The VapBC Toxin–Antitoxin System Enhances Shigella flexneri Fitness Through Coordination of Metabolic Stress Adaptation and Virulence

Shigella flexneri is a facultative intracellular pathogen that causes bacillary dysentery by invading and replicating within intestinal epithelial cells. Successful intracellular survival requires the bacterium to balance metabolic adaptation with the sustained expression of virulence programs by the type III secretion system (T3SS). Toxin–‘antitoxin (TA) systems, including the highly conserved type II VapBC module, are classically associated with plasmid maintenance via post-segregational killing, but their broader roles during infection remain poorly understood. Here, we investigate the function of the VapBC system during S. flexneri infection. We show that the vapBC operon is activated in response to intracellular stress and that VapC-dependent cleavage of initiator tRNA ^fMet occurs specifically during infection. The stringent response, primarily mediated by SpoT, appears to influence operon responsiveness by maintaining vapB expression levels, while iron limitation emerges as a strong activator of vapC transcription and activity. Deletion of vapBC impairs T3SS activation and bacterial dissemination, despite normal invasion, and induces a strong host interferon response, including the upregulation of guanylate-binding proteins (GBPs), known to restrict bacterial spread. Transcriptomic profiling of the ΔvapBC mutant reveals downregulation of core metabolic genes and upregulation of envelope stress and other TA modules, indicating a loss of intracellular homeostasis. These findings uncover a novel role for VapBC in promoting Shigella fitness by coordinating stress adaptation, virulence expression, and immune evasion, thereby sustaining the bacterium’s intracellular lifestyle.