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A Phosphoproteomic Analysis of Mycobacterial PknG-Mediated Host Immune Evasion
Seanantha S Baros-Steyl1, Kehilwe C Nakedi1, Tariq A Ganief1,2
1Division of Chemical & Systems Biology, Department of Integrative Biomedical Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa.
Abstract:
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.
Insights
Pathogenic mycobacteria use Protein Kinase G (PknG) to evade immune cells. This study reveals PknG reprograms macrophage functions like cytoskeletal organization and cell death, aiding mycobacterial survival.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Pathogenic mycobacteria, including Mycobacterium tuberculosis, evade host immune responses for persistence.
- Protein Kinase G (PknG) is implicated in mycobacterial evasion by inhibiting phagosome-lysosome fusion, but its precise mechanism remains unclear.
Purpose of the Study:
- To investigate the role of PknG in early macrophage infection events.
- To identify host phosphoproteins regulated by PknG during Mycobacterium bovis BCG infection.
Main Methods:
- RAW 264.7 macrophages were infected with wild-type and PknG knockout Mycobacterium bovis BCG strains.
- Phosphoproteomic analysis using TiO2-based phosphopeptide enrichment and LC-MS/MS was performed.
- Differential expression and functional analyses were conducted on identified phosphosites.
Main Results:
- 3003 phosphosites across 1638 host proteins were identified.
- 143 phosphosites were significantly altered between wild-type and mutant infections, with 95 increased in the presence of PknG.
- PknG activity was shown to interfere with host cytoskeletal organization, phagosome maturation, and programmed cell death.
Conclusions:
- PknG plays a critical role in reprogramming macrophage functions to facilitate mycobacterial survival.
- PknG interferes with host cell processes including cytoskeleton dynamics, phagosome maturation, and apoptosis.
- This study provides a foundation for understanding PknG's host substrate interactions and its role in mycobacterial pathogenesis.
