Pseudomonas aeruginosa (Pa) is a major human pathogen whose virulence is predicated on its ability to form
biofilms - slimy layers of polysaccharides and bacteria that confer resistance to antibiotics and immune
clearance. Pa biofilms are particularly problematic in Cystic Fibrosis (CF), where they are a major cause of
mortality. We have uncovered novel roles for Pf bacteriophages in chronic biofilm infections. We recently
reported that Pf phages produced by Pa organize host and microbial polymers present in sputum into a liquid
crystal. This crystalline architecture makes CF sputum more viscous and more adhesive. It also traps antibiotics
like tobramycin and prevents them from reaching the bacteria living within.
Along with these effects on biofilm formation and function, Pf phages directly interfere with host immunity. Purified
Pf phage inhibits phagocytosis and hampers the ability to clear bacterial infections. In preliminary work with a
cohort of Pa infected CF patients, we found associations between Pf phage and Pa lung infection burden,
chronicity of Pa infection, declines in pulmonary function during exacerbation, and antibiotic resistance to anti-
Pseudomonal antibiotics. Given the high abundance of Pf in CF sputum we also postulate that Pf liquid crystals
may affect host mucociliary clearance by attachment and impairment of the epithelial ciliary brush function.
In light of these exciting preliminary data, we hypothesize that Pf phage cause worse clinical outcomes in CF by
disrupting bacterial clearance mechanisms and promoting bacterial tolerance to antibiotics. We will test this
hypothesis in experiments with the following aims:
In Aim 1 we will determine how Pf phage impacts cell function in the airways. Our hypothesis is that Pf phage
interferes with phagocytosis, impairs mucus transport and disrupts the function of the ciliary brush. To test this,
we will conduct a number of in vitro experiments to investigate the mechanisms of Pf pathogenecity.
In Aim 2 we will elucidate how Pf phage production drives Pa antibiotic tolerance. Our hypothesis is that the
liquid crystalline organization of Pa biofilms prevents diffusion of antibiotics and promotes the emergence of
resistant strains. To test this, we will assess how Pf phage-mediated sequestration impacts the bioactivity of
antibiotics and the emergence of antibiotic resistant Pa isolates over time.
In Aim 3 we will characterize how Pf phage impacts clinical outcomes in CF patients. Our hypothesis is that
higher Pf phage titers are associated with crystalline sputum and worse clinical outcomes. To test this, we will
perform longitudinal studies to determine how Pa colonization influences pulmonary function and the occurrence
of exacerbations. We will also investigate in CF patients for the presence of antibody responses directed at Pf
and correlate their presence with clinical outcomes.
This proposal represents a bold and radically unconventional approach to Pa biofilm infections and CF
pathobiology. If successful, this work will identify a novel therapeutic target in CF and other settings where Pa
biofilm infections cause disease.