(p)ppGpp is required for Virulence ofShigella flexneri

Abstract

ABSTRACTInfection by the enteric pathogenShigella flexnerirequires transit through the gastrointestinal tract and invasion of and replication within the cells of the host colonic epithelium. This process exposes the pathogen to a range of diverse microenvironments. Further, the unique composition and physical environment of the eukaryotic cell cytosol represents a stressful environment forS. flexneri, and extensive physiological adaptations are needed for the bacterium to thrive. In this work, we show that disrupting synthesis of the stringent response alarmone (p)ppGpp inS. flexneridiminished expression of key virulence genes, includingipaA, ipaB, ipaCandicsA, and it reduced bacterial invasion and intercellular spread. Deletion of the (p)ppGpp synthase generelAalone had no effect onS. flexnerivirulence, but disruption of bothrelAand the (p)ppGpp synthase/hydrolase genespoTresulted in loss of (p)ppGpp synthesis and virulence. While therelA spoTdeletion mutant was able to invade a cultured human epithelial cell monolayer, albeit at reduced levels, it was unable to maintain the infection and spread to adjacent cells, as indicated by loss of plaque formation. Complementation withspoTon a plasmid vector restored plaque formation. Thus, SpoT alone is sufficient to provide the necessary level of (p)ppGpp for virulence. These results indicate that (p)ppGpp is required forS. flexnerivirulence and adaptation to the intracellular environment, adding to the repertoire of signaling pathways that affectShigellapathogenesis.