GlnK Regulates the Type III Secretion System by Modulating NtrB-NtrC Homeostasis in Pseudomonas aeruginosa

Bacterial pathogens exploit host-derived nutrients to coordinate metabolism and virulence determinants to optimize fitness in vivo. In <i>Pseudomonas aeruginosa</i>, GlnK is a central regulator of nitrogen metabolism. It senses the intracellular nitrogen status by integrating 2-oxoglutarate (2-OG) and glutamine signals, which in turn triggers its uridylylation and conformational changes. This reversible post-translational modification modulates its interaction with target proteins, thereby precisely regulating carbon-nitrogen metabolic homeostasis and enabling adaptive nitrogen metabolism in response to host-derived nutrient cues. In this study, we found that <i>glnK</i> is upregulated during infection in a mouse pneumonia model. By growing bacteria in mouse bronchoalveolar lavage fluid (BALF), we demonstrated that the expression of <i>glnK</i> is activated by the NtrB-NtrC two-component regulatory system in response to the host nutrient environment. Mutation of <i>glnK</i> impairs bacterial virulence. Transcriptomic analysis revealed downregulation of the type III secretion system (T3SS) genes in the <i>glnK</i> mutant. Further studies revealed a role of GlnK in maintaining the homeostasis of the NtrB-NtrC system through a negative feedback mechanism, which is required for the expression of the T3SS genes. Collectively, these findings reveal a role of GlnK in interconnecting carbon-nitrogen balance and the T3SS in response to the host environment.