Antimicrobial peptides and antibiotic diffusion in sputum

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. Project Number: 1R21AI194044-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Paul Bollyky | Institution: STANFORD UNIVERSITY, STANFORD, CA | Award Amount: $423,500 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZRG1 RCCS-A (03)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI19404401