Intact and Degenerate Diguanylate Cyclases regulateShigellaCyclic di-GMP

Abstract

AbstractThe intracellular human pathogenShigellainvades the colonic epithelium to cause disease. Prior to invasion, this bacterium navigates through different environments within the human body, including the stomach and the small intestine. To adapt to changing environments,Shigellauses the bacterial second messenger c-di-GMP signaling system, synthesized by diguanylate cyclases (DGCs) encoding GGDEF domains.Shigella flexneriencodes a total of 9 GGDEF or GGDEF-EAL domain enzymes in its genome, but 5 of these genes have acquired mutations that presumably inactivated the c-di-GMP synthesis activity of these enzymes. In this study, we examined individualS. flexneriDGCs for their role in c-di-GMP synthesis and pathogenesis. We individually expressed each of the 4 intact DGCs in anS. flexneristrain where these 4 DGCs had been deleted (Δ4DGC). We found that the 4S. flexneriintact DGCs synthesize c-di-GMP at different levelsin vitroand during infection of tissue-cultured cells. We also found thatdgcFanddgcIexpression significantly reduces invasion and plaque formation, anddgcFexpression increases acid sensitivity, and that these phenotypes did not correspond with measured c-di-GMP levels. However, deletion of these 4 DGCs did not eliminateS. flexneric-di-GMP, and we found thatdgcE, dgcQ,anddgcN, which all have nonsense mutations prior to the GGDEF domain, still produce c-di-GMP. TheseS. flexneridegenerate DGC genes are expressed as multiple proteins, consistent with multiple start codons within the gene. We propose that both intact and degenerate DGCs contribute toS. flexneric-di-GMP signaling.