A Phosphoproteomic Analysis of Mycobacterial PknG-Mediated Host Immune Evasion

Pathogenic mycobacteria, such as Mycobacterium
tuberculosis, 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 Mycobacterium bovis 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.