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PNAS Nexus
PNAS Nexus
pnasnexus
PNAS Nexus
2752-6542
Oxford University Press US

10.1093/pnasnexus/pgae390
pgae390
Biological, Health, and Medical Sciences
AcademicSubjects/MED00010
AcademicSubjects/SCI00010
AcademicSubjects/SOC00010
PNAS_Nexus/med-microbio
The lysogenic filamentous Pseudomonas bacteriophage phage Pf slows mucociliary transport
https://orcid.org/0000-0001-8969-6232
Burgener Elizabeth B Department of Pediatrics, Center for Excellence in Pulmonary Biology, Stanford University, Stanford, CA 94305, USA
Division of Pediatric Pulmonology and Sleep Medicine, Children's Hospital of Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, CA 90027, USA

Cai Pamela C Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA

https://orcid.org/0000-0002-8845-2524
Kratochvil Michael J Department of Materials Science and Engineering, Stanford University, Stanford, CA 94305, USA

Rojas-Hernandez Laura S Department of Pediatrics, Center for Excellence in Pulmonary Biology, Stanford University, Stanford, CA 94305, USA

https://orcid.org/0000-0002-0510-6862
Joo Nam Soo Department of Pediatrics, Center for Excellence in Pulmonary Biology, Stanford University, Stanford, CA 94305, USA
Cystic Fibrosis Research Laboratory, School of Humanities and Sciences, Stanford University, Stanford, CA 94305, USA

Gupta Aditi Department of Pediatrics, Center for Excellence in Pulmonary Biology, Stanford University, Stanford, CA 94305, USA

https://orcid.org/0000-0001-7123-3037
Secor Patrick R Division of Biological Sciences, University of Montana, Missoula, MT 59812, USA

https://orcid.org/0000-0002-9801-6304
Heilshorn Sarah C Department of Materials Science and Engineering, Stanford University, Stanford, CA 94305, USA

https://orcid.org/0000-0002-0585-1942
Spakowitz Andrew J Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA

https://orcid.org/0000-0002-4038-1392
Wine Jeffrey J Cystic Fibrosis Research Laboratory, School of Humanities and Sciences, Stanford University, Stanford, CA 94305, USA

https://orcid.org/0000-0003-2499-9448
Bollyky Paul L Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University, Stanford, CA 94305, USA

https://orcid.org/0000-0001-5515-3053
Milla Carlos E Department of Pediatrics, Center for Excellence in Pulmonary Biology, Stanford University, Stanford, CA 94305, USA

Discher Dennis Editor
To whom correspondence should be addressed: Email: eburgener@chla.usc.edu
Competing Interest: The authors declare no competing interests.

9 2024
09 9 2024
09 9 2024
3 9 pgae39013 5 2024
20 8 2024
19 9 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.
2024
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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.

bacteriophage
mucociliary transport
cystic fibrosis
Stanford Training Program in Pulmonary Biology NHLBI 10.13039/100000050 T32HL129970, 1K23HL169902-01 Francis Family Foundation 10.13039/100005834 Cystic Fibrosis Foundation 10.13039/100000897 BURGEN20Q0, BURGEN21A0-KU National Science Foundation Graduate Research Fellowship 10.13039/100023581 National Science Foundation 10.13039/100000001 Physics of Living Systems Program PHY-2102726 CFF foundation JOO19G0 NIH 10.13039/501100012264 R01AI138981, K24AI166718-01A1, and R21GM147838 Emerson Collective 10.13039/100019329 R01AI138981 MILLA23P0 1R01HL148184 Ross Mosier Laboratories Gift Fund
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pmcSignificance Statement

Pf phage is a nonlytic bacteriophage of Pseudomonas aeruginosa found in chronic infections that creates liquid crystalline biofilms. This confers increased adhesion and viscosity to biofilms, thus making clinical infections challenging to clear. We show that Pf phage also impairs mucus transport of the airway epithelium, which is critical for defense against invading pathogens, in both a primary epithelial cell culture model and an ex vivo porcine tracheal model. Notably, Pf phage abrogates the rescue of defective mucus transport by cystic fibrosis transmembrane conductance regulator modulator drugs. This demonstrates yet another mechanism by which P. aeruginosa infections persist within the airway, subverting host defenses. This further highlights the potential of Pf phage as a novel therapeutic target for treating chronic P. aeruginosa infections.

Introduction

In cystic fibrosis (CF), defective or absent cystic fibrosis transmembrane conductance regulator (CFTR) protein results in an anion permeability defect that leads to thick and dehydrated secretions. Retained airway secretions become colonized with bacteria, establishing a cycle of inflammation and infection with progressive tissue destruction and eventual death or need for lung transplantation (1–3).

In the past decade, CF care has been transformed by highly effective CFTR modulator therapies, drugs that target defective CFTR protein and restore its function (4). These medications significantly improve mucociliary transport (5) and induce dramatic improvements in lung function and quality of life (6). However, even with these groundbreaking therapies, airway inflammation and infection persist, notably infection with Pseudomonas aeruginosa (P. aeruginosa) (7–10).

P. aeruginosa is a common pathogen infecting the airways of people with CF and is associated with accelerated decline in lung function and mortality (11–14). A significant effort is placed in preventing acquisition of P. aeruginosa and attempting eradication after initial detection (15–17). However, P. aeruginosa forms biofilms that facilitate persistence of the bacteria in the airway (18–20). Consequently, a significant proportion of people with CF are chronically infected by the time they reach adulthood (21).

The lysogenic filamentous bacteriophage, Pf phage, infects P. aeruginosa and incorporates into the bacterial chromosome as a prophage. Pf phage expression and production are significantly increased in the biofilm growth state (22–24). Unlike lytic bacteriophage, Pf phage does not lyse P. aeruginosa upon replication; instead, long filamentous virions are extruded from the bacteria without killing it (25). Pf phage can be highly abundant in CF sputum, at concentrations up to 1010 virions/mL (26, 27). We have previously shown that the presence of Pf phage in the sputum is associated with poor outcomes in a cross-sectional study of people with CF (27).

In vitro Pf phage contributes to P. aeruginosa biofilm formation by assembling biofilms and polymers commonly present in the CF airway (DNA, mucins, actin, and alginate produced by mucoid P. aeruginosa) into a liquid crystalline structure (26). This higher-order structure is spontaneously created by crowding (depletion) forces between Pf phage virions and polymers, providing additional adhesion and viscosity to P. aeruginosa biofilms (28). The impact of Pf phage-induced liquid crystals on mucociliary clearance is unknown.

We hypothesized that Pf phage carriage would provide a selective advantage to P. aeruginosa by disrupting clearance from the airway by impairing mucociliary transport. First, we demonstrate liquid crystalline structures in epithelial cell culture mucus treated with Pf phage. Then, we demonstrate both in vitro and in an ex vivo model that Pf phage slows mucociliary transport. Our data provide support for a role of Pf phage in the pathogenicity of P. aeruginosa and as a novel therapeutic target for chronic P. aeruginosa airway infection.

Results

Pf phage organizes mucus in ways that entangle cilia in an in vitro culture system

Direct visualization of the apical surface of primary CF nasal epithelial cell cultures at air–liquid interface (ALI) treated with Pf phage by polarized light microscopy demonstrated areas of birefringent liquid crystal along the ciliated surface (Fig. S1A), while no birefringence was seen in the negative, wild-type (WT) cultures (Fig. S1B). Additionally, mucus collected from the apical surface of the Pf phage-treated culture also revealed crystalline structures (Fig. S1C), which demonstrate areas of birefringence (Fig. S1D). These findings are consistent with Pf phage organizing the apical mucus into high-order birefringent, liquid crystalline structures (28).

We obtained additional details by scanning electron microscopy (SEM) imaging in both WT (Fig. 1A and B) and CF (Fig. 1C and D) cultures. Matting and clumping of cilia with visible net-like structures were observed in both WT and CF cultures exposed to Pf phage (Fig. 1B and D). Together, these data suggest that Pf phage interacts with the mucin polymers and induces the formation of structures that in contact with the cilia produce an entangling effect. This observed adherence of Pf phage to the cilia is consistent with the biophysical properties we have described previously in Pf phage liquid crystals (26, 28).

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).

Pf phage slows mucociliary transport and blocks rescue by CFTR modulators

To determine the effects of the Pf phage-induced entanglement of the ciliated surface on mucociliary function, we applied Pf phage to the apical surface of the nasal epithelial cell cultures at ALI. While ciliary beat frequency is significantly slower in CF vs. WT cultures, we found that ciliary beat frequency is not affected in a consistent manner by Pf phage in WT and CF cells (Fig. 2A), including those that were treated with the combination of CFTR modulators: elexacaftor, tezacaftor, and ivacaftor (ETI), to rescue CFTR function (Fig. 2B). There was no difference in active area of ciliary beating between all conditions (Fig. 2C).

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.

However, when we measured mucus transport at the apical surface, we found a significant impairment of transport in the presence of Pf phage. The mucociliary transport was dramatically decreased in the presence of Pf phage in WT cell cultures (Figs. 3A and S2–S3). There was no significant decrease in the CF cell cultures as the control CF cultures started with very low mucociliary transport velocities. However, the lower velocity seen in CF cells was rescued by ETI treatment, but this effect was abrogated by the presence of Pf phage (Fig. 3B).

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.

We conducted additional experiments to evaluate other phage including the Escherichia coli filamentous phage, Fd, which is very similar in structure to Pf phage, and a lytic P. aeruginosa phage, DMS3vir, and found that they also decreased mucociliary transport but less dramatically than Pf phage (Fig. 3C).

Further, by multiple particle tracking analysis of both WT cultures, both effective diffusivity (Fig. 3D) and mean squared distance (Fig. 3E) of particle transport were significantly decreased by the presence of Pf phage.

These data demonstrate that the effect of Pf phage on mucociliary transport is not due to slowing of ciliary beat frequency, but rather to an impediment to the mobilization of the mucus on the epithelial surface.

Pf phage impacts mucociliary transport in ex vivo newborn piglet trachea treated with Pf phage

To translate our findings, we sought to test the effects of Pf phage on a validated ex vivo system for assessing mucociliary transport. To this end, we made use of our previously reported method to measure mucociliary transport velocity (MCV) in explanted newborn piglet tracheas (29) (Fig. 4A).

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 (*) T10–90 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.

Monitoring the cephalad particle movement recorded over 30 minutes, we observed that MCV in Pf phage-treated tracheas was significantly lower (P = 0.03) than control conditions (n = 4 piglet tracheas per condition) (Figs. 4B, C and S4A, B). These data establish that Pf phage impairs mucociliary transport when present in the mucosal surface of an intact airway.

Discussion

We have identified impacts of Pf phage on mucociliary transport. We previously reported that Pf phage promotes higher-order crystalline structures in mixtures of biologically relevant polymers (26, 28, 30). We now report that Pf phage-induced crystalline structures directly entangle respiratory cilia and impair mucociliary transport in primary epithelial cell culture as well as an ex vivo porcine trachea model. These findings provide additional mechanistic insight into how Pf phage may impact lung disease in CF and potentially in other clinical settings.

The effects of Pf phage on mucociliary transport are consistent with our previous work of demonstrating Pf phage-induced liquid crystals (26, 28, 30). Here, we demonstrate Pf phage is capable of forming liquid crystalline structures within mucus that adheres to the cell culture surface and the SEM images reveal Pf phage entangling ciliary axonemes (Fig. 1B and D). Interestingly, in the epithelial cell cultures we find ciliary beat frequency is not affected by Pf phage (Fig. 2). This suggests that the liquid crystal may increase mucus viscosity and adhesiveness and thus effectively become anchored on the mucosal surface rendering the ciliary beat ineffective at mobilizing the mucus. This is supported by the negative effects found on MCV by adding Pf phage in both our in vitro and ex vivo systems (Figs. 3 and 4).

There is precedent for interactions of bacteriophage with polymers on mucosal surfaces from prior work identifying attachment of bacteriophage capsids to mucus (31). This is postulated to be a mechanism evolved by bacteriophage as a predator to take advantage of the mucosal surface to gain increased exposure to potential bacterial prey and may explain the similar yet less dramatic effect noted with DMS3vir, a tailed lytic P. aeruginosa bacteriophage (Fig. 3C). The highly polymeric nature of CF airway secretions likely allows Pf phage to form liquid crystalline structures that are highly adherent within the airway (26). This we postulate serves as an additional mechanism that P. aeruginosa utilizes to persist as a well-established biofilm within the CF airway.

Perhaps of greatest interest for clinicians treating individuals with CF, the presence of Pf phage abrogates the normalization of mucociliary transport in the CF cultures treated with the highly effective CFTR modulator, ETI (Fig. 3B). Whether the effects observed in the CF condition can be reverted simply by exchanging for WT-like mucus (i.e. similar solids concentrations) is answered by our experiment with ETI-treated cells. ETI has been shown in similar in vitro cultures to have a significant effect on mucus properties (32). We then find that under this WT-like mucus conditions, Pf still has a negative effect on mucociliary transport and negates the positive effects of ETI, with the mucociliary transport velocities similar to that of CF cultures without ETI treatment. This suggests that for individuals with chronic P. aeruginosa infection being treated with ETI, the presence of Pf phage may affect their ability to clear P. aeruginosa from their airways. Given recent data demonstrating persistence of P. aeruginosa among a proportion of individuals with CF treated with ETI (9, 10), it will be important to further investigate the implications of Pf phage in the CF airway in future studies.

There are limitations to this study. First, our in vitro systems do not permit full exploration of the effects of Pf phage in the presence of P. aeruginosa and biofilms or in the presence of immune cells. While the CF airway environment is quite complex and heterogeneous, here we aimed to interrogate primarily the effect of Pf phage in its interaction with the mucociliary system. Thus, our experimental procedures as described allowed us to isolate the effects of Pf phage more clearly. Second, our ex vivo model does not include all the components of an intact organ, but for all practical purposes has demonstrated to be of great value to identify important changes in mucociliary transport. Whether these effects translate directly to the CF airway will require further studies in intact animal models of chronic airway infection. These aspects are the focus of current efforts.

In summary, we find that the P. aeruginosa bacteriophage, Pf phage, forms liquid crystals in the mucus on airway cultures and slows mucociliary transport in two airway models. Together with work published by our group and others, demonstrating effects of Pf phage also on bacterial fitness (22, 33–36), antibiotic tolerance (26, 27, 30, 37, 38), and host immunity (30, 39), these data suggest that Pf phage promotes chronic infection in multiple ways. Here, we identify an additional mechanism that P. aeruginosa may utilize to persist in the CF airway, as well as identifying Pf phage as a potential therapeutic target to control chronic P. aeruginosa infections. These results may be relevant to diseases outside of CF where P. aeruginosa infections are problematic, such as ventilator-associated pneumonia, tracheostomy-associated airway infection, bronchiectasis, and primary ciliary dyskinesia, among others.

Materials and methods

Human nasal epithelial cells

Nasal epithelial cells were obtained from pwCF and WT, healthy controls by brushing both inferior turbinates and following an established standard operating procedure. The CF cells were obtained from a 14-year-old male patient with 3876delA (p.Lys1250ArgfsX9 or c.3744delA)/3876delA (p.Lys1250ArgfsX9 or c.3744delA) mutations for the experiments in Figs. 2A and 3A and C. Additional cells were obtained from an 18-year-old female with F508del (p.Phe508del or c.1521_1523del)/F508del (p.Phe508del or c.1521_1523del) mutations for the experiments in Figs. 2A and 3B as they would be responsive to ETI. The WT cells were obtained from a 60-year-old healthy male volunteer with known WT CFTR. This project was approved by the Stanford University IRB protocol (protocol #37232). Informed consent was obtained from all subjects.

Human nasal epithelial cell cultures at ALI

Human nasal epithelial cells were cultured at ALI as per previously published protocol (40, 41). Briefly, the cells from the nasal sample were dissociated, seeded onto collagen-coated, 0.4-μm pore polyester membrane inserts (Corning Inc.), and expanded with PneumaCult Ex-Plus media (StemCell Technologies) added to both the basal and apical chambers (42). Once cells reached confluence, an ALI was generated by removing the apical medium and replacing the basal medium with PneumaCult ALI media (StemCell Technologies). Once cultures were fully ciliated and active mucociliary activity was visualized, typically after 3 weeks, we proceeded to experimental measurements. For the CF cultures with ETI-responsive mutations, rescue CFTR function was introduced by treatment with a combination of elexacaftor 3 μM and tezacaftor 3 μM for 48 hours prior to and supplemented by the addition of ivacaftor 10μM at time of measurements. All inserts were washed with warm phosphate-buffered saline (PBS) to the apical surface to remove mucus and debris 48 hours prior to experimental manipulations as we have described before (43). Pf phage or vehicle control was then applied to the apical surface at a final concentration of 109 virions/mL and incubated at 37 °C for 24 hours prior to imaging. Once in vitro experiments were completed, filter inserts were either imaged with polarized light microscopy to evaluate for birefringence or fixed in 4% glutaraldehyde in cacodylate buffer for SEM.

Scanning electron microscopy

For imaging of ALI culture inserts by SEM, we followed our published methods (40). In brief, Transwell filters were fixed by submersion in a fixative mix of 2% glutaraldehyde, 4% paraformaldehyde in 0.1 M Na-cacodylate buffer titrated to pH 7.4, and kept at 4 °C overnight. The samples were osmicated, dehydrated, and then dried with a Tousimis AutoSamdri-815 critical point dryer. The samples were mounted luminal side up and sputter-coated with 100Å layer of Au/Pd. Images were acquired with a Hitachi S-3400N VP-SEM microscope operated at 10–15 kV, with a working distance of 7–10 mm and using secondary electron detection.

In vitro ciliary beat frequency and MCV of human nasal epithelial cell cultures at ALI

Once the ALI cultures had reached full maturity as evidenced by the presence of active ciliary beat, inserts were washed as noted above and then imaged at 500× on an inverted microscope with a heated stage with controlled environmental conditions at 37 °C and at a frame rate of 120 fps (Basler Ace, Basler AG, Germany). The images were acquired and analyzed with the SAVA system (Ammons Engineering, Clio, MI, USA) to determine ciliary beat frequency. Five regions were evaluated per filter insert and ciliary beat frequency averaged. Next, the inserts were cut from their support, placed on a concave well slide filled with media so that only the basal side was exposed to media, and 20 μL of a 0.1% suspension of 2-μm fluorescent polystyrene beads (Thermo Scientific R0200) was added to the apical surface. The slide was then placed on a custom-built system that includes a heated stage at 37 °C and imaged from above with a digital microscope fitted with a high-speed camera (Keyence Inc., Elmwood Park, NJ, USA). The images were acquired on three regions per filter and with tracking of 10–20 beads per region at a frame rate of 1,000 fps. Image files were exported to ImageJ to analyze particle movement with the MTrackJ plug-in (v. 1.5.1) (44). The extracted frame-by-frame coordinates were then used to estimate individual particle distance traveled and MCV. In addition, the coordinates and time lag between frames were used to define time scales (τ) following multiple particle transport methodology (45) and then determine the mean squared displacement of the particle for all possible time durations. The effective diffusivity (Deff) was then calculated from the mean squared displacement and time scales extracted (45). The mean squared displacement and effective diffusivity data generated for all particles tracked under Pf + and Pf− conditions were analyzed by Loess-smoothed regression to generate ensemble plots of mean squared displacement and effective diffusivity vs. time scales with 95% CIs to allow for comparisons between Pf+ and Pf− conditions.

MCV measurement in ex vivo piglet trachea

Tracheas from WT, Yorkshire, newborn piglets (2–5 days old) were freshly obtained from the swine facility at UC Davis. All methods using animal tracheae were carried out in accordance with relevant guidelines and regulations of Stanford University, and animal protocols received institutional approval (Stanford IACUC protocol # 10048). Piglet tracheas were transported in cold PhysioSol (Hospira Inc, IL, USA) and then transferred to ice-cold Krebs-Ringer bicarbonate (KRB) buffer gassed with 95% O2 and 5% CO2 and kept at 4 °C until use. The KRB buffer contained (in mM) 115 NaCl, 25 NaHCO3, 2.4 K2HPO4, 0.4 KH2PO4, 1.2 MgCl2, 1.2 CaCl2, 10 glucose, and 1.0 µM indomethacin, adjusted to pH 7.2 and ∼290 mOsm at room temperature. Indomethacin was added to reduce tissue exposure to endogenously released prostaglandins.

Measurement of MCV was taken as we have previously reported (46–48). Briefly, each whole-length piglet trachea was cut open along the mid-dorsal line and mounted mucosal side up onto a Sylgard elastomer platform. The prepared trachea was placed into a sealed, humidified chamber bubbled continuously with gas (95% O2/5% CO2) with the serosal surface bathed with KRB buffer. For the initial 30-minute stabilization period, the tissue was submerged in the bath as the temperature was gradually increased to 37 °C. Then. excess apical and bath solution was drained. The tissue was then stimulated for 30 minutes with the combined application of cholinergic (0.3 µM carbachol) and β-adrenergic (10 µM formoterol) applied to the serosal bath to initiate mucus secretion from submucosal glands and surface epithelia (46) in the presence of apical Pf phage (∼1 mL of 1010 Pf phage virions/mL in PBS or PBS as a control) designed to maximize contact between secreted mucus and apically added Pf phage. After 30-minute mucosal incubation, the excessive mucosal Pf phage was drained, and bath was refreshed with the combined agonists. After additional 10-minute stabilization period, MCV measurements using time-lapse imaging began by placing dry Xerox ink particles (∼10 µm) apically on the caudal end. The images of cephalad particle movements were automatically captured (four images at 20-second intervals every 5 min) by an Aven time-lapse digital camera and associated software (Ann Arbor, MI, USA). MCV (in millimeter per minute) was determined by measuring the fastest moving group of particles using NIH ImageJ software (46).

Phage purification and quantification

Pf4 phage was isolated and purified from PA01, DMS3vir from PA14 and Fd phage from E. coli. Phage purification was performed as previously described (30). Briefly, bacteria at mid-log phase growth were infected with stocks of phage and cultured in LB broth for 48 hours at 37 °C in a shaking incubator. Centrifugation at 6,000×g for 5 minutes was performed to remove bacteria. The supernatant was treated with 1 μg/mL DNase (Roche, catalog # 4716728001) for 2 hours at 37 °C, followed by treatment with 250 μg/mL of RNAse A (Thermo Fisher Scientific, catalog # EN0531) for 4 hours at 37 °C. Vacuum filtration through a 0.22-μM filter was performed to sterilize supernatant. Pf4 phage was precipitated from the supernatant by adding 0.3 M NaCl and 4% polyethylene glycol (PEG) 8000 (Millipore Sigma, catalog # P2139). Phage solutions were incubated overnight at 4 °C. Phage was then pelleted by centrifugation at 10,000×g for 20 minutes. The supernatant was subjected to a second round of PEG precipitation as above. The purified phage pellets were suspended in sterile PBS and dialyzed in 10-kDa molecular weight cut off tubing (Fisher Scientific, catalog # 88243) against PBS and quantified by plaque assay, and Pf phage preparation titers were confirmed by qPCR using our previously described methods (26, 30, 49).

Statistical analyses

Graphing and statistical analyses were performed using GraphPad Prism 6 (GraphPad Software), R (R Computing), and SAS (v.9.4, SAS Institute Cary, NC, USA). Comparisons of ciliary beat frequency and MCV by particle tracking were made by one-way ANOVA with Tukey's adjustment of multiple comparisons for individual comparisons. Comparison of average MCV in porcine model was based on simple two-sample t test.

Supplementary Material

pgae390_Supplementary_Data

Supplementary Material

Supplementary material is available at PNAS Nexus online.

Funding

E.B.B. was supported by the National Heart, Lung, and Blood Institute of the National Institue of Health (T32HL129970, 1K23HL169902-01), the Francis Family Foundation (Parker B Franis Fellowship), and the Cystic Fibrosis Foundation (BURGEN20Q0, BURGEN21A0-KU). P.C.C. was funded by a National Science Foundation (Graduate Research Fellowship). A.J.S. was supported by the National Science Foundation (Physics of Living Systems Program) (PHY-2102726). N.S.J. was funded by the Cystic Fibrossis Foundation (JOO19G0). P.L.B. was supported by National Institutes of Health (R01AI138981, K24AI166718-01A1, and R21GM147838) and a grant from the Emerson Collective. P.R.S. was supported by National Institutes of Health (R01AI138981). C.E.M. was supported by grants from the Cystic Fibrosis Foundation (MILLA22Y0, MILLA23P0), the National Heart, Lung, and Blood Institute (1R01HL148184), and the Ross Mosier Laboratories Gift Fund.

Author Contributions

Conceptualization of study and individual experiments by E.B.B., P.C.C., M.J.K., P.R.S., S.C.H., A.J.S., P.L.B., and C.E.M. Collection of data and performance of experiments by E.B.B., N.S.J., L.S.R.-H., A.G., and C.E.M. Analysis of data E.B.B., N.S.J., P.C.C., M.J.K., J.J.W., P.L.B., and C.E.M. Editing of manuscript E.B.B., L.S.R.-H., N.S.J., P.C.C., M.J.K., A.G., P.R.S., S.C.H., A.J.S., J.J.W., P.L.B., and C.E.M.

Data Availability

All data are included in Tables S1 and S2.
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