Heterodimerization of staphylococcal phage φ2638A endolysin isoforms and their functional role in bacterial lysis

TL;DR

A phage produces two versions of an enzyme that work together to efficiently break down bacteria during infection.

Contribution

The study reveals that a short isoform of endolysin enhances phage lytic efficiency by forming a heterodimer with the full-length isoform.

Findings

The SV isoform of Ply2638A forms a heterodimer with the full-length endolysin during phage infection.

Phages producing only one isoform show delayed progeny release and impaired lytic activity.

Co-expression of both isoforms enhances cell lysis efficiency and progeny release.

Abstract

Bacteriophage endolysins targeting Gram-positive bacteria typically feature a modular architecture of one or more enzymatically active domains (EADs) and cell wall binding domains (CBDs). Several endolysins also feature internal translational start sites (iTSSs) that produce short variant (SV) isoforms alongside the full-length (FL) endolysin. While the lytic activity of endolysins and their isoforms has been extensively studied as exogenous agents, the purpose behind producing the SV isoform during the phage infection cycle remains to be explored. In this study, we used staphylococcal phage φ2638A as a model to determine the interplay between its FL endolysin, Ply2638A, and its SV isoform during phage infection. X-ray crystallography structures and AlphaFold-generated models enabled elucidation of individual functions of the M23 endopeptidase, central amidase, and SH3b domains of Ply2638A. Production of the SV isoform (amidase and SH3b) was confirmed during phage infection and shown to form a heterodimer complex with Ply2638A via interamidase domain interactions. Using genetically engineered phage variants, we show that production of both isoforms provides an advantage during phage infection as phages producing only one isoform presented delayed progeny phage release as well as impaired lytic activity, which was partly restored through complementation of the missing isoform protein. Interestingly, when applied as an antimicrobial against Staphylococcus aureus in culture, the activity of Ply2638A remained constant regardless of SV isoform complementation. We propose that the SV isoform enhances the efficiency of cell lysis and progeny release at the end of the lytic cycle, providing a functional explanation for iTSSs conservation across diverse phage genomes.

Phage φ2638A co-expresses a short variant endolysin from an internal translational start site (iTSS), which heterodimerizes with the full-length isoform; this co-expression enhances the efficiency of cell lysis and progeny release, providing a functional explanation for the conservation of iTSSs across diverse phage genomes.

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