Microbial Pathogenesis
Research Article
15 December 2023

(p)ppGpp is required for virulence of Shigella flexneri


Infection by the enteric pathogen Shigella flexneri requires 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. Furthermore, the unique composition and physical environment of the eukaryotic cell cytosol represents a stressful environment for S. 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 in S. flexneri diminished expression of key virulence genes, including ipaA, ipaB, ipaC, and icsA, and it reduced bacterial invasion and intercellular spread. Deletion of the (p)ppGpp synthase gene relA alone had no effect on S. flexneri virulence, but disruption of both relA and the (p)ppGpp synthase/hydrolase gene spoT resulted in loss of (p)ppGpp synthesis and virulence. While the relA spoT deletion 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 with spoT on 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 for S. flexneri virulence and adaptation to the intracellular environment, adding to the repertoire of signaling pathways that affect Shigella pathogenesis.

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cover image Infection and Immunity
Infection and Immunity
Volume 92Number 116 January 2024
eLocator: e00334-23
Editor: Denise Monack, Stanford University, Palo Alto, California, USA
PubMed: 38099658


Received: 22 August 2023
Accepted: 16 November 2023
Published online: 15 December 2023


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  1. Shigella
  2. (p)ppGpp
  3. alarmone
  4. virulence



Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA
Author Contributions: Conceptualization, Investigation, Methodology, and Writing – original draft.
Charles L. Turnbough, Jr.
Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA
Author Contributions: Conceptualization, Investigation, and Writing – review and editing.
Juan Carlos Salazar
Programa de Microbiología y Micología, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile
Author Contribution: Resources.
Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA
John Ring LaMontagne Center for Infectious Disease, The University of Texas at Austin, Austin, Texas, USA
Author Contributions: Conceptualization, Funding acquisition, Supervision, and Writing – review and editing.


Denise Monack
Stanford University, Palo Alto, California, USA


The authors declare no conflict of interest.

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