A previously uncharacterized Fur-family metalloregulator integrates iron- and manganese-sensing to control virulence gene regulatory networks in Brucella.

Intracellular survival of bacterial pathogens relies on specific adaptations to the stress found within the eukaryotic host cell environment. These processes depend on the activity of transcriptional regulatory networks that ensure proper spatiotemporal expression of all genes participating in the subversion of eukaryotic cellular functions. Such transcriptional networks often involve complex regulatory mechanisms, including both activators and repressors as well as functional redundancy among transcription factors (TFs), making the discovery of novel virulence regulators challenging. In this study, we dissected a bacterial gene expression network in order to identify new TFs impacting the virulence of Brucella, a widespread intracellular pathogen. By combining in silico and experimental approaches, we identified TF-binding sites resembling the ferric-uptake regulator (Fur) binding motif adjacent to binding sites of VjbR, the main activator of Brucella virulence. Genetic and molecular analyses revealed that Fur4, a previously uncharacterized Fur family regulator, is responsible for iron (Fe)- and manganese (Mn)-dependent repression of different loci including VjbR target virulence genes such as the operon encoding the VirB Type-IV Secretion System. Our results showed that Fur4 acts in concert with the Mn-uptake regulator Mur as well as other Mn/Fe-responsive TF, affecting bacterial gene expression both in vitro and during intracellular trafficking of Brucella within macrophage cells. These findings reveal a novel metal-dependent regulatory mechanism modulating the expression of Brucella virulence determinants, enhancing our understanding of how pathogens integrate environmental signals to fine-tune gene expression during intracellular infection.