6S RNA facilitates bacterial virulence and adaptation at the epithelial barrier

Enteropathogenic bacteria must adapt dynamically to the complex gastrointestinal environment to successfully colonize host tissue and evade immune defenses. Using Yersinia pseudotuberculosis as a model, we performed in vivo spatial transcriptomics to investigate bacterial gene expression as it translocates from cecal lumen to associated lymphoid tissue in the mouse intestine. By optimizing bacterial RNA enrichment, we achieved near-complete transcriptome coverage and identified compartment-specific transcriptional profiles. Bacteria in lymphoid tissue exhibited elevated expression of virulence-associated type III secretion system (T3SS) genes and markers of increased replication, alongside a higher plasmid copy number. Oxygen availability emerged as an environmental cue for T3SS induction. Importantly, we discovered a role for the non-coding 6S RNA in accelerating virulence gene expression. Tissue-localized bacteria had significantly upregulated 6S RNA levels, and deletion of the ssrS gene encoding 6S RNA impaired T3SS gene expression and effector secretion. Further, spatial analyses of bacterial gene expression in foci of infected lymphoid tissue revealed heterogeneous expression patterns with significantly elevated expression of T3SS and 6S RNA in bacteria located close to surrounding phagocytes. Together these findings demonstrate that Y. pseudotuberculosis undergoes rapid transcriptional reprogramming upon epithelial barrier crossing coordinated by environmental sensing, where 6S RNA accelerates tissue colonization by promoting efficient expression of T3SS.