Characterizing metabolic drivers of Clostridioides difficile infection with activity-based hydrazine probes
Many enzymes require post-translational modifications or cofactor machinery for primary function. As these catalytically essential moieties are highly regulated, they act as dual sensors and chemical handles for context-dependent metabolic activity. Clostridioides difficile is a major nosocomial pat...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-01-01
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| Series: | Frontiers in Pharmacology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fphar.2023.1074619/full |
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| author | Katelyn A. Bustin Arwa Abbas Xie Wang Michael C. Abt Joseph P. Zackular Joseph P. Zackular Megan L. Matthews |
| author_facet | Katelyn A. Bustin Arwa Abbas Xie Wang Michael C. Abt Joseph P. Zackular Joseph P. Zackular Megan L. Matthews |
| author_sort | Katelyn A. Bustin |
| collection | DOAJ |
| description | Many enzymes require post-translational modifications or cofactor machinery for primary function. As these catalytically essential moieties are highly regulated, they act as dual sensors and chemical handles for context-dependent metabolic activity. Clostridioides difficile is a major nosocomial pathogen that infects the colon. Energy generating metabolism, particularly through amino acid Stickland fermentation, is central to colonization and persistence of this pathogen during infection. Here using activity-based protein profiling (ABPP), we revealed Stickland enzyme activity is a biomarker for C. difficile infection (CDI) and annotated two such cofactor-dependent Stickland reductases. We structurally characterized the cysteine-derived pyruvoyl cofactors of D-proline and glycine reductase in C. difficile cultures and showed through cofactor monitoring that their activity is regulated by their respective amino acid substrates. Proline reductase was consistently active in toxigenic C. difficile, confirming the enzyme to be a major metabolic driver of CDI. Further, activity-based hydrazine probes were shown to be active site-directed inhibitors of proline reductase. As such, this enzyme activity, via its druggable cofactor modality, is a promising therapeutic target that could allow for the repopulation of bacteria that compete with C. difficile for proline and therefore restore colonization resistance against C. difficile in the gut. |
| first_indexed | 2024-04-10T20:16:48Z |
| format | Article |
| id | doaj.art-b38433cd984a468ab5c288cc5af539db |
| institution | Directory Open Access Journal |
| issn | 1663-9812 |
| language | English |
| last_indexed | 2024-04-10T20:16:48Z |
| publishDate | 2023-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Pharmacology |
| spelling | doaj.art-b38433cd984a468ab5c288cc5af539db2023-01-26T05:16:41ZengFrontiers Media S.A.Frontiers in Pharmacology1663-98122023-01-011410.3389/fphar.2023.10746191074619Characterizing metabolic drivers of Clostridioides difficile infection with activity-based hydrazine probesKatelyn A. Bustin0Arwa Abbas1Xie Wang2Michael C. Abt3Joseph P. Zackular4Joseph P. Zackular5Megan L. Matthews6Department of Chemistry, University of Pennsylvania, Philadelphia, PA, United StatesDivision of Protective Immunity, Children’s Hospital of Pennsylvania, Philadelphia, PA, United StatesDepartment of Chemistry, University of Pennsylvania, Philadelphia, PA, United StatesDepartment of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United StatesDivision of Protective Immunity, Children’s Hospital of Pennsylvania, Philadelphia, PA, United StatesDepartment of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United StatesDepartment of Chemistry, University of Pennsylvania, Philadelphia, PA, United StatesMany enzymes require post-translational modifications or cofactor machinery for primary function. As these catalytically essential moieties are highly regulated, they act as dual sensors and chemical handles for context-dependent metabolic activity. Clostridioides difficile is a major nosocomial pathogen that infects the colon. Energy generating metabolism, particularly through amino acid Stickland fermentation, is central to colonization and persistence of this pathogen during infection. Here using activity-based protein profiling (ABPP), we revealed Stickland enzyme activity is a biomarker for C. difficile infection (CDI) and annotated two such cofactor-dependent Stickland reductases. We structurally characterized the cysteine-derived pyruvoyl cofactors of D-proline and glycine reductase in C. difficile cultures and showed through cofactor monitoring that their activity is regulated by their respective amino acid substrates. Proline reductase was consistently active in toxigenic C. difficile, confirming the enzyme to be a major metabolic driver of CDI. Further, activity-based hydrazine probes were shown to be active site-directed inhibitors of proline reductase. As such, this enzyme activity, via its druggable cofactor modality, is a promising therapeutic target that could allow for the repopulation of bacteria that compete with C. difficile for proline and therefore restore colonization resistance against C. difficile in the gut.https://www.frontiersin.org/articles/10.3389/fphar.2023.1074619/fullcofactorABPPClostridioides difficile infectiondruggable modalityStickland fermentationhydrazine pharmacophore |
| spellingShingle | Katelyn A. Bustin Arwa Abbas Xie Wang Michael C. Abt Joseph P. Zackular Joseph P. Zackular Megan L. Matthews Characterizing metabolic drivers of Clostridioides difficile infection with activity-based hydrazine probes Frontiers in Pharmacology cofactor ABPP Clostridioides difficile infection druggable modality Stickland fermentation hydrazine pharmacophore |
| title | Characterizing metabolic drivers of Clostridioides difficile infection with activity-based hydrazine probes |
| title_full | Characterizing metabolic drivers of Clostridioides difficile infection with activity-based hydrazine probes |
| title_fullStr | Characterizing metabolic drivers of Clostridioides difficile infection with activity-based hydrazine probes |
| title_full_unstemmed | Characterizing metabolic drivers of Clostridioides difficile infection with activity-based hydrazine probes |
| title_short | Characterizing metabolic drivers of Clostridioides difficile infection with activity-based hydrazine probes |
| title_sort | characterizing metabolic drivers of clostridioides difficile infection with activity based hydrazine probes |
| topic | cofactor ABPP Clostridioides difficile infection druggable modality Stickland fermentation hydrazine pharmacophore |
| url | https://www.frontiersin.org/articles/10.3389/fphar.2023.1074619/full |
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