A Phosphoproteomic Analysis of Mycobacterial PknG-Mediated Host Immune Evasion

Pathogenic mycobacteria, such as <i>Mycobacterium tuberculosis</i>, modulate the host immune system to evade clearance and promote long-term persistence, leading to disease progression or latent infection. Understanding how these mycobacteria evade elimination is key to uncovering the molecular mechanisms of infection. Protein kinase G (PknG) in pathogenic mycobacteria plays a critical role in avoiding macrophage clearance by inhibiting phagosome-lysosome fusion; however, the exact mechanism is not completely understood. To investigate the role of PknG during early events of macrophage infection, RAW 264.7 macrophages were infected with <i>Mycobacterium bovis</i> BCG wild-type and PknG knockout mutant strains. Phosphoproteomic analysis, including TiO₂-based phosphopeptide enrichment and LC-MS/MS, identified 3003 phosphosites across 1638 host proteins. Differential expression analysis revealed 143 phosphosites significantly altered between wild-type and mutant infections, with 95 exhibiting increased phosphorylation in the presence of PknG. Additionally, 34 phosphosites were exclusively phosphorylated in the presence of PknG. Functional analysis demonstrated that PknG kinase activity reprograms normal macrophage function by interfering with host cytoskeletal organization, phagosome maturation, and programmed cell death, establishing a new role for PknG in directing the fate of mycobacteria within macrophages. Differentially phosphorylated proteins in this study serve as a foundation for further validation and the assignment of PknG host substrate assignment.