Simultaneous inhibition of bacterial virulence and anti-phage defense systems by synergistic bacteriophage counter-defense proteins.

Bacteriophages have evolved diverse inhibitors targeting key bacterial processes, including virulence and anti-phage defense systems, which could inspire novel antimicrobial strategies and enhance phage therapy approaches. In this study, we characterize Dap2, a protein encoded by a Pseudomonas aeruginosa phage PaoP5, which disrupts host virulence by sequestering the type III secretion system (T3SS) transcriptional activator ExsA, thus suppressing bacterial pathogenicity. Furthermore, Dap2 also directly binds the host Lon protease to prevent degradation of the phage-encoded HNH endonuclease. Deletion of dap2 in PaoP5 strongly impairs phage genome packaging due to insufficient levels of HNH. Finally, Dap2 synergizes with its genomically adjacent partner Dap1, a previously identified HNH-binding protein providing partial Lon resistance, to completely protect HNH against degradation. Together, these findings reveal a dual-function phage protein that simultaneously modulates bacterial virulence and anti-phage immunity, and showcase a synergistic mechanism for complete neutralization of bacterial defense system against which individual components provide only partial protection.