Antimicrobial peptides and antibiotic diffusion in sputum

ABSTRACT SUMMARY
Inhaled antibiotics are critical to the health of many patients, including those with cystic fibrosis (CF), a genetic
disease characterized by thick, polymer-rich sputum and devastating chronic airway infections. Inhaled
antibiotics are particularly important for suppression of Pseudomonas aeruginosa, a bacterial pathogen and
major contributor to morbidity and mortality in CF.
Unfortunately, the effectiveness of inhaled antibiotics is limited by poor antibiotic penetration of sputum
biofilms – slimy communities of bacteria and polymers that colonize the airways of pwCF. We need improved
approaches and therapeutic targets to enable diffusion of antibiotics through infected sputum.
We have identified a microbial factor that prevents antibiotic diffusion in sputum. Pf bacteriophages (phages),
are viruses produced by P. aeruginosa. Unlike most phages that lyse (kill) their bacterial hosts, Pf phages are
produced without lysis. Instead, Pf phages function as structural elements in bacterial biofilms, including in CF
airways. Pf phages organizes polymers present in both sputum and biofilms into a liquid crystalline state. The
formation of these crystalline networks is driven by entropic, charge-based interactions between phages and
polymers present in sputum. These biophysical assemblies prevent antibiotic diffusion and shield the bacteria
within from antibiotic killing. We and others have reported that Pf phage is found in over 80% of adult CF patients.
Moreover, Pf phage is associated with chronic P. aeruginosa lung infection, declines in pulmonary function, and
resistance to several anti-Pseudomonal antibiotics.
It may be possible to disrupt these crystalline structures by targeting Pf phage. Our preliminary data suggest
that the antimicrobial peptide cathelicidin or LL-37, a component of the innate immune system, disrupts
crystalline networks formed by Pf phage in vitro. Our model is that LL-37 and other cationic peptides bind to
anionic Pf phage capsid proteins in ways that prevent liquid crystal formation and promote antibiotic diffusion.
It may be possible to bundle conventional antibiotics with antimicrobial peptides to promote the diffusion of
inhaled antibiotics in sputum. Unfortunately, however, the concentrations of LL-37 required to disrupt crystalline
biofilms are toxic to human cells, Nonetheless, it may be possible to synthesize other cationic peptides that
likewise promote antibiotic penetration but are non-toxic to cells.
Here, we propose to identify peptides and small molecules that bind Pf, disrupt crystalline biofilms, and
facilitate antibiotic diffusion in sputum. First, we will perform high-throughput screens to identify these molecules.
Then, we will then evaluate these candidates in physiologically relevant and highly quantitative assays. Together,
these aims represent a bold and radically unconventional approach to improving the efficacy of inhaled antibiotics
against Pseudomonas airway infections in CF and other settings.

Grant Number: 1R21AI194044-01
NIH Institute/Center: NIH
Principal Investigator: Paul Bollyky