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A Phosphoproteomic Analysis of Mycobacterial PknG-Mediated Host Immune Evasion
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Resumo(s)
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.
Descrição
Funding Information: This work is based on the research supported, in part, by the National Research Foundation (NRF) of South Africa (grant numbers: 467126 and 95984). S.S.B. thanks the NRF for a doctoral bursary. K.C.N. thanks the NRF and UCT/CSIR for doctoral bursaries. N.C.S. thanks the South African Medical Research Council for a Junior Research Fellowship. J.M.B. thanks the NRF for a South African Research Chair grant. Publisher Copyright: © 2025 The Authors. Published by American Chemical Society
Palavras-chave
mass spectrometry Mycobacterium tuberculosis phosphoproteomics phosphorylation post-translational modifications serine/threonine protein kinases Biochemistry General Chemistry SDG 3 - Good Health and Well-being