking was initiated 30 minutes after treatments applied, and images were automatically captured every 5 minutes (four images/20 s intervals) using a digital camera. B) Time course of first 30 minutes of recorded MCV in pig tracheas (n = 4 per condition). Control in open circles and Pf phage-treated in solid squares (P = 0.04 for difference between treatments). C) Boxplots with the average MCV over 30 minutes, mean (*) T_10–90 MCV (in millimeter per minute): control 13.2 ± 0.2; Pf 8.2 ± 0.5 (P = 0.0462). Statistical comparison by t test. P-value * < 0.05. MCV, mucociliary transport velocity; PBS, phosphate-buffered saline; Pf, Pf phage.