An uncharacterized small protein MicN mediates the transcriptional reprogramming of Salmonella through regulating the RpoS-RNA polymerase interaction

Salmonella adapts its metabolism upon entry into macrophages to survive in the hostile host environment. However, the specific regulatory mechanisms involved remain largely unknown. In this study, we identify a previously uncharacterized small protein, MicN, that is essential for the survival of Salmonella within macrophages and significantly influences its pathogenicity in vivo. The expression of MicN induces substantial alterations in the metabolic pathways of Salmonella, notably resulting in promoting a transition to a low-energy metabolic state. We determined the crystal structure of MicN, revealing the protein conformation characterized by a high density of negatively charged regions on its surface. Employing a pull-down assay, we established that MicN primarily interacts with RNA polymerase (RNAP). Computational modeling of the interaction between MicN and RNA polymerase subunits suggested a strong likelihood of binding between MicN and RpoS. Further validation through both in vivo and in vitro experiments confirmed the direct interaction of MicN and RpoS. The predicted MicN-RpoS structure indicated that the binding of MicN to RpoS modifies RpoS's interaction with RNAP core enzyme, and functional assays confirmed that MicN indeed changes the binding affinity of RpoS to RNA polymerase. This research provides the first insight into how Salmonella utilizes specific small proteins to finely tune transcriptional reprogramming, thereby establishing a foundational understanding of the intracellular survival mechanisms of pathogens and paving the way for the development of novel therapeutic strategies.