The Shigella species are Gram-negative, facultative intracellular pathogens that invade the colonic epithelium and cause significant diarrheal disease. Despite extensive research on the pathogen, a comprehensive understanding of how Shigella initiates contact with epithelial cells remains unknown. Shigella maintains many of the same Escherichia coli adherence gene operons; however, at least one critical gene component in each operon is currently annotated as a pseudogene in reference genomes. These annotations, coupled with a lack of structures upon microscopic analysis following growth in laboratory media, have led the field to hypothesize that Shigella is unable to produce fimbriae or other traditional adherence factors. Nevertheless, our previous analyses have demonstrated that a combination of bile salts and glucose induces both biofilm formation and adherence to colonic epithelial cells. The goal of this study was to perform transcriptomic and genetic analyses to demonstrate that adherence gene operons in Shigella flexneri strain 2457T are functional, despite the gene annotations. Our results demonstrate that at least three structural genes facilitate S. flexneri 2457T adherence for epithelial cell contact and biofilm formation. Furthermore, our results demonstrate that host factors, namely, glucose and bile salts at their physiological concentrations in the small intestine, offer key environmental stimuli required for adherence factor expression in S. flexneri This research may have a significant impact on Shigella vaccine development and further highlights the importance of utilizing in vivo-like conditions to study bacterial pathogenesis.IMPORTANCE Bacterial pathogens have evolved to regulate virulence gene expression at critical points in the colonization and infection processes to successfully cause disease. The Shigella species infect the epithelial cells lining the colon to result in millions of cases of diarrhea and a significant global health burden. As antibiotic resistance rates increase, understanding the mechanisms of infection is vital to ensure successful vaccine development. Despite significant gains in our understanding of Shigella infection, it remains unknown how the bacteria initiate contact with the colonic epithelium. Most pathogens harbor multiple adherence factors to facilitate this process, but Shigella was thought to have lost the ability to produce these factors. Interestingly, we have identified conditions that mimic some features of gastrointestinal transit and that enable Shigella to express adherence structural genes. This work highlights aspects of genetic regulation for Shigella adherence factors and may have a significant impact on future vaccine development.

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