Ebselen Not Only Inhibits Clostridioides difficile Toxins but Displays Redox-Associated Cellular Killing

Marreddy, Ravi K. R.; Olaitan, Abiola Olumuyiwa; May, Jordan N; Dong, Min; Hurdle, Julian G.

doi:10.1128/spectrum.00448-21

Cited by 12 publications

(10 citation statements)

References 44 publications

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“…Complementation of R20291Δ feoB1 with WT feoB1 , ectopically expressed from its own promoter, increased the synthesis of both toxins (10.42 ± 0.66 ng/ml; and 6.77 ± 0.18 ng/ml, for TcdA and TcdB, respectively ( Fig 1C ). Comparable results were attained in cytopathic assays that measure toxin-induced cell rounding of mammalian cells and detects picomolar amounts of toxins [21]. Against Vero cells, R20291Δ feoB1 had toxin titers of ≤10 1 , which was 1000-fold lower than the titers of WT cultures (10 4 toxin titer; Fig.…”

Section: Resultsmentioning

confidence: 90%

“…Interestingly, cultures at OD ~0.6 showed statistically significant, but moderate, increases in transcription of prd operon genes prdA , prdB , prdC and prdF (1.46-1.61-fold) that encode proteins involved in the Stickland metabolism of proline ( Table 1 ). Upregulation of Stickland enzymes is also consistent with there being a shift to energy generating and redox balancing processes, which may include enzymes that do not rely on ferredoxin [21]. Furthermore, activation of Stickland reduction of proline is associated with repression of toxin production [13].…”

Section: Resultsmentioning

confidence: 98%

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The Ferrous Iron Transporter FeoB1 is Essential for Clostridioides difficile Toxin Production and Pathogenesis in Mice

Deshpande

Olaitan

McKelvey

et al. 2022

Preprint

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Ferrous iron is the dominant form of iron in the oxygen-limited large intestine, the site for Clostridioides difficile infection (CDI). We investigated the extent to which C. difficile requires the ferrous iron transporter 1 (FeoB1), a main permease for iron acquisition, using in vitro and in vivo approaches. Construction of feoB1 deletion mutant in C. difficile R20291 (i.e., R20291ΔfeoB1) decreased intracellular iron content by ~25%. This was accompanied by reduced synthesis of TcdA and TcdB toxins and downregulation of tcdA and tcdB genes, which was reflected by a ~1000-fold reduction in cytopathy against Vero cells. Complementation with WT feoB1 restored toxin production. R20291ΔfeoB1 was avirulent in mice with underlying colitis. In this model, mice are prone to develop severe CDI, but mice infected with the mutant lacked diarrheal symptoms, had lesser inflammatory responses, bacterial bioburdens and cecal toxin titers. Transcriptional and biochemical analyses revealed ΔfeoB1 altered metabolic pathways that negatively impact toxin production, including downregulation of the oxidative branch of the Kreb cycle and an associated cellular accumulation of pyruvate. Hence FeoB1 influences multiple bioenergetic and redox pathways, which in turn inhibits toxin biosynthesis. Targeting FeoB1 could be a potential therapeutic strategy to impede the colonization and pathogenesis of C. difficile, even in the setting of colitis where CDI is more severe and intestinal bleeding may occur.AUTHOR SUMMARYClostridioides difficile is a major cause of antibiotic associated diarrhea that result in significant morbidity and mortality in hospitals. The ability of C. difficile to cause disease and damage the gut is directly related to the production of toxin A and toxin B. Without these toxins, C. difficile is not virulent. There is need to identify drug targets and therapeutic strategies that prevent C. difficile from producing toxins A and B. For C. difficile to colonize the large bowel, it must acquire ferrous iron, which is thought to be the main form of iron within the oxygen-limited large bowel environment. We show that genetic deletion of the ferrous iron transporter FeoB1 stops C. difficile from producing significant amounts of toxins under in vitro conditions. Importantly, the mutant R20291ΔfeoB1 was avirulent when tested in a mouse model that favors the development of severe infections. Avirulence of R20291ΔfeoB1 was related to the strain having significant metabolic changes that negatively impact toxin production. For example, cells could not properly metabolize pyruvate, which became accumulated in cells; pyruvate is known to reduce the production of toxins A and B. Our study provides new insights into how FeoB1 affects C. difficile colonization and disease development, indicating FeoB1 could be a drug target to stop toxin production in this bacterium.

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Section: Resultsmentioning

confidence: 90%

Section: Resultsmentioning

confidence: 98%

The Ferrous Iron Transporter FeoB1 is Essential for Clostridioides difficile Toxin Production and Pathogenesis in Mice

Deshpande

Olaitan

McKelvey

et al. 2022

Preprint

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“…ATP synthase in C. difficile is thought to play a key role generating ATP from the sodium/proton gradient generated by the RNF complex (29). Reduction of toxin production in C. difficile was reported to result from dissipation of the membrane potential (43), and an imbalance of the NAD + /NADH ratio (44). Our findings thus add to the growing indication that proteins involved in energy metabolism could be targets for inhibitors of toxin production.…”

Section: Discussionmentioning

confidence: 99%

“…MICs were determined in BHI broth by two-fold serial dilution in 96-well microtiter plates. MICs were recorded as the lowest concentration of test compound that inhibited growth, following anaerobic incubation for 24 h. Growth kinetics in BHI broth were determined as described previously (44), using exponentially growing cells at OD 600 ≈ 0.2. Automated growth was recorded every 60 min for 20 h with shaking before each read in an Infinite M Plex microplate reader (Tecan) in an anaerobic chamber (Coy Laboratory Products).…”

Section: Methodsmentioning

confidence: 99%

The licorice metabolite enoxolone attenuates Clostridioides difficile pathophysiology by corrupting its metabolic and toxin production networks

Marreddy

Picker

Phelps

et al. 2022

Preprint

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Toxins TcdA and TcdB are the main virulence factors of Clostridioides difficile, a leading cause of hospital-acquired diarrhea. We investigated the therapeutic potential of inhibiting the biosynthesis of TcdA and TcdB. Accordingly, screening of structurally diverse phytochemicals with medicinal properties identified 18β-glycyrrhetinic acid (enoxolone) as an inhibitor of TcdA and TcdB biosynthesis. Enoxolone also inhibited sporulation. In a CDI colitis model, enoxolone when combined with vancomycin protected mice from becoming moribund and the combination was more effective than vancomycin alone, a standard of care antibiotic for CDI. While enoxolone alone reduced the in vivo load of toxins, the monotherapy did not protect mice from CDI. Affinity based proteomics identified ATP synthase subunit alpha (AtpA) and adenine deaminase (Ade) as possible molecular targets for enoxolone. Silencing of mRNA for Ade and AtpA also reduced toxin biosynthesis, while molecular interaction analysis showed that enoxolone directly bound to Ade. Ade converts adenine to hypoxanthine as an early step in the purine salvage pathway. Metabolomics revealed enoxolone caused cells to accumulate adenosine and deplete hypoxanthine and ATP. Accordingly, supplementation with hypoxanthine partly restored toxin production. Enoxolone also impacted phosphate metabolism by reducing the amounts of cellular phosphate. Thus, supplementation with triethyl phosphate as a source of phosphate also partly restored toxin production. When hypoxanthine and triethyl phosphate were combined, toxin production was fully restored in the presence of enoxolone. Taken together, studies with enoxolone revealed metabolic pathways that affect C. difficile toxin production and could represent potential anti-virulence drug targets.IMPORTANCEClostridioides difficile, a leading cause of hospital-acquired diarrhea, produces two co-regulated toxins (TcdA and TcdB) that are the focus of most anti-virulence discovery efforts for C. difficile infection (CDI). Exploration of an alternate anti-virulence strategy led to the discovery that the licorice metabolite enoxolone inhibits C. difficile virulence by blocking the cellular biosynthesis of TcdA and TcdB. Blockage of toxin production by enoxolone was associated with multiple effects on cells, including inhibiting adenine deaminase and ATP synthase leading to disruption of purine biosynthesis and phosphate metabolism. In mice infected with C. difficile, the efficacy of enoxolone in combination with vancomycin was superior to vancomycin alone. These findings contribute to establishing toxin biosynthesis inhibition as a newer therapeutic concept for CDI.

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“…A more general influence of ebselen on the virulence potential was recently suggested [ 43 ]. The study reevaluated the lack of antimicrobial properties against C. difficile , compared with typical activity reported for other species.…”

Section: Specific Bacterial Protein Targets For Ebselenmentioning

confidence: 99%

Antibacterial Activity of Ebselen

Maślanka

Mucha

2023

IJMS

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Ebselen is a low-molecular-weight organoselenium compound that has been broadly studied for its antioxidant, anti-inflammatory, and cytoprotective properties. These advantageous properties were initially associated with mimicking the activity of selenoprotein glutathione peroxidase, but the biomedical impact of this compound appear to be far more complex. Ebselen serves as a substrate or inhibitor with multiple protein/enzyme targets, whereas inhibition typically originates from the covalent modification of cysteine residues by opening the benzisoselenazolone ring and S–Se bond formation. The inhibition of enzymes of various classes and origins has been associated with substantial antimicrobial potential among other activities. In this contribution, we summarize the current state of the art regarding the antibacterial activity of ebselen. This activity, alone and in combination with commercial pharmaceuticals, against pathogens, including those resistant to drugs, is presented, together with the molecular mechanism behind the reactivity. The specific inactivation of thioredoxin reductase, bacterial toxins, and other resistance factors is considered to have certain therapeutic implications. Synergistic action and sensitization to common antibiotics assisted with the use of ebselen appear to be promising directions in the treatment of persistent infections.

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Ebselen Not Only Inhibits Clostridioides difficile Toxins but Displays Redox-Associated Cellular Killing

Cited by 12 publications

References 44 publications

The Ferrous Iron Transporter FeoB1 is Essential for Clostridioides difficile Toxin Production and Pathogenesis in Mice

The Ferrous Iron Transporter FeoB1 is Essential for Clostridioides difficile Toxin Production and Pathogenesis in Mice

The licorice metabolite enoxolone attenuates Clostridioides difficile pathophysiology by corrupting its metabolic and toxin production networks

Antibacterial Activity of Ebselen

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