Targeting Multidrug-Resistant Pseudomonas aeruginosa with R Pyocins

PROJECT SUMMARY Pseudomonas aeruginosa is an opportunistic human pathogen classified as a serious threat by the Centers for Disease Control and Prevention and a high-priority pathogen according to the World Health Organization. P. aeruginosa is exceptionally adept at colonizing human tissues due to its efficient efflux pumps, ability to form biofilms, and the presence of genomic and mobile genetic elements encoding antimicrobial resistance genes. P. aeruginosa poses a major risk to patients with cystic fibrosis (CF), where it causes chronic, often decades-long infections that destroy respiratory tissues. Multidrug-resistant (MDR) strains are of utmost concern to CF pa- tients, as infections with these strains increase their risk of death up to eightfold. Further compounding the prob- lem is the rise in antibiotic resistance in Pseudomonas and other pathogens. Recent large-scale studies showed limited effectiveness of currently used antibiotics, and even novel combinations of β-lactams/β-lactamase inhib- itors were ineffective against over 1/4th of clinical isolates of P. aeruginosa. Our preliminary data demonstrate that most infectious, high-risk, and globally widespread multilocus se- quence types of P. aeruginosa (e.g. ST111) produce phage tail-like protein complexes called R pyocins. Previous work showed that R pyocins have strain-specific killing, making them key contributors to Pseudomonas intra- specific competition. We found that the R5 pyocin encoded by an ST111 isolate kills ~80% of MDR CF isolates, which is better than any antimicrobial combination we tested. Pyocins bind to the core of the lipopolysaccharide (LPS) on the bacterial surface, ignoring the function of efflux pumps. Unlike phages, pyocins carry no DNA and can be administered as purified proteins, eliminating concerns about mutations in the human host. R pyocins can also kill bacteria in biofilms, a key factor in CF infections. R pyocins specifically kill P. aeruginosa without applying pressure on other bacteria to develop and spread resistance. Finally, our preliminary data show that deletion of two LPS biosynthesis genes makes P. aeruginosa universally sensitive to R5 pyocins. This approach offers a pathway for development of an inhibitory compound that will sensitize P. aeruginosa to pyocins. Taken together, these observations position R5 pyocins as a promising new treatment for P. aeruginosa that outperforms most conventional therapies. In this grant, we propose to comprehensively evaluate a panel of R pyocins from different pyocin subtypes, identifying those with the widest killing spectrum. We will also ex- amine the relationships between sensitivity to R pyocins, LPS mutations, and cognate pyocin production, as our preliminary data demonstrate that these relationships may be more complex than suggested by the current liter- ature. We will also produce high-purity recombinant pyocins in E. coli to test their activity and toxicity in bacterial and relevant human cells. Finally, we will define evolutionary trajectories that lead to R pyocin resistance. Overall, our research will provide a thorough evaluation of the clinical potential of R pyocins and will pinpoint key LPS proteins whose inhibition may further expand pyocins’ killing range.