Substrate Characterization of Mycobacterial Kinase PknG Revealing PknG‐KARS‐MAPK Phosphorylation Signaling Axis in Host

Tuberculosis is the second leading infectious disease globally, severely threatening human health. The emergence of drug‐resistant Mycobacterium tuberculosis makes the development of new therapeutic targets particularly urgent. Serine/threonine protein kinase PknG can help the pathogen escape macrophage clearance by phosphorylating host proteins. This study adopted multi‐omics approaches to investigate the regulatory mechanism of mycobacterial PknG on host cell processes. The results showed that host lysine‐tRNA ligase (KARS) is a potential substrate of PknG; PknG, by catalyzing the phosphorylation of KARS at T592 and T595 sites, affects the phosphorylation levels in the MAPK/NF‐κB signaling pathway, thereby regulating the immune response. Furthermore, PknG overexpression ( Ms PknG‐OE) can lead to differential impacts on the lungs, characterized by elevated mRNA levels of Tnfa , Il1b , and Il6 , and more severe histopathological changes. Flow cytometry showed that M. smeg ‐PknG Mtb significantly reduced splenic CD3 + and CD8 + T cells, exhibiting an immunosuppressive trend; whereas M. smeg ‐PknG Ms increased the proportions of MDSCs and multiple regulatory T‐cell subsets while decreasing the proportion of CD8+ T cells, suggesting enhanced regulatory immunity and altered T‐cell subset composition. This study systematically elucidated the mechanism by which mycobacteria regulate the host immune network through the PknG‐KARS axis, and pointed out that the T592 and T595 sites of KARS could be potential targets for treating infection.