# Intact and mutated Shigella diguanylate cyclases increase c-di-GMP

## TL;DR

This study shows that both intact and mutated DGC enzymes in Shigella contribute to c-di-GMP signaling, which is important for the bacterium's adaptation and pathogenesis.

## Contribution

The study reveals that degenerate DGC pseudogenes in Shigella still produce c-di-GMP, challenging the assumption that mutations inactivate these enzymes.

## Key findings

- Intact DGCs in Shigella synthesize c-di-GMP at varying levels in vitro and during infection.
- Degenerate DGCs with nonsense mutations still produce c-di-GMP, suggesting functional contributions despite mutations.
- Expression of certain DGCs affects invasion, plaque formation, and acid sensitivity, independent of c-di-GMP levels.

## Abstract

The intracellular human pathogen Shigella invades 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, Shigella uses the bacterial second messenger cyclic di-GMP (c di-GMP) signaling system, synthesized by diguanylate cyclases (DGCs) encoding GGDEF domains. Shigella flexneri encodes a total of 9 GGDEF or GGDEF-EAL domain enzymes in its genome, but five of these genes have acquired mutations that presumably inactivated the c-di-GMP synthesis activity of these enzymes. In this study, we examined individual S. flexneri DGCs for their role in c-di-GMP synthesis and pathogenesis. We individually expressed each of the four intact DGCs in a S. flexneri strain, where these four DGCs had been deleted (Δ4DGC). We found that the 4 S. flexneri intact DGCs synthesize c-di-GMP at different levels in vitro and during infection of tissue-cultured cells. We also found that dgcF and dgcI expression significantly reduces invasion and plaque formation, and dgcF expression increases acid sensitivity, and that these phenotypes did not correspond with measured c-di-GMP levels. However, deletion of these four DGCs did not eliminate S. flexneri c-di-GMP, and we found that dgcE, dgcQ, and dgcN, which all have nonsense mutations prior to the GGDEF domain, still produce c-di-GMP. These S. flexneri degenerate DGC pseudogenes are expressed as multiple proteins, consistent with multiple start codons within the gene. We propose that both intact and degenerate DGCs contribute to S. flexneri c-di-GMP signaling.