Concentration-dependent dual activity of a phage-encoded lysin modulates infection dynamics in mycobacteriophage D29

Lytic enzymes encoded by bacteriophages are conventionally understood to serve a single function in the phage life cycle: to enable progeny release through host cell lysis. Here, we identify an unexpected dual functionality in LysB, a lytic cassette protein of mycobacteriophage D29, which exerts opposing effects on phage propagation depending on the stage of infection. In our model, during the early phase of phage expansion, LysB's activity is limited to infected cells, hydrolyzing their outer membrane to facilitate efficient virion release. However, as the infection progresses, LysB accumulates in the extracellular environment, eventually reaching levels sufficient to act on uninfected bystander cells, rendering them resistant to infection. Consistent with this model, deletion of lysB impairs progeny release at low multiplicity of infection yet enhances phage propagation at later stages of plaque expansion. We further show that exogenous treatment of host cells with LysB inhibits phage infection in a concentration-dependent manner, likely by removing cell-surface determinants required for phage adsorption. Together, these findings demonstrate LysB's ability to modulate infection dynamics at the population level, revealing a potential new mechanism for phage-encoded host resilience. Engineered phages that lack this self-limiting activity may offer a path toward more effective antibacterial agents for therapeutic applications.IMPORTANCELytic bacteriophages are often viewed as purely destructive viruses that rapidly eliminate their bacterial hosts. Yet, as in other predator-prey relationships, how these phages avoid exhausting their host populations remains poorly understood. Here, we report an example in which a phage-encoded protein that drives lysis also acts to limit it. LysB of mycobacteriophage D29 facilitates efficient virion release during infection but accumulates extracellularly as infection progresses, eventually blocking phage adsorption on uninfected cells in a concentration-dependent manner. The result is an intrinsic negative feedback on phage infection that might act to prevent host extinction. Given that rebounding infections pose a major challenge in the management of bacterial diseases, understanding and counteracting the mechanisms that underpin host resilience is crucial for the therapeutic application of phages.