The VirF(21):VirF(30) protein ratio is affected by temperature and impacts Shigella flexneri host cell invasion

Shigella spp, the etiological agents of bacillary dysentery in humans, have evolved an intricate regulatory strategy to ensure fine-tuned expression of virulence genes in response to environmental stimuli. A key component in this regulation is VirF, an AraC-like transcription factor, which at the host temperature (37°C) triggers, directly or indirectly, the expression of > 30 virulence genes important for invasion of the intestinal epithelium. Previous work identified two different forms of VirF with distinct functions: VirF(30) activates virulence gene expression, while VirF(21) appears to negatively regulate virF itself. Moreover, VirF(21) originates from either differential translation of the virF mRNA or from a shorter leaderless mRNA (llmRNA). Here we report that both expression of the virF(21) llmRNA and the VirF(21):VirF(30) protein ratio are higher at 30°C than at 37°C, suggesting a possible involvement of VirF(21) in minimizing virulence gene expression outside the host (30°C). Ectopic elevation of VirF(21) levels at 37°C indeed suppresses Shigella´s ability to infect epithelial cells. Finally, we find that the VirF(21) C-terminal portion, predicted to contain a Helix-Turn-Helix motif (HTH2), is required for the functionality of this negative virulence regulator.