Exploiting the bacteriophage arsenal to discover novel classes of anti-biofilm agents

PROJECT SUMMARY Bacteria often grow non-planktonically in aggregates termed biofilms, a growth state in which they are highly tolerant of antibiotics and host immune defenses, and are even protected from the viruses that target bacteria, phage. These aggregates are encased within a complex extracellular matrix that typically includes exopolysaccharides (EPS), extracellular DNA (eDNA), and secreted proteins. Biofilm-involved infections are a particularly grave danger from the opportunistic pathogen, Pseudomonas aeruginosa, that chronically infects the lungs of people with cystic fibrosis, chronic wounds, and medical devices. Thus, alternative therapies are urgently needed, and phage therapy, the treatment of bacterial infections with phage, is one promising option. Biofilms are tolerant to many phages, which reduces the effectiveness of phage therapy. However, there are numerous phages that have evolved mechanisms to infect biofilm-associated bacteria. Mining these anti- biofilm strategies used by phages represents an exciting opportunity to improve phage therapy and to identify proteins that could be developed as stand-alone therapeutics. Previous studies have revealed that some phages encode depolymerase proteins associated with their tail spikes; however, the assays used for identifying these proteins are limited in scope, and as a result the diversity of antibiofilm strategies remains largely unexplored. In general, known depolymerases target the o-antigen LPS and capsule rather than bona fide matrix components such as EPS. Furthermore, there are few studies that investigate phage and phage- derived anti-biofilm proteins using in vitro biofilm models. As a result, we have very little knowledge of how phages are normally tolerated by biofilms and what mechanisms they use to overcome this barrier. We propose two unique strategies to isolate biofilm-interacting phages coupled with innovative methods to identify the phage proteins responsible for the interactions. We next propose dissecting the mechanism by which the phages and phage-derived anti-biofilm proteins interact with the biofilm matrix using in vitro biofilm models coupled with imaging and biophysical approaches such as solid-state NMR. These studies will begin to assign functions to previously unknown, hypothetical phage genes that make up the enormous amount of uncharacterized viral genetic material. Additionally, the proposed research will open new exciting avenues for future studies with additional biofilm-forming pathogens. We anticipate the outcome of this work will move the field forward by shedding unprecedented light on basic phage and biofilm biology and provide essential information to better treat biofilm-associated infections.