Phage-Encoded Cationic Antimicrobial Peptide Required for Lysis

Abstract

Most phages of Gram-negative hosts encode spanins for disruption of the outer membrane, the last step in host lysis. However, bioinformatic analysis indicates that ∼15% of these phages lack a spanin gene, suggesting they have an alternate way of disrupting the OM. Here, we show that the T7-like coliphage phiKT causes the explosive cell lysis associated with spanin activity despite not encoding spanins. A putative lysis cassette cloned from the phiKT late gene region includes the hypothetical novel gene 28 located between the holin and endolysin genes and supports inducible lysis in E. coli K-12. Moreover, induction of an isogenic construct lacking gene 28 resulted in divalent cation-stabilized spherical cells rather than lysis, implicating gp 28 in OM disruption. Additionally, gp 28 was shown to complement the lysis defect of a spanin-null λ lysogen. Gene 28 encodes a 56-amino acid cationic protein with predicted amphipathic helical structure and is membrane-associated after lysis. Urea and KCl washes did not release gp 28 from the particulate, suggesting a strong hydrophobic membrane interaction. Fluorescence microscopy supports membrane localization of the gp 28 protein prior to lysis. Gp 28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. Synthesized gp 28 behaved similar to LL-37 in standard assays mixing peptide and cells to measure bactericidal and inhibitory effects. Taken together, these results indicate that phiKT gp 28 is a phage-encoded cationic antimicrobial peptide that disrupts bacterial outer membranes during host lysis and thus establishes a new class of phage lysis proteins, the disruptins. Significance We provide evidence that phiKT produces an antimicrobial peptide for outer membrane disruption during lysis. This protein, designated as a disruptin, is a new paradigm for phage lysis and has no similarities to other known lysis genes. Although many mechanisms have been proposed for the function of antimicrobial peptides, there is no consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic system to support such studies. Therefore, the phiKT disruptin may represent the first genetically tractable antimicrobial peptide, facilitating mechanistic analyses.

Probing the Structural Topology and Dynamic Properties of gp28 Using Continuous Wave Electron Paramagnetic Resonance Spectroscopy

“…The MIC (minimum inhibitory concentration) value of gp28 after synthesis was tested, as in the literature it had been previously shown to be bactericidal. 12 Similar MIC values were observed for the wild type (WT) and spin-labeled gp28 construct (Supplementary Figure S2). However, MIC studies on additional spin-labeled gp28 constructs would be helpful to understand the antimicrobial activity of gp28 peptide, and the functional effects of individual Cys mutations cannot be ruled out.…”

“…As an amphipathic peptide, gp28 should exist in both a soluble and membrane-bound state. 12 For a more complete understanding of the peptide, we wanted to model it in both states as well as the transition between them as adsorption occurs. Because gp28 does not have a published structure, we needed to develop a structural model.…”

“…2,10,11 However, according to a recent bioinformatic study of 677 genomes of Gram-negative bacteria-infecting phages, about 15% of these phages lack spanins but are still able to lyse the bacterial cell. 12 The findings indicate that some phages disrupt the OM differently and may involve different proteins. Termed Disruptins, this new class of proteins are discussed in terms of a new model for the coordination of phage lysis.…”

Genomic Diversity of Bacteriophages Infecting the Genus Acinetobacter

“…A growing body of evidence has shown the ability of recombinant Acinetobacter phage endolysins to naturally degrade cells after exogenous application due to the presence of highly positively charged C-terminal domains with predicted amphipathic helical structures [73,74]. Another possibility might be as reported in the coliphage ϕKT phage, which encodes an independent protein-encoding gene that disrupts the membrane using a mechanism similar to antimicrobial peptides [75].…”

Endolysin Regulation in Phage Mu Lysis

“…The holin initiates lysis by forming holes in the cytoplasmic membrane, which allows the endolysin, a muralytic enzyme, to attack the peptidoglycan (PG). Other MGL lysis proteins have been identified, including spanins and disruptins to attack the outer membrane and antiholins to regulate the timing of holin function ( 2 , 3 ). Among the paradigm phages used to study most of the fundamental processes of prokaryotic biology, phage Mu stands out because its lysis system has not been delineated ( 4 , 5 ).…”