The small protein YjiS acts as a virulence suppressor in Salmonella 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 Salmonella pathogenicity, we previously identified YjiS, a 54 amino acid protein, which is strongly induced during this pathogen’s intracellular infection stage and exhibits features of a virulence suppressor. Here, we set out to further characterize the virulence-related role of YjiS. Cell culture infection assays with Salmonella mutants lacking or overexpressing YjiS corroborated previous functional genomics data and additionally suggested this small protein to counteract 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 effects related to oxidative stress and endo-/exocytosis. Bacterial cell fractionation experiments demonstrated YjiS to associate with the inner membrane, without being itself membrane-inserted. Accordingly, proteins interacting with YjiS in pull-down experiments were enriched for inner membrane processes. Together, our study points to a role for Salmonella YjiS as a membrane-associated suppressor of pathogen dissemination.