Deciphering the role of flavodoxins in the intestinal pathogen Clostridioides difficile
Final Report Abstract
Flavodoxins are small electron-transferring proteins that have a low redox potential and the capability to catalyze one-electron-transfer reactions. With a few exceptions, genuine flavodoxins are restricted to prokaryotes. In some species, they have been shown to be involved in essential metabolic functions, to substitute for ferredoxins under iron restriction or to increase resilience to oxidative stress. However, in the majority of species their relevance is not understood. The intestinal human pathogen Clostridioides difficile features 8 putative flavodoxins and hence a much more than average number of these small electron carrier proteins. One of the flavodoxins FldX has been shown to be induced under iron limitation, otherwise the cellular function of flavodoxins in C. difficile is not known yet. In-depth in-silico analyses revealed the typical three-layered αβα-sandwich structure of the 8 flavodoxins. However, one flavodoxin was predicted to additionally contain iron-sulfur clusters excluding it from the list of bona fide flavodoxins. Interestingly, structures of the remaining flavodoxins do not in every case resemble the one of short-chain flavodoxins as it is described for Gram-positive bacteria, but show additional -helices or loops comparable to long-chain flavodoxins of Gram-negative bacteria. C. difficile flavodoxins show very low similarity among each other, but homologs can be found in other bacteria featuring an anaerobic lifestyle. Strikingly, the flavodoxins are conserved across known C. difficile strains. Transcriptional analyses revealed expression of 6 out of 7 tested flavodoxins during exponential growth. Furthermore, strong induction of flavodoxin fldX was not only caused by iron limitation, but also in the presence of oxygen. Transcription of flavodoxin CD2825 was induced by hydrogen peroxide. Targeted mass spectrometry (MS) analysis could prove FldX induction also on protein level. The cellular location of the protein could be pinpointed to the membrane by immunogold labelling and transmission electron microscopy suggesting interaction of FldX with membrane-bound proteins. A polyclonal anti-FldX antibody was used to pull down FldX and interacting proteins from crude protein extracts of different physiological conditions. Precipitated proteins have been identified and relatively quantified via MS. These data are currently evaluated and will provide information on the cellular function of FldX. A gene deletion of FldX was not possible in our hands pointing at an essential function of this flavodoxin in C. difficile. Deletion mutants of flavodoxins wrbA, CD2825, CD2207 and CD2684 could be constructed and will be investigated by comprehensive MS-based proteomics to deduce their cellular function. In summary, 6 flavodoxins are actively transcribed and 2 of them are inducible at infection-relevant conditions turning flavodoxins of C. difficile into potential targets of novel therapeutic strategies.
Publications
-
at the 12th International Conference on the Molecular Biology and Pathogenesis of the Clostridia (Clostpath), 13 – 16/09 2021, digital, Tracking the expression of eight different flavodoxins of Clostridioides difficile under iron limitation and oxidative stress
Daniel Troitzsch, Silvia Dittmann, Fabian Marek, Timon Alexander Möller, Linda Trän & Susanne Sievers
-
at the annual conference of the VAAM, 17 – 20/03 2022, (digital) Tracking the expression of eight different flavodoxins of Clostridioides difficile under iron limitation and oxidative stress
Robert Knop, Daniel Troitzsch, Silvia Dittmann, Fabian Marek, Timon Alexander Möller, Linda Trän & Susanne Sievers
-
Invited talk at MICALIS Institute INRAE, 13/05 2022, Jouy-en-Josas, France; Pinpointing hubs of adaptation in the human pathogen Clostridioides difficile as potential new therapeutic targets
Sievers, S.
-
Invited talk at the Proteomics Spring School, 21/03 2022, Greifswald, Germany; A proteomics approach to understand the patho-physiology of Clostridioides difficile
Sievers, S.
-
Invited talk Réseau CD France, Université de Paris, 12/05 2022, Paris, France; Pinpointing hubs of adaptation in the human pathogen Clostridioides difficile as potential new therapeutic targets
Sievers, S.
-
poster at 2nd Summer School “Infection Biology”, 26 – 28/09 2022, Greifswald, Germany; Tracking the expression of eight different flavodoxins of Clostridioides difficile under iron limitation and oxidative stress
Robert Knop, Daniel Troitzsch, Silvia Dittmann, Timon Alexander Möller, Linda Trän & Susanne Sievers
-
Lecture at the „Kinder- und JugendUni 2023“ at the University of Greifswald Multikulti im Darm: Warum wir Mikroorganismen zum Gesundbleiben brauchen
Sievers, S.
-
Poster at the 13th International Conference on the Molecular Biology and Pathogenesis of the Clostridia (Clostpath), 19- 23/09 2023, Banff, Canada, The putative flavodoxin FldX and its role in the oxidative stress response in Clostridioides difficile
Robert Knop, Daniel Troitzsch, Silvia Dittmann, Thaddäus Echelmeyer, Johanna Pukall, Lisa Hill, Valeria Kamozina & Susanne Sievers
-
Characterizing the flavodoxin landscape in Clostridioides difficile. Microbiology Spectrum, 12(3).
Troitzsch, Daniel; Knop, Robert; Dittmann, Silvia; Bartel, Jürgen; Zühlke, Daniela; Möller, Timon Alexander; Trän, Linda; Echelmeyer, Thaddäus & Sievers, Susanne
