Shigella Flexneri Diguanylate Cyclases Regulate Pathogenesis

Date of Award


Degree Name

Doctor of Philosophy


Biological Sciences

First Advisor

Benjamin J. Koestler, Ph.D.

Second Advisor

Karim Essani, Ph.D.

Third Advisor

Terri G. Kinzy, Ph.D.

Fourth Advisor

Frederick Stull, Ph.D.


The intracellular human pathogen Shigella invades the colonic epithelium to cause disease. Prior to colon invasion, this bacterium navigates through different environments within the human body, including acidic conditions in the stomach and the small intestine which contains antimicrobial molecules including bile acids. To adapt to changing environments, Shigella uses the bacterial second messenger c-di-GMP signaling system. C-di-GMP is synthesized by diguanylate cyclases (DGCs) encoding GGDEF domains. Shigella encodes 10 DGCs in its genome, but 6 of these genes have acquired mutations that presumably inactivated the c-di-GMP synthesis activity of these enzymes. In this study, we first expressed VCA0956, a constitutively active c-di-GMP synthesizing diguanylate cyclase (DGC) from Vibrio cholerae, in S. flexneri to determine if virulence phenotypes were regulated by c-di-GMP. We found that expressing VCA0956 in S. flexneri increased c-di-GMP levels, and this corresponds with increased biofilm formation, and reduced acid resistance, host cell invasion, and plaque size. We also found that individual S. flexneri DGC mutants exhibit reduced biofilm formation, reduced host cell invasion and plaque size, as well as increased resistance to acid shock. We individually expressed each of the 4 intact DGCs in an S. flexneri strain where these 4 DGCs had been deleted (4DGC). We found that the 4 S. flexneri intact DGCs (dgcC, dgcF, dgcI and dgcP) 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. However, deletion of these 4 DGCs did not eliminate S. flexneri c-di-GMP, and we found that S. flexneri still encode other DGCs with mutations (annotated as degenerate). Interestingly, dgcN, dgcE and dgcQ, which both have nonsense mutations prior to the GGDEF domain, still produce c-di-GMP. S. flexneri degenerate DGCs are expressed as multiple proteins, consistent with multiple start codons within the gene. We propose that, in addition to intact DGCs, S. flexneri degenerate DGCs contribute to c-di-GMP signaling.

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