Shigella flexneri undergoes niche-specific adaptation during infection.

Shigella flexneri is a leading cause of diarrheal disease worldwide, yet bacterial factors required within distinct host niches remain poorly understood. Here, we used genome-wide transposon sequencing in an infant rabbit model of shigellosis to identify genes promoting bacterial fitness in vivo. The screen identified virulence factors on the large virulence plasmid and novel chromosomal fitness factors. Characterization of zitB, encoding a cation diffusion facilitator family Zn transporter, revealed a niche-specific fitness contribution. The ΔzitB mutant showed no growth defect in vitro in rich media or in vivo during the early infection phase (8 hpi) in epithelial cells. However, bacterial burden was reduced during the late infection phase (24 hpi) in vivo, when bacteria interact with immune cells, in both competitive and mono-infection assays. Reduced bacterial burden was associated with increased MARCO-specific macrophages, suggesting impaired colonization and killing by the ΔzitB mutant. Consistently, ΔzitB fitness was impaired in THP-1-derived macrophages. Metal chelation increased bacterial burden, indicating a role for macrophage-mediated metal toxicity. The ΔzitB mutant also showed increased sensitivity to zinc and copper. Finally, macrophage depletion in vivo restored bacterial burden to wild-type levels. Together, these findings identify ZitB as a niche-specific bacterial factor promoting S. flexneri fitness during macrophage-associated metal stress.