Biology subjects

Koestler, B. J.

Publications and source records attributed to Koestler, B. J..

4 recordsLinked to original sources

bioRxiv2026

Shigella's c-di-GMP specific PDEs Modulate Biofilm and Virulence Phenotypes

To establish infection and cause disease, the intracellular pathogen Shigella must successfully navigate a series of host defenses and distinct microenvironments within the human body. One way Shigella navigates these enviroments is by using the secondary messenger c-di-GMP, which regulates many different bacterial behaviours. C-di-GMP is synthesized by diguanylate cyclases (DGCs) and broken down by c-di-GMP specific phosphodiesterases (PDEs). In this study, we investigated how Shigellas c-di-GMP specific PDEs impact c-di-GMP turn-over and subsequently biofilm and virulence phenotypes. We knocked out each of Shigellas six c-di-GMP specific PDEs to determine how these PDEs impact biofilm, virulence and c-di-GMP levels within the bacterial cell. We found that these PDEs negatively regulate c-di-GMP levels while modulating Shigellas virulence and biofilm behaviour. We also noted that altering expression of these Shigella PDEs changes bacterial cell size. Transcriptome analysis revealed that a Shigella {Delta}pdeB strain showed reduced expression of many genes, including the virulence genes ipgD and ipgE, as well as genes associated with lipid metabolism. We confirmed that a Shigella {Delta}pdeB strain had altered levels of stearic acid, and expression of pdeB alters Shigella antibiotic susceptibility. This study highlights the complexities of c-di-GMP signaling in regulating numerous Shigella pathways.

microbiology↗

bioRxiv2024

FhlA is a Formate Binding Protein

Escherichia coli uses glycolysis and mixed acid fermentation and produces formate as by product. One system E. coli uses for formate oxidation is formate hydrogen lyase complex (FHL). The expression of the FHL complex is dependent on formate and regulated by the transcriptional regulator FhlA. The structure of FhlA is composed of three domains. The N-terminal domain is putatively responsible for formate binding and FhlA oligomerization as a tetramer, the central portion of FhlA contains a AAA+ domain that hydrolyzes ATP, and the C-terminal domain binds DNA. Formate enhances FhlA-mediated expression of FHL; however, FhlA direct interaction with formate has never been demonstrated. Formate-protein interactions are challenging to assess, due to the small and ubiquitous nature of the molecule. Here, we have developed three techniques to assess formate-protein interaction. We use these techniques to confirm that FhlA binds formate in the N-terminal domain in vitro, and that this interaction is partially dependent on residues E183 and E363, consistent with previous reports. This study is a proof of concept that these techniques can be used to assess other formate-protein interactions.

microbiology↗

bioRxiv2024

Intact and Degenerate Diguanylate Cyclases regulate Shigella Cyclic di-GMP

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 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 5 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 4 intact DGCs in an S. flexneri strain where these 4 DGCs had been deleted ({Delta}4DGC). 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 4 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 genes 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.

microbiology↗

bioRxiv2022

Vibrio cholerae alkalizes its environment via citrate metabolism to inhibit enteric growth

Vibrio cholerae is a Gram-negative pathogen, living in constant competition with other bacteria in both marine environments and during human infection. One competitive advantage of V. cholerae is the ability to metabolize diverse carbon sources such as chitin and citrate. We observed that when V. cholerae strains were grown on a medium with citrate, the mediums chemical composition turned into a hostile alkaline environment for Gram-negative bacteria such as Escherichia coli and Shigella flexneri. We found that although the ability to exclude competing bacteria was not contingent on exogenous citrate, V. cholerae citrate metabolism mutants {Delta}oadA-1, {Delta}citE, and {Delta}citF mutants were not able to inhibit S. flexneri or E. coli growth. Lastly, we demonstrated that while the V. cholerae mediated increased medium pH was necessary for the enteric exclusion phenotype, secondary metabolites such as bicarbonate (protonated to carbonate in the raised pH) from the metabolism of citrate enhanced the ability to inhibit the growth of E. coli. These data provide a novel example of how V. cholerae outcompetes other Gram-negative bacteria.

microbiology↗