Phage peptide promotes bacterial cell death via inner and outer membrane targets

ABSTRACT Cationic antimicrobial peptides (CAMPs) play a major role in mammalian innate immunity by preferentially targeting bacterial membranes through charge-dependent interactions. While CAMPs are extensively studied in eukaryotic systems, less is known about phage-encoded CAMPs and their role in bacterial lysis. Here, we characterize gp28, a 56-aa cationic peptide encoded by the T7-like coliphage phiKT to define its mechanism of membrane disruption and bacteriostatic activity. Biochemical fractionation reveals that gp28 is asymmetrically distributed across bacterial membranes. Supporting its direct role in causing cell death, gp28 induces permeability changes at the inner membrane. Using synthetic gp28 peptide, we also show that gp28 activity is blocked by lipopolysaccharide and divalent cation competition. This led us to discover that gp28 is also active against Gram-positive bacteria in a surface charge-dependent manner. Finally, we demonstrate gp28 has broad bacteriostatic activity against multiple Gram-negative species, including clinical Uropathogenic E. coli (UPEC) isolates. Together, our results support a model in which gp28 exploits charge-based interactions with bacterial surface molecules to gain access to membranes, then destabilizes the envelope to promote host cell death. These findings expand our understanding of phage lysis strategies and highlight phage-derived peptides as potential scaffolds for antimicrobial development. SIGNIFICANCE STATEMENT Cationic antimicrobial peptides produced by eukaryotes target and disrupt bacterial membranes. These peptides are integral members of the innate immune system and potential antibacterials. Due to their cellular origin, the genetics to understand their mechanisms of action are cumbersome. Here, we demonstrate functional similarity between a bacteriophage peptide called gp28 and cationic antimicrobial peptides. Gp28 partitions between both membranes of Gram-negative bacteria and reduces their integrity. We show that gp28 employs charge-based interactions to associate with and disrupt membranes, suggesting that gp28 targets charged polymers on the membrane surface. Additionally, gp28 inhibits the growth of Gram-negative and Gram-positive bacteria, and renders them more susceptible to antibiotics. Our studies provide a foundation for future genetics and development of phage peptide-based antimicrobials.