Conserved and acquired: Decoding YbjX and VirK in the pathogenicity of <i>Shigella flexneri</i>

The pathogenicity of <i>Shigella</i> spp. human pathogens responsible for bacillary dysentery, results from the combination of factors encoded both on the chromosome and on the virulence plasmid acquired during pathoadaptation. While many key elements have been extensively investigated, several remain poorly characterized. Among these, YbjX and VirK exhibit high structural similarity and are encoded by genes located on the chromosome and the virulence plasmid, respectively. We provide a molecular and functional characterization of <i>Shigella flexneri</i> YbjX and VirK. We defined the <i>ybjX</i> and <i>virK</i> promoter regions and confirmed that their expression is regulated by the PhoPQ two-component system. Localization studies demonstrated that both proteins are cytoplasmic. <i>In silico</i> analysis predicted a similar structure for the two proteins, resembling members of the Gcn5-related N-acetyltransferase superfamily. Functionally, lack of VirK resulted in increased permeability of the OM, sensitivity to cationic antimicrobial peptides, and an intensified release of proinflammatory cytokines by infected macrophages (THP-1-derived) and epithelial cells (Caco-2). The deletion of <i>ybjX</i> alone didn't confer detectable phenotypes. When both genes were deleted at the same time a complementary function of YbjX was revealed, as all the phenotypes described above were reinforced. Our findings underscore a synergistic role for YbjX and VirK in OM integrity and the modulation of the host response, suggesting a prominent role of VirK and a supporting role of YbjX. From an evolutionary perspective, our work suggests that the retention of <i>ybjX</i> and the acquisition of <i>virK</i> reinforced bacterial survival and fitness during the infection of the host.