Functional characterization of the DUF1127-containing small protein YjiS of Typhimurium.

Bacterial small proteins impact diverse physiological processes, however, technical challenges posed by small size hampered their systematic identification and biochemical characterization. In our quest to uncover small proteins relevant for pathogenicity, we previously identified YjiS, a 54 amino acid protein, which is strongly induced during this pathogen's intracellular infection stage. Here, we set out to further characterize the role of YjiS. Cell culture infection assays with mutants lacking or overexpressing YjiS suggested this small protein to delay bacterial escape from macrophages. Mutant scanning of the protein's conserved, arginine-rich DUF1127 domain excluded a major effect of single amino acid substitutions on the infection phenotype. A comparative dual RNA-seq assay uncovered the molecular footprint of YjiS in the macrophage response to infection, with host effects related to oxidative stress and the cell cortex. Bacterial cell fractionation experiments demonstrated YjiS to associate with the inner membrane and proteins interacting with YjiS in pull-down experiments were enriched for inner membrane processes. Among the YjiS interactors was the two-component system SsrA/B, the master transcriptional activator of intracellular virulence genes and a suppressor of flagellar genes. Indeed, in the absence of YjiS, we observed elevated expression of motility genes and an increased number of flagella per bacterium. Together, our study points to a role for YjiS as a membrane-associated timer of pathogen dissemination.