Proteomic Profiling Reveals RisR as a Novel Regulatory Element in the Virulence Network of <i>Bordetella pertussis</i>

<i>Bordetella pertussis</i>, the causative agent of whooping cough, regulates its virulence through the BvgAS two-component system, which controls the transition between virulent (Bvg⁺) and avirulent (Bvg^-) phases. In the virulent phase, virulence-activated genes (<i>vag</i>s) are expressed, while in the avirulent phase, these are repressed. Virulence-repressed genes (<i>vrg</i>s) are induced under the control of the response regulator RisA. Phosphorylation of RisA by the nonoperonic kinase RisK is essential for <i>vrg</i>s expression. Adjacent to <i>risK</i> lies <i>risR</i>, a gene encoding a putative response regulator, whose function remained unexplored. To investigate the RisR's role in <i>B. pertussis</i> biology, we performed comparative proteomic analyses between wild-type and an isogenic <i>risR</i>-deficient strain, grown under both virulent and avirulent conditions. Our data show that RisR modulates the abundance of proteins encoded by <i>vag</i>s and <i>vrg</i>s, suggesting a previously unrecognized interplay between RisR and the BvgAS-RisAK regulatory network. Moreover, RisR regulates the abundance of proteins important for bacterial fitness, including those involved in iron acquisition and intracellular adaptation. Functional assays further support the role of RisR in promoting intracellular survival. This study provides the first proteome-wide characterization of the RisR regulon and identifies it as a novel regulator of <i>B. pertussis</i> virulence and adaptation.