Two-component signal transduction systems (TCSs) are widespread types of protein machinery, typically consisting of a histidine kinase membrane sensor and a cytoplasmic transcriptional regulator that can sense and respond to environmental signals. TCSs are responsible for modulating genes involved in a multitude of bacterial functions, including cell division, motility, differentiation, biofilm formation, antibiotic resistance, and virulence. Pathogenic bacteria exploit the capabilities of TCSs to reprogram gene expression according to the different niches they encounter during host infection. This review focuses on the role of TCSs in regulating the virulence phenotype of Shigella, an intracellular pathogen responsible for severe human enteric syndrome. The pathogenicity of Shigella is the result of the complex action of a wide number of virulence determinants located on the chromosome and on a large virulence plasmid. In particular, we will discuss how five TCSs, EnvZ/OmpR, CpxA/CpxR, ArcB/ArcA, PhoQ/PhoP, and EvgS/EvgA, contribute to linking environmental stimuli to the expression of genes related to virulence and fitness within the host. Considering the relevance of TCSs in the expression of virulence in pathogenic bacteria, the identification of drugs that inhibit TCS function may represent a promising approach to combat bacterial infections.

The tripartite complex AcrAB-TolC is the major RND pump in Escherichia coli and other Enterobacteriaceae, including Shigella, the etiological agent of bacillary dysentery. In addition to conferring resistance to many classes of antibiotics, AcrAB plays a role in the pathogenesis and virulence of several bacterial pathogens. Here, we report data demonstrating that AcrAB specifically contributes to Shigella flexneri invasion of epithelial cells. We found that deletion of both acrA and acrB genes causes reduced survival of S. flexneri M90T strain within Caco-2 epithelial cells and prevents cell-to-cell spread of the bacteria. Infections with single deletion mutant strains indicate that both AcrA and AcrB favor the viability of the intracellular bacteria. Finally, we were able to further confirm the requirement of the AcrB transporter activity for intraepithelial survival by using a specific EP inhibitor. Overall, the data from the present study expand the role of the AcrAB pump to an important human pathogen, such as Shigella, and add insights into the mechanism governing the Shigella infection process.

The pathogenicity of Shigella 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 Shigella flexneri YbjX and VirK. We defined the ybjX and virK promoter regions and confirmed that their expression is regulated by the PhoPQ two-component system. Localization studies demonstrated that both proteins are cytoplasmic. In silico 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 ybjX 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 ybjX and the acquisition of virK reinforced bacterial survival and fitness during the infection of the host.