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            -Proline Reductase Underlies Proline-Dependent Growth of Clostridioides difficile

Johnstone, Michael; Self, William T.

doi:10.1128/jb.00229-22

Cited by 10 publications

(9 citation statements)

References 70 publications

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“…8). In line with previous studies (5, 23, 24), C. difficile WT displayed proline-dependent growth, leading to higher cell density at higher proline levels. In contrast, the growth of the Δ prdH -mutant was found to not respond to proline.…”

Section: Resultssupporting

confidence: 92%

“…Surprisingly and against first intuition, the mutant showed similar growth rates as the wildtype strain at the highest proline concentrations, irrespective of the amount of proline present in the medium. A similar observation was made in a recent study using a PrdB-knockout (23), which suggests that PrdH is indeed essential for formation of active Prd and hence proline-dependent growth.…”

Section: Resultssupporting

confidence: 86%

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Formation of the pyruvoyl-dependent proline reductase Prd fromClostridioides difficilerequires the maturation enzyme PrdH

Behlendorf,

Diwo,

Neumann-Schaal

et al. 2023

Preprint

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Stickland fermentation, the coupled oxidation and reduction of amino acid pairs, is a major pathway for obtaining energy in the nosocomial bacteriumClostridioides difficile. D-proline is the preferred substrate for the reductive path, making it not only a key component of the general metabolism but also impacting on the expression of the clostridial toxins TcdA and TcdB. D-proline reduction is catalyzed by the proline reductase Prd, which belongs to the pyruvoyl-dependent enzymes. These enzymes are translated as inactive proenzymes and require subsequent processing to install the covalently bound pyruvate. Whereas pyruvoyl formation by intramolecular serinolysis has been studied in unrelated enzymes, details about pyruvoyl generation by cysteinolysis such as in Prd are lacking. Here we show that Prd maturation requires a small dimeric protein that we have named PrdH. PrdH is co-encoded with the PrdA and PrdB subunits of Prd and also found in species producing similar reductases. By producing stable variants of PrdA and PrdB, we demonstrate that PrdH-mediated cleavage and pyruvoyl formation in the PrdA subunit require PrdB, which can be harnessed to produce active recombinant Prd for subsequent analyses. We further created PrdA- and PrdH-mutants to get insight into the interaction of the components and into the processing reaction itself. Finally, we show that deletion ofprdHinC. difficilerenders the corresponding mutant blind to proline, suggesting that this processing factor is essential for proline utilization. Due to the link between Stickland fermentation and pathogenesis, we suggest PrdH may be an attractive target for drug development.Significance StatementEnergy conservation via Stickland fermentation was first described in the 1930s, yet information about the key enzyme of this process, Prd, is scarce, despite the fact that its central role in both metabolism and toxin production make it a promising potential drug target. Here we show how a small, previously overlooked protein that we named PrdH mediates the formation of the catalytically essential pyruvoyl-group in the active center of Prd.In vivostudies inC. difficileemphasize its critical importance in the utilization of proline. The known interplay between proline reduction and toxin production leads us to suggest PrdH as a potential drug target. Moreover, our findings open the door for further structural and functional studies with recombinantly produced active Prd.

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Section: Resultssupporting

confidence: 92%

Section: Resultssupporting

confidence: 86%

Formation of the pyruvoyl-dependent proline reductase Prd fromClostridioides difficilerequires the maturation enzyme PrdH

Behlendorf,

Diwo,

Neumann-Schaal

et al. 2023

Preprint

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“…Furthermore we see positive partial correlations between 5-aminovalerate and tcdA and tcdB which indicates that as their expression increases, 5-aminovalerate concentration also increases. This is consistent with the known biology of C. difficile, which ferments proline to 5-aminovalerate as a preferred mechanism of ATP generation [45,46,47,48,49,50,51].…”

Section: Sparse Graphical Models Of Interactions Between Toxins and M...supporting

confidence: 86%

Distilling Mechanistic Models From Multi-Omics Data

Erwin,

Fletcher,

Sweeney

et al. 2023

Preprint

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High-dimensional multi-omics data sets are increasingly accessible and now routinely being generated as part of medical and biological experiments. However, the ability to infer mechanisms of these data remains low due to the abundance of confounding data. The gap between data generation and interpretation highlights the need for strategies to harmonize and distill complex multi-omics data sets into concise, mechanistic descriptions. To this end, a four step analysis approach for multiomics data is herein demonstrated, comprising: filling missing data and harmonizing data sources, inducing sparsity, developing mechanistic models, and interpretation. This strategy is employed to generate a parsimonious mechanistic model from high-dimensional transcriptomics and metabolomics data collected from a murine model of Clostridioides difficile infection. This approach highlighted the role of the Stickland reactor in the production of toxins during infection, in agreement with recent literature. The methodology present here is demonstrated to be feasible for interpreting multi-omics data sets and it, to the authors knowledge, one of the first reports of a successful implementation of such a strategy.

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“…We Stickland metabolism is an important method of energy production for C. difficile, though the investigation of individual Stickland metabolites on growth and physiology thus far has focused primarily on proline (Bouillaut et al, 2013;Neumann-Schaal et al, 2019;Bouillaut et al, 2019;Johnstone and Self, 2022). Results from other groups have demonstrated that glycine and the grd genes of C. difficile are important for enhancing growth, despite differences in the strains investigated (Karasawa et al, 1995;Bouillaut et al, 2013;Collery et al, 2017;Johnstone and Self, 2022). However, glycine catabolism is secondary to preferred amino acids such as proline, which represses grd expression (Bouillaut et al, 2013).…”

Section: Clnr-dependent Regulation Of Pgrdxmentioning

confidence: 99%

“…Glycine is catabolized by the glycine reductase (GR) pathway, which is encoded by the grd genes (Andreesen, 2004). While glycine fermentation is known to occur in C. difficile, the importance of this pathway to C. difficile development and pathogenesis is not known (Bouillaut et al, 2013;Hofmann et al, 2018;Johnstone and Self, 2022).…”

Section: Introductionmentioning

confidence: 99%

Glycine fermentation byC. difficilepromotes virulence, spore formation, and is induced by host cathelicidin

Rizvi,

Vargas-Cuebas,

Edwards

et al. 2023

Preprint

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The amino acid glycine is enriched in the dysbiotic gut and is suspected to contribute to Clostridioides difficile infection. We hypothesized that the use of glycine as an energy source contributes to colonization of the intestine and pathogenesis of C. difficile. To test this hypothesis, we deleted the glycine reductase genes grdAB, rendering C. difficile unable to ferment glycine, and investigated the impact on growth and pathogenesis. We found that the grd pathway promoted growth, toxin production, sporulation, and pathogenesis of C. difficile in the hamster model of disease. Further, we determined that the grd locus is regulated by host cathelicidin (LL-37) and the cathelicidin-responsive regulator, ClnR, indicating that the host peptide signals to control glycine catabolism. The induction of glycine fermentation by LL-37 demonstrates a direct link between the host immune response and the bacterial reactions of toxin production and spore formation.

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d -Proline Reductase Underlies Proline-Dependent Growth of Clostridioides difficile

Abstract: Stickland metabolism is a core facet of C. difficile physiology that likely plays a major role in host colonization. Here, we carefully delineate the effects of each amino acid on the growth of C. difficile with respect to the selenoenzymes d -proline reductase and glycine reductase.

Cited by 10 publications

References 70 publications

Formation of the pyruvoyl-dependent proline reductase Prd fromClostridioides difficilerequires the maturation enzyme PrdH

Formation of the pyruvoyl-dependent proline reductase Prd fromClostridioides difficilerequires the maturation enzyme PrdH

Distilling Mechanistic Models From Multi-Omics Data

Glycine fermentation byC. difficilepromotes virulence, spore formation, and is induced by host cathelicidin

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