Phage-encoded cationic antimicrobial peptide used for outer membrane disruption in lysis
Spanins are required for the last step in bacteriophage lysis: the disruption of the outer membrane. Bioinformatic analysis has shown that ~15% of phages lack a spanin gene, which suggests an alternate mechanism of outer membrane disruption. To address this, we selected virulent podophage {phi}KT as a spaninless exemplar and tested {phi}KT genes for outer membrane disruption during lysis. Hypothetical novel gene 28 causes outer membrane disruption when co-expressed with {phi}KT lysis genes and complements the lysis defect of a {lambda} spanin mutant. Gp28 is a 56 aa cationic peptide with predicted amphipathic helical structure and is associated with the particulate fraction after lysis. Urea and KCl washes did not release gp28 from the particulate, suggesting a strong hydrophobic interaction with the membrane. Super high-resolution microscopy supports a primarily outer membrane localization for the peptide. Additionally, holin function is not required for gp28-mediated lysis. Gp28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. In standard assays to measure bactericidal and inhibitory effects of antimicrobial peptides on bacterial cells, synthesized gp28 performed equivalently to LL-37. The studies presented here suggest that {phi}KT Gp28 disrupts bacterial outer membranes during lysis in a manner akin to antimicrobial peptides.\n\nSignificanceHere we provide evidence that {phi}KT produces an antimicrobial peptide for outer membrane disruption during lysis. The disruptin is a new paradigm for phage lysis, and has no similarities to other known lysis genes. Many mechanisms have been proposed for the function of antimicrobial peptides, however there is not a consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic selection system to support such studies. Therefore, the {phi}KT disruptin may represent the first genetically tractable antimicrobial peptide.