Dataset Information

Characterizing metabolic drivers of Clostridioides difficile infection with activity-based hydrazine probes

ABSTRACT: 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, for the first time, structurally assigned proline reductase and glycine reductase as cysteine-derived pyruvoyl-dependent enzymes 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.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Clostridioides Difficile Atcc 43255

SUBMITTER: Megan Matthews

LAB HEAD: Megan L. Matthews

PROVIDER: PXD039143 | Pride | 2023-03-11

REPOSITORIES: Pride

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Publications

Characterizing metabolic drivers of <i>Clostridioides difficile</i> infection with activity-based hydrazine probes.

Bustin Katelyn A KA Abbas Arwa A Wang Xie X Abt Michael C MC Zackular Joseph P JP Matthews Megan L ML

Frontiers in pharmacology 20230126

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. <i>Clostridioides difficile</i> 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. ...[more]

PMID: 36778002

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Project description:A major risk factor for Clostridioides difficile infection (CDI) is the perturbation of the gut microbiota by antibiotic administration, and antibiotic therapy, the standard treatment option for CDI, exacerbates the imbalance of the gut microbiota, leading to a very high recurrent CDI (rCDI) incidence rate. Therefore, CDI treatment based on live biotherapeutic products (LBPs) has recently emerged for long-term CDI management and preventive treatment However, there is limited research and understanding of how these LBPs improve CDI symptoms, which raises the need to elucidate the therapeutic mechanisms of LBPs. To fill this knowledge gap, here, we holistically analyzed and investigated the mechanisms involved in the inhibitory effect of probiotics on C. difficile at the molecular level through a multiomics approach on screened strain from probiotics that inhibit C. difficile growth. First, Bifidobacterium species were co-cultured with C. difficile, and B. longum was screened based on its ability to inhibit the growth of C. difficile. Then, the antimicrobial activity of B. longum against C. difficile was confirmed by spot-on-lawn assay and organic acid quantification. Next, we performed proteomic and metabolomic analysis on C. difficile co-cultured with B. longum, and observed physiological changes associated with the inhibition of C. difficile growth and toxin production. It was found that lactate produced by B. longum up-regulated the lactate dehydrogenase complex of C. difficile, leading to a decrease in intracellular ATP synthesis and a subsequent decrease in (deoxy)ribonucleoside triphosphate synthesis. Furthermore, proteinaceous stress induced by B. longum was also identified through the up-regulation of ribosomal proteins, molecular chaperones, and chaperonins, and the down-regulation of translation-related proteins. Finally, we found that B. longum suppressed butyrate metabolism and toxin production by producing and replenishing proline consumed by C. difficile. Therefore, this study will not only expand our understanding of the mechanisms of probiotics' inhibition of C. difficile, but also contribute to the development of LBPs based on molecular mechanisms for treating CDI.

Project description:Clostridium difficile is an anaerobic spore-forming rod-shaped gram-positive bacterium that can infect both humans and animals. Most studies on the pathogenesis of C. difficile have focused on its toxins and their effect on the host cells. Recently, we utilized microarrays to identify conserved and divergent genes associated with virulence in C. difficile isolates from humans and animals. Our data provided the first clue toward a complex mechanism underlying host adaptation and pathogenesis. Microarray technology offers an efficient high-throughput tool to study the transcriptional profiles of pathogens and infected host cells. Transcriptomes of C. difficile after exposure to environmental and antibiotic stresses and those of human epithelial colorectal Caco-2 cells upon TcdA treatment have been analyzed. To our knowledge, there are still no reports on the transcriptomic study of host-pathogen interactions for C. difficile infection (CDI). In vitro analyses of interplay between host and pathogen are essential to unravel the mechanisms of infection and to investigate the host response to infection. We therefore employed microarrays to study both bacterial and human cellular transcriptome kinetics during CDI to Caco-2 cells. Here we present a large-scale analysis of transcriptional profiles to reveal molecular determinants playing a role in C. difficile pathogenesis and the host response. We found that there were 254 and 224 differentially-expressed genes after CDI in C. difficile and Caco-2 cells, respectively. These genes are clustered according to their functional categories and their potential roles in pathogenesis and host response are discussed. Our results will not only increase our understanding on the host-pathogen interaction, but may also provide targets for drug development. Clostridium difficile: Control vs Infection (time course) mRNA with genomic DNA of tested and reference strains Caco-2 cells: Control vs Infected with Clostridium difficile Time-course experiments of Caco-2 cells infected with C. difficile for 30, 60 and 120 min

Dataset Information

Characterizing metabolic drivers of Clostridioides difficile infection with activity-based hydrazine probes

Publications

Characterizing metabolic drivers of <i>Clostridioides difficile</i> infection with activity-based hydrazine probes.

Similar Datasets

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