The filamentous Pseudomonas phage Pf disrupts airway basal cell proliferation

Abstract

Filamentous phages made by Pseudomonas (Pa) spontaneously organize polymers into crystalline structures and function as a scaffold in Pa biofilms. We have detected the phage Pf in high levels in sputum from Pa infected Cystic Fibrosis (CF) patients. The presence of Pf phage in CF sputum we find associated with chronic Pa infection, detriments in lung function, and resistance to antibiotics. We hypothesize that Pf can also affect cellular repair mechanisms in the airway. To test this we isolated basal cells from respiratory samples obtained from healthy donors. Cells were seeded onto collagen coated 96-well plates and allowed to attach overnight. We then added to the wells Pf phage at MOIs of log 3, log 5 or log 7, or control media or a mix of Pf and LPS (1 μg/mL) to emulate Pa infection conditions. Cells were then incubated at 37^oC and 5% CO₂ and monitored by live imaging with phase contrast for 72 hours using the Incucyte system (Sartorius Lab Instruments, Goettingen). Images were analyzed for cell number and confluence following manufacturer recommendations. We find that after the first 24 hours, there is a dose dependent and significant effect of Pf decreasing proliferation compared to the control conditions. There was also an additive effect of LPS, with the combination of Pf log 7 and LPS basically halting any proliferation. In a separate set of experiments, we seeded cells onto collagen coated coverslips and after allowing the cells to attach overnight exposed them to log 7 fluorescently tagged Pf. Fluorescent microscopy revealed intracellular presence of Pf. We are conducting further experiments with cells from CF donors and other phages, as well as interrogating potential mechanisms involved.