Unravelling the link between reductive corticosteroid metabolism and energy conservation in gut bacteria

• Applicant: Dr. Christian Jacoby
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 542537779

The aim of this project is to test the hypothesis that reductive corticosteroid metabolism in gut bacteria uses an Fe-S flavoenzyme-based strategy to (i) produce essential metabolites in the host and (ii) determine whether this is a widespread mechanism for energy conservation in gut bacteria. To confirm this hypothesis experimentally, the recently isolated bacterial strain Clostridium HCS.1 will be used as a model organism. The experiments will involve the identification of genes from reductive corticosteroid metabolism, their heterologous production and biochemical characterization in E. coli. In particular, the substrate spectrum of the enzymes will be analyzed to determine whether they convert not only corticosteroids but also other steroid hormones into 3ß and 5ß variants. In addition, the products of reductive steroid metabolism will be studied in mouse models to test the hypothesis that the production of reductive corticosteroids may lead to increased blood pressure by inhibiting the function of the host's own 11-hydroxysteroid dehydrogenase 2, which converts active corticosteroids to inactive corticosteroids such as cortisone. The hypothesis that reductive corticosteroid metabolism influences energy metabolism in bacteria will also be tested. Previous studies and preliminary results suggest that an interaction between reductive corticosteroid metabolism enzymes, the Rnf complex and bifurcating hydrogenases, contributes to ATP production by generating a proton gradient across the bacterial membrane. In this process, electrons are likely to be transferred to NAD+ and oxidized ferredoxin through the oxidation of hydrogen by bifurcating hydrogenases. The latter acts as an electron donor for the Rnf complex, which transfers the electrons to NAD+. The reductive metabolism of corticosteroids serves to regenerate NADH. To confirm this experimentally, knockout mutants of HCS.1 will be generated that lack either the gene rnfB, which encodes the ferredoxin-binding subunit of the Rnf complex, the large subunit of a potential electron-transfer hydrogenase, or one of the enzymes involved in reductive steroid degradation. The generated mutants will be analyzed for their ability to produce ATP upon addition of cortisol or other steroids by measuring the amount of ATP produced. In addition, growth experiments with the wild-type strain and the mutants will be performed to assess phenotypic changes in vitro and in mouse models through growth competition experiments.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professor Sam Light, Ph.D.