Chemical genetic analysis of enoxolone inhibition of Clostridioides difficile toxin production reveals adenine deaminase and ATP synthase as antivirulence targets

Abstract

Toxins TcdA and TcdB are the main virulence factors of Clostridioides difficile, a leading cause of hospital-acquired diarrhea. Despite their importance, there is a significant knowledge gap of druggable targets for inhibiting toxin production. To address this, we screened nonantibiotic phytochemicals to identify potential chemical genetic probes to discover antivirulence drug targets. This led to the identification of 18β-glycyrrhetinic acid (enoxolone), a licorice metabolite, as an inhibitor of TcdA and TcdB biosynthesis. Using affinity-based proteomics, potential targets were identified as ATP synthase subunit alpha (AtpA) and adenine deaminase (Ade, which catalyzes conversion of adenine to hypoxanthine in the purine salvage pathway). To validate these targets, a multifaceted approach was adopted. Gene silencing of ade and atpA inhibited toxin biosynthesis, while surface plasmon resonance and isothermal titration calorimetry molecular interaction analyses revealed direct binding of enoxolone to Ade. Metabolomics demonstrated enoxolone induced the accumulation of adenosine, while depleting hypoxanthine and ATP in C. difficile. Transcriptomics further revealed enoxolone dysregulated phosphate uptake genes, which correlated with reduced cellular phosphate levels. These findings suggest that enoxolone's cellular action is multitargeted. Accordingly, supplementation with both hypoxanthine and triethyl phosphate, a phosphate source, was required to fully restore toxin production in the presence of enoxolone. In conclusion, through the characterization of enoxolone, we identified promising antivirulence targets that interfere with nucleotide salvage and ATP synthesis, which may also block toxin biosynthesis.

Keywords: ATP synthase; adenine deaminase; phosphate metabolism; purine metabolism; sporulation; toxins.

Figure 1

Screening for inhibitors of Clostridioides difficile toxin biosynthesis. R20291 in BHI in 96 well plates was exposed to 100 μM of phytochemicals for 24 h to measure: (A) effects on growth (A₆₀₀ nm); growth inhibitors are indicated in red and were triaged by comparison with the DMSO control (n = 3 biological replicates). B, toxins were quantified by cytopathic cell rounding; inhibitors of toxin synthesis are indicated in green (n = 4 biological replicates). Controls were vancomycin (0.5 μM) and glucose 1% (w/v). The purple line indicates cutoff criteria for compound selection. C, toxins (TcdA and TcdB) in culture supernatants were quantified by ELISA; enoxolone (100 μM) inhibited synthesis of TcdA by 81.18 ± 12.68% and TcdB by 47.40 ± 9.02% (n = 3 biological replicates; statistical significance was assessed by one-way ANOVA with Tukey’s test; ∗p < 0.05, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001 in Graphpad prism 9.3.1). D, cell rounding assay showing dose-response curves of toxin biosynthesis inhibitors parthenolide, tannic acid, kahweol, and enoxolone (n = 4 biological replicates). Respectively, EC₅₀s and Hill slopes were 27.93 μM and −3.75; >80 μM and −2.24; >80 μM and −4.56; and 7.77 μM and −4.33; the negative Hill slopes are indicative of downhill inhibition curves, where increases in drug concentration caused a decrease in cell rounding, with narrow thresholds between effective and ineffective drug concentrations; this suggests the compounds have complex modes of actions. Toxins, namely TcdB, were quantified by cell rounding against Vero cells and used for biological replicates. Data in all plots are shown as mean ± SEM. BHI, brain heart infusion; DMSO, dimethyl sulfoxide.

Figure 2

Characterization of enoxolone (ENX) activity against Clostridioides difficile.A, dose response of ENX against R20291. Exponential cultures (A₆₀₀ nm ≈ 0.3) were exposed to DMSO or 2 fold increasing concentrations of ENX (n = 3 biological replicates). After 24 h, TcdA and TcdB were quantified by ELISA. Respectively, EC₅₀s and Hill slopes were for TcdA (11.38 mM and −4.04) and TcdB (14.29 and −3.96), which are indicative of downhill inhibition curves, where increasing drug concentration significantly decreases toxin production about a narrow threshold between effective and ineffective drug concentrations; this suggests a complex mode of action. B, effect of ENX on mRNA levels of tcdA and tcdB, as determined by RT-qPCR. Cultures (A₆₀₀ ≈ 0.3; n = 4 biological replicates) were exposed to ENX, and mRNA analyzed after 9 h; the fold change was calculated relative to the DMSO control. Statistical significance was assessed from ΔCt values, comparing DMSO and the different ENX treated samples by two-way ANOVA with Tukey’s test: ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗∗p < 0.0001 in Graphpad prism 9.3.1. C, effect of ENX on growth of R20291. Exponential cells (A₆₀₀ nm ≈ 0.2; n = 4 biological replicates) were treated with DMSO or ENX at ½×, 11× and 22× EC₅₀ of 8 μM. D, effect of ENX on sporulation. Exponential cells (A₆₀₀ nm ≈ 0.3) were exposed to compound or DMSO for 5 days; total viable counts and spores were then enumerated; ENX was used at ½x, 11× and 22× EC₅₀ of 8 μM. Controls were vancomycin (VAN; 1 μM) and acridine orange (AO = 116 μM); n = 4 biological replicates are shown as mean ± SEM and statistical significance was by one-way ANOVA with Tukey’s test: ∗∗p < 0.01 and ∗∗∗p < 0.001 in Graphpad prism 9.3.1. DMSO, dimethyl sulfoxide.

Figure 3

Effect of silencing genes for which proteomics suggested to encode targets of enoxolone. Analyzed were R20291 with the empty vector control (pMSPT) or R20291 with pMSPT expressing antisense RNA (asRNA) to ade, gapN, or atpA; 0.032 μg/ml of anhydrotetracycline (ATc) was used for induction. A, growth kinetics were analyzed for the strains i.e., pMSPT (black), atpAi (red), gapNi (green), and adei (blue) in microdilution 96-well plate (n = 4 biological replicates). As a growth control, R20291 bearing pMSPT without ATc exposure was analyzed. B, TcdA was analyzed at 24 h after induction of asRNA. Data from three biological replicates are shown as mean ± SEM and analyzed by two-way ANOVA with Tukey’s test: ∗p < 0.05 in Graphpad prism 9.3.1. C, mRNA levels of toxin genes (tcdA and tcdB) were analyzed by reverse transcriptase quantitative PCR (n = 3 biological replicates) from exponential cultures (A₆₀₀ nm ≈ 0.3) treated with 0 or 0.032 μg/ml of ATc for 6 h. The fold change was calculated for mRNA from drug free and ATc exposed cultures. Significance was analyzed by comparing the ΔCt values of samples with ATc at 0 μg/ml versus those with ATc 0.032 μg/ml by two-way ANOVA with Tukey’s test: ∗p < 0.05 and ∗∗p < 0.01 in Graphpad prism 9.3.1. Ade, adenine deaminase; atpA, ATP synthase subunit alpha; GapN, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase.

Figure 4

Enoxolone (ENX) binds to adenine deaminase and inhibits purine metabolism.A, molecular interaction analysis using surface plasmon resonance (SPR). Dose response sensograms with different concentrations of ENX (top panel) shown as a representative of four replicates. The bottom panel shows the corresponding analysis of response at equilibrium of the experiment in the top panel (K_d = 52 μM for the replicate and 33.73 ± 14.14 μM overall). B, schematic representation of purine salvage pathway adapted from Kyoto Encyclopedia of Genes and Genomes (KEGG number T00998 for strain R20291), showing adenine deaminase’s role in converting adenine to hypoxanthine. Enzymes involved in this pathway are indicated in blue color. Ade = adenine deaminase; Add = adenosine deaminase; DeoD = purine nucleoside phosphorylase; CDR20291_2424 = putative membrane-associated 5′-nucleotidase/phosphoesterase; Adk = adenylate kinase; GuaD = guanine deaminase; GuaB = inosine-5′-monophosphate dehydrogenase; GuaA = GMP synthase [glutamine-hydrolyzing]; Gmk = guanylate kinase. C, LC-MS/MS analysis of purine metabolites. C. difficile R20291 cells (A₆₀₀ ≈ 0.3) was exposed to ENX (16 μM) or 1% (v/v) DMSO for 3 h. Heatmaps generated with ClustVis software shows relative quantities of metabolites; red and blue color intensities indicate levels of metabolites that were increased or decreased, respectively. DMSO, dimethyl sulfoxide; LC-MS/MS, liquid chromatography with tandem mass spectrometry. Statistical significance was assessed by two-tailed unpaired t-tests; ∗p < 0.05, p < 0.01 in Graphpad prism 9.3.1.

Figure 5

Relationship between enoxolone (ENX) effects on growth, toxin production, and purine metabolism.A, growth kinetics of exponential R20291 (n = 3 biological replicates) in 1% (v/v) DMSO or ENX (32 μM) with or without various purine derivatives (250 μM). B, quantification of TcdA in R20291 cultures exposed to purine derivatives (250 μM) in presence of DMSO or ENX (32 μM); data from six biological replicates, shown as mean ± SEM, were statistically analyzed by two-way ANOVA with Tukey’s test ∗∗p < 0.01 and ∗∗∗p < 0.001 in Graphpad prism 9.3.1. DMSO, dimethyl sulfoxide.

Figure 6

Analysis of effects of enoxolone on cellular ATP, toxin production, and global transcriptome.A, quantification of intracellular pools of AMP, ADP, and ATP in Clostridioides difficile R20291 cells exposed to 16 μM of enoxolone. Cells in early exponential growth phase (A₆₀₀ ≈ 0.3) were exposed to DMSO or enoxolone (ENX) in presence of 250 μM triethyl phosphate (red bars) or 250 μM hypoxanthine (blue bars). Cultures (n = 4 biological replicates) were harvested 3 h after exposure to ENX, and metabolites were quantified through HPLC. The data in the plot are representative of three biological replicates, and the error bars indicate mean ± SEM and significance were determined relative to DMSO control (one-way ANOVA with Dunnett’s test ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 in Graphpad prism 9.3.1). B, C. difficile R20291 (A₆₀₀ ≈ 0.3) was exposed to ENX (black bars) or Bz-423 (red bars). TcdA quantification was performed on culture supernatants after 24 of exposure. Data from three biological replicates are shown as mean ± SEM (one-way ANOVA with Tukey’s test ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 done using Graphpad prism version 9.3.1). C, gene expression was analyzed by quantifying mRNA in R20291 (n = 3 biological replicates) exposed to 16 μM (1× EC₅₀) ENX. Exponentially growing cells (A₆₀₀ ≈ 0.3) were exposed to ENX or DMSO for 30 min before RNA was extracted for RNA-seq. D, mRNA levels for the genes involved in phosphate metabolism were analyzed by reverse transcriptase quantitative PCR, and the fold change was calculated as the difference in mRNA levels of control versus ENX-treated cells; mean ± SEM are shown. Statistical significance was assessed by comparing ΔCt values of DMSO and ENX treatment by two-tailed paired t-tests; ∗∗p < 0.01 in Graphpad prism 9.3.1. DMSO, dimethyl sulfoxide.

Figure 7

Analysis of effects of enoxolone and ATP synthase on cellular phosphate (Pi) metabolism and toxin production.A, Clostridioides difficile at A₆₀₀ ≈ 0.3 was treated with 8, 16, or 32 μM of enoxolone (ENX), or 1 μM of vancomycin (VAN), or 55 μM of glucose (GLU) and harvested after 3 h. Intracellular Pi was analyzed from whole cell lysates (n = 3 biological replicates; with exception of GLU/HYP and DMSO/TEP, all other samples had two technical replicates within the three biological replicates). Significance was assessed relative to DMSO control by two-way ANOVA with Dunnett’s test ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; in Graphpad prism 9.3.1. B, TcdA quantification from 24 h old cultures of C. difficile R20291 cells exposed to 250 μM metabolite in presence of DMSO (black bars) or 32 μM ENX (red bars). The data in the plot are representative of minimum three biological replicates, and the error bars indicate mean ± SEM and the data significance is representative of two-way ANOVA with Tukey’s test ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 in Graphpad prism 9.3.1. C, relationship between AtpA activity and cellular phosphates, determined from phosphate levels in C. difficile R20291 cells carrying empty vector or the vector encoding an antisense RNA (asRNA) to atpA or ade. Cells were grown to A₆₀₀ ≈ 0.3, and asRNA induced with indicated concentrations of ATc. Cells were harvested after 3 h of exposure to ATc and their phosphate contents were measured from whole cell lysates (n = 3 biological and two technical replicates; effects of gene silencing on toxin production are in Figs. 3 and S4). Significance was assessed for phosphate levels relative to uninduced cells (ATc 0 μg/ml) by two-way ANOVA with Tukey’s test ∗∗p < 0.01 in Graphpad prism 9.3.1. ade, adenine deaminase; ATc, anhydrotetracycline; atpA, ATP synthase subunit alpha; DMSO, dimethyl sulfoxide; TEP, triethyl phosphate.

Figure 8

Proposed model for enoxolone mode of action against Clostridioides difficile. (Left) scheme depicting the molecular events during the regular growth of C. difficile. (Right) represents adaptation of C. difficile to enoxolone, which hampers multiple processes in cells including ATP, phosphate, and purine metabolisms. The protein designated with a question mark, represents an unknown effector that might be a downstream factor causing repression of toxin genes. While effectors of toxin gene expression are known (e.g., TcdR, CcpA, and CodY), it is not unknown whether enoxolone suppression of toxin production works through these or other regulators. CcpA, catabolite control protein A.