TY - JOUR

T1 - A dimeric holin/antiholin complex controls lysis by phage T4

AU - Schwarzkopf, Jan Michel Frederik

AU - Mehner-Breitfeld, Denise

AU - Brüser, Thomas

N1 - Publisher Copyright: Copyright © 2024 Schwarzkopf, Mehner-Breitfeld and Brüser.

PY - 2024/9/5

Y1 - 2024/9/5

N2 - Lytic phages control the timepoint of host cell lysis by timing the holin-mediatedrelease of cell wall-degrading endolysins. In phage T4, the antiholin RI inhibitsthe holin T, thereby preventing the early release of the T4 endolysin and lysis.The antiholin achieves lysis inhibition (LIN) in response to phage superinfections,thereby increasing the chance for lysis in an environment with a lower phageconcentration. The holin T consists of a small N-terminal cytoplasmic domain,a transmembrane helix, and a periplasmic C-terminal domain. The antiholin istargeted to the periplasm by a cleavable signal peptide. Recently, the periplasmicsoluble domains of the holin and the antiholin were found to form T2/RI2tetramers in crystals. To investigate the functional relevance of this complex,we reconstituted LIN in a phage-free system, using only RI, T, and endolysin,and combined targeted mutagenesis with functional analyses. Inactivation ofthe RI signal peptide cleavage site did not abolish LIN, indicating that RI canfunction in a membrane-bound state, which argued against the tetramer. Thisled to analyses showing that only one of the two T/RI interfaces in the tetrameris physiologically relevant, which is also the only interaction site predictedby AlphaFold2. Some holin mutations at this interaction site prevented lysis,suggesting that the RI interaction likely acts by blocking the holin oligomerization required for hole formation. We conclude that LIN is mediated by a dimeric T/RI complex that, unlike the tetramer, can be easily formed when both partners are membrane-anchored.

AB - Lytic phages control the timepoint of host cell lysis by timing the holin-mediatedrelease of cell wall-degrading endolysins. In phage T4, the antiholin RI inhibitsthe holin T, thereby preventing the early release of the T4 endolysin and lysis.The antiholin achieves lysis inhibition (LIN) in response to phage superinfections,thereby increasing the chance for lysis in an environment with a lower phageconcentration. The holin T consists of a small N-terminal cytoplasmic domain,a transmembrane helix, and a periplasmic C-terminal domain. The antiholin istargeted to the periplasm by a cleavable signal peptide. Recently, the periplasmicsoluble domains of the holin and the antiholin were found to form T2/RI2tetramers in crystals. To investigate the functional relevance of this complex,we reconstituted LIN in a phage-free system, using only RI, T, and endolysin,and combined targeted mutagenesis with functional analyses. Inactivation ofthe RI signal peptide cleavage site did not abolish LIN, indicating that RI canfunction in a membrane-bound state, which argued against the tetramer. Thisled to analyses showing that only one of the two T/RI interfaces in the tetrameris physiologically relevant, which is also the only interaction site predictedby AlphaFold2. Some holin mutations at this interaction site prevented lysis,suggesting that the RI interaction likely acts by blocking the holin oligomerization required for hole formation. We conclude that LIN is mediated by a dimeric T/RI complex that, unlike the tetramer, can be easily formed when both partners are membrane-anchored.

KW - Protein-Protein Interaktionen

KW - Membranproteine

KW - Bacteriophagen

KW - Holine

KW - Antiholine

KW - Phage T4

KW - Lysisinhibition

KW - Escherichia coli

KW - protein-protein interaction

KW - membrane protein

KW - bacteriophage

KW - holin

KW - antiholin

KW - phage T4

KW - lysis inhibition

KW - Escherichia coli

KW - protein–protein interaction

UR - http://www.scopus.com/inward/record.url?scp=85204743971&partnerID=8YFLogxK

U2 - 10.3389/fmicb.2024.1419106

DO - 10.3389/fmicb.2024.1419106

M3 - Article

VL - 15

JO - Frontiers in Microbiology

JF - Frontiers in Microbiology

SN - 1664-302X

M1 - 1419106

ER -