Inhibition of selenoprotein synthesis is not the mechanism by which auranofin inhibits growth of Clostridioides difficile

Clostridioides difficile infections (CDIs) are responsible for a significant number of antibiotic-associated diarrheal cases. The standard-of-care antibiotics for C. difficile are limited to fidaxomicin and vancomycin, with the recently obsolete metronidazole recommended if both are unavailable. No new antimicrobials have been approved for CDI since fidaxomicin in 2011, despite varying rates of treatment failure among all standard-of-care drugs. Drug repurposing is a rational strategy to generate new antimicrobials out of existing therapeutics approved for other indications. Auranofin is a gold-containing anti-rheumatic drug with antimicrobial activity against C. difficile and other microbes. In a previous report, our group hypothesized that inhibition of selenoprotein biosynthesis was auranofin’s primary mechanism of action against C. difficile. However, in this study, we discovered that C. difficile mutants lacking selenoproteins are still just as sensitive to auranofin as their respective wild-type strains. Moreover, we found that selenite supplementation dampens the activity of auranofin against C. difficile regardless of the presence of selenoproteins, suggesting that selenite’s neutralization of auranofin is not because of compensation for a chemically induced selenium deficiency. Our results clarify the findings of our original study and may aid drug repurposing efforts in discovering the compound’s true mechanism of action against C. difficile.

Clostridioides difficile (formerly Clostridium difficile) is a Gram-positive, endospore-forming strict anaerobe and the leading cause of antibiotic-associated diarrhea (~15-25% of cases) 1,2 .C. difficile infections (CDIs) typically occur in patients with gut dysbiosis and can lead to severe clinical complications such as pseudomembranous colitis and toxic megacolon 3 .During infection, C. difficile causes disease and induces inflammation by producing two large exotoxins-TcdA and TcdB-which damage the intestinal lining through the glucosylation of Rho-family GTPases in host epithelial cells 4 .According to a recent CDC report, CDIs were responsible for approximately 223,900 hospitalized patient cases and 12,800 deaths in 2017 5 .Moreover, CDIs have contributed to approximately $1 billion in U.S. healthcare costs 5 .
The standard-of-care antibiotics for treating CDI are fidaxomicin and vancomycin 6,7 .If neither drug is available, metronidazole is recommended as an alternative 6,7 , though this former first-line antibiotic is regarded as obsolete due to its high rates of treatment failure 8 .In fact, CDI recurrence occurs in ~15-30% of patients treated with metronidazole and vancomycin despite their effectiveness in inhibiting C. difficile growth 8,9 .On the other hand, fidaxomicin is a narrow-spectrum antimicrobial with greater potency and is typically associated with comparatively lower recurrence rates 10,11 , though treatment failure has also been reported 12 .Moreover, while not the primary issue encountered in CDI management, antimicrobial resistance is still a cause for concern as drug-resistant clinical isolates have been reported for all three antibiotics [13][14][15][16] .Overall, the current repertoire for treatment is quite limited, especially since fidaxomicin was the last CDI drug approved by the U.S. Food and Drug Administration (FDA) in 2011 17 .If no new alternatives are added to the current list of standard-of-care antibiotics, the rising rates of recurrence and antibiotic resistance could outpace efforts to keep CDI under reasonable control.
Auranofin is an FDA-approved anti-rheumatic gold (Au) compound that possesses antimicrobial activity against C. difficile 18,19 .Many reports have highlighted its inhibitory activity against C. difficile vegetative cells and sporulation, its ability to reduce toxin levels and protect Caco-2 cells against their lethal effects, and its efficacy in preventing CDI and disease recurrence in mouse and hamster models [20][21][22][23][24] .While the mechanism of action auranofin sensitivity of an array of C. difficile strains deficient in some or all selenoproteins (Table 1) using a modified version of the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method 33 .While CLSI recommends the agar dilution method for antimicrobial susceptibility testing of anaerobes 33 , we chose broth microdilution because of its practicality and less cumbersome methodology.Moreover, broth microdilution has been reported to perform similarly to agar dilution in susceptibility tests of C. difficile 34,35 , though we are aware that others have observed substantial differences between both methods and argue against routine testing with broth microdilution 36,37 .Since our goal was to compare relative differences in minimum inhibitory concentrations (MICs) between strains rather than report standardized values that could be translated to the clinic, broth microdilution was therefore deemed appropriate for this study.
Briefly, we cultured each strain in supplemented brain heart infusion (BHIS) broth containing varying concentrations of auranofin at 37 °C for 48 h.At the end of the growth period, we established each strain's MIC of auranofin by measuring the optical density of each culture at 600 nm (OD 600 ).With this method, we first determined the auranofin sensitivity of wild-type strains R20291 (MIC = 2 µg/mL) and JIR8094 (MIC = 8 µg/ mL) (Figs.1A and 2A).The standard-of-care CDI therapeutics, fidaxomicin and vancomycin, were also included as positive controls for the assay.Accordingly, R20291 was inhibited by 0.125 µg/mL fidaxomicin and 1 µg/mL vancomycin while JIR8094 was inhibited by 0.016 µg/mL fidaxomicin and 4 µg/mL vancomycin (Supplementary Fig. S1).
Based on our laboratory's previous work, it was proposed that auranofin inhibits the growth of C. difficile by forming a complex with Se, thereby depleting the amount of bioavailable Se for trafficking and eventual incorporation into selenoproteins 19 .Thus, if auranofin's activity arises from the inhibition of selenoprotein biosynthesis, a strain lacking selenoproteins (i.e., a selD mutant) would theoretically be resistant to the compound and harbor a significantly elevated MIC compared to wild type.Despite this assumption, we surprisingly found that the MICs for R20291 (Fig. 1A), KNM6 (ΔselD) (Fig. 1B), and KNM9 (ΔselD::selD + ) (Fig. 1C) were all equally 2 µg/mL auranofin, suggesting that the compound's activity does not stem from targeting selenoproteins.To determine if this phenomenon was strain dependent, we repeated the assay with JIR8094 and LB-CD7 (selD::ermB) and likewise saw no increase in the MIC.However, while the wild-type strain JIR8094 exhibited an MIC of 8 µg/ mL (Fig. 2A), the selD::ermB strain was actually more susceptible to auranofin as it failed to grow at 4 µg/mL (Fig. 2B).This slight increase in sensitivity was surprising as it seemed to suggest a complex relationship between auranofin's antimicrobial activity and the selenoproteins in JIR8094.Out of curiosity, we evaluated two Prd Table 1.Bacterial strains used in this study.

Bacterial strain
Description (relevant genotype) Reference/source R20291 Wild type, ribotype 027 mutants-LB-CD4 (prdB::ermB) and LB-CD8 (prdR::ermB)-and one Grd mutant-LB-CD12 (grdA::ermB)using the same assay in order to determine which reductase plays a greater role in this phenomenon, if any.Interestingly, we discovered that all three mutants exhibited the same MIC of 4 µg/mL as the selD::ermB strain (Fig. 2C,D,E).While these data seem to suggest that a mutation in either of these selenoproteins renders C. difficile JIR8094 more sensitive to auranofin, a simple two-fold difference in MIC is likely not enough evidence for this.Regardless, these data clearly show that auranofin inhibits the growth of C. difficile in the absence of selenoproteins.
Selenite supplementation neutralizes auranofin's activity against C. difficile even in the absence of selenoproteins.We previously demonstrated that supplementing the culture medium with Se  www.nature.com/scientificreports/(either as sodium selenite or L-selenocysteine) exhibits a protective effect against auranofin, which we had interpreted as excess Se overcoming the apparent nutritional deficiency caused by the formation of Au-Se adducts 19 .Since auranofin still inhibits the growth of selD mutants as well as wild-type strains, we wanted to determine if selenite supplementation would still influence auranofin's antibacterial activity.When we repeated the previous assay using BHIS broth augmented with 5 µM selenite, we surprisingly observed a two-fold increase in the MICs of all strains (with the exception of JIR8094) (Figs. 3 and 4), suggesting that excess selenite dampened auranofin's activity regardless of whether selenoproteins were present.To verify if this response could be exacerbated at higher doses, we repeated the same assay with 50 µM selenite.Under these conditions, all strains grew regardless of the auranofin concentration (Figs. 3 and 4).These results clearly demonstrate that selenite's protective effect against auranofin cannot be explained as simply overcoming a Se deficiency imposed by the compound.While this phenomenon could potentially be interpreted as chemical inactivation by Se, it must be noted that Thangamani et al. reported no selenite-dependent neutralization of auranofin's activity against methicillin-resistant Staphylococcus aureus 38 , suggesting that there are different species-specific mechanisms at play.Finally, given the fact that selenite exhibits varying toxicity to some bacteria 39,40 , we wanted to determine if this was potentially acting as a confounding variable in our experiments.When we cultured our strains in BHIS broth containing varying selenite concentrations, we subsequently observed no difference in growth yields even up to 100 µM (Supplementary Figs.S2 and S3).This result correlates with a publication that reports a staggering MIC of 27 mM sodium selenite against two C. difficile isolates 41 .

Discussion
In this work, we unexpectedly discovered that auranofin inhibits the growth of C. difficile mutants lacking selenoproteins.This result was perplexing as we originally thought that auranofin's antimicrobial activity against C. difficile was mainly due to the inhibition of Se metabolism 19 .Our idea had been supported by several lines of evidence: (i) auranofin prevented the uptake of 75 Se and its incorporation into selenoproteins in both C. difficile and anaerobically grown Escherichia coli; (ii) the anaerobic growth yield of an E. coli ΔselD mutant was unaffected by auranofin compared to wild type; and (iii) auranofin exhibited little to no activity against Clostridium perfringens and Clostridium tetani (i.e., clostridia that lack selenoproteins) 19 .Additionally, we had found that the oral pathogen Treponema denticola-an organism with a strict Se requirement for growth 42 -was also susceptible to auranofin, as the compound likewise prevented the uptake and incorporation of 75 Se into its selenoproteins 43 .Consistent with our initial observations of auranofin's activity against C. difficile 19 , the compound's growth inhibition of T. denticola could be attenuated by supplementation with either sodium selenite or L-selenocysteine 43 .
Clearly, our idea of targeting Se metabolism in C. difficile was predicated on the assumption that the pathogen required Se for growth in the same manner as T. denticola, when in reality, genetic techniques have revealed that selenoproteins are actually not essential to C. difficile 28 .Thus, when dealing with organisms that carry dispensable selenoproteins (e.g., E. coli and C. difficile), Se metabolism becomes a poor candidate for a drug target.Moreover, it is obvious that auranofin's effects in these bacteria are far more complex than initially assumed; for example, it is unknown why an E. coli selD mutant gains slight resistance to auranofin while a C. difficile selD mutant exhibits no appreciable change in sensitivity.Further research should focus on fully characterizing the compound's multiple modes of action in order to truly understand their effects in different pathogens.
As of now, auranofin's mechanism of action against C. difficile is unknown, but a promising candidate may exist within the thioredoxin (Trx) system, which utilizes disulfide reductase activity to protect cytosolic components against oxidative stress and maintain thiol redox homeostasis 44 .The Trx system is comprised of Trx, Trx reductase (TrxR), and NADPH 44 .Trx reduces aberrant disulfides in the cell using a thiol-disulfide exchange mechanism that inevitably causes itself to be oxidized; TrxR utilizes electrons from NADPH to reduce Trx, allowing it to resume its surveillance of the cytosol for more oxidized substrates 44 .Interestingly, auranofin is known to be a selective inhibitor of TrxR in mammalian cells and parasites [45][46][47] .Likewise, auranofin has been shown to inhibit bacterial TrxR in some clinical pathogens such as Mycobacterium tuberculosis, S. aureus, and Helicobacter pylori [48][49][50] .Harbut et al. 48even proposed that auranofin's poor activity against several Gram-negative bacteria is actually due to the presence of the glutathione system, which can provide compensatory disulfide reductase activity in the event of a compromised Trx system.Thus, in bacteria lacking glutathione (i.e., most Gram-positives), auranofin-dependent inhibition of TrxR is expected to be lethal.It is therefore tempting to believe that auranofin could be exhibiting a similar mechanism in C. difficile due to two important observations: (i) a trxR gene exists within the grd operon 25 , and (ii) the cysteine-to-glutathione biosynthesis pathway is reportedly absent from the genome 51 .Alternatively, Thangamani et al. claimed that auranofin likely possesses multiple modes of action, as the compound was able to inhibit several biosynthetic pathways in S. aureus (e.g., DNA, protein, and cell wall syntheses) 38 .Moreover, the authors suggest that auranofin's weak activity against Gram-negatives may instead be due to the presence of the outer membrane and efflux pumps, rather than the redundant activity of the glutathione system 38 .Specifically, they showed that several Gram-negative pathogens were only susceptible to auranofin when the permeabilizing agent polymyxin B nonapeptide was present; moreover, an E. coli double mutant lacking both TrxR (trxB) and glutathione reductase (gor) did not differ in auranofin sensitivity compared to wild type 38 .Overall, these data imply that inhibition of TrxR-akin to inhibition of selenoprotein synthesis in C. difficile-may not be the only mechanism that this compound utilizes against bacteria.A classic technique to determine the mechanism of action of an antimicrobial involves the careful isolation of spontaneous drugresistant mutants in vitro; however, numerous groups have clearly reported an inability to generate spontaneous auranofin-resistant mutants of several bacterial species using this method 38,48,50,[52][53][54] .Likewise, our attempts to isolate spontaneous auranofin-resistant C. difficile mutants were met with failure, which further supports the idea of auranofin possessing multiple modes of action.

Materials and methods
Bacterial strains and growth maintenance.All C. difficile strains used in this study are listed in Table 1.
Growth experiments were performed in a Coy anaerobic chamber under an atmosphere of ~1.0%H 2 , 5% CO 2 , and >90% N 2 .Strains were routinely maintained on BHIS agar (37 g/L brain heart infusion, 5 g/L yeast extract, 0.1% L-cysteine).When indicated, overnight cultures were prepared by inoculating 5 mL BHIS broth with single colonies of the appropriate strains followed by 16-24 h of incubation at 37 °C.
Broth microdilution assay.MICs were determined using a modified broth microdilution assay as per the CLSI M11 33 .Briefly, auranofin was dissolved in 100% dimethyl sulfoxide (DMSO) and subsequently diluted to achieve working stocks at 20× concentration in 50% DMSO.Similarly, fidaxomicin was dissolved in 100% DMSO while vancomycin hydrochloride was dissolved in deionized water.Diluted test compounds (5 μL) were

Figure 1 .
Figure 1.A C. difficile ΔselD mutant has the same sensitivity to auranofin as wild type.C. difficile strains (A) R20291, (B) KNM6, and (C) KNM9 were grown in BHIS broth augmented with 2.5% DMSO and varying concentrations of auranofin at 37 °C for 48 h.The OD 600 of each culture was recorded at 48 h.The experiment was performed twice.Data points represent the means of triplicate cultures while error bars represent standard deviations.

Figure 2 .
Figure 2. Mutations in selenophosphate synthetase, proline reductase, or glycine reductase do not confer resistance to auranofin.C. difficile strains (A) JIR8094, (B) LB-CD7, (C) LB-CD4, (D) LB-CD8, and (E) LB-CD12 were grown in BHIS broth augmented with 2.5% DMSO and varying concentrations of auranofin at 37 °C for 48 h.The OD 600 of each culture was recorded at 48 h.The experiment was performed twice.Data points represent the means of triplicate cultures while error bars represent standard deviations.

Figure 3 .
Figure 3. Selenite supplementation decreases auranofin sensitivity even in the absence of selenoproteins.C. difficile strains (A) R20291, (B) KNM6, and (C) KNM9 were grown in selenite-supplemented BHIS broth augmented with 2.5% DMSO and varying concentrations of auranofin at 37 °C for 48 h.Sodium selenite was added to give a final concentration of 5 µM (red open circle) or 50 µM (red filled circle).The OD 600 of each culture was recorded at 48 h.The experiment was performed twice.Data points represent the means of triplicate cultures while error bars represent standard deviations.

Figure 4 .
Figure 4. Selenite supplementation decreases auranofin sensitivity in a manner independent of selenophosphate synthetase, proline reductase, or glycine reductase.C. difficile strains (A) JIR8094, (B) LB-CD7, (C) LB-CD4, (D) LB-CD8, and (E) LB-CD12 were grown in selenite-supplemented BHIS broth augmented with 2.5% DMSO and varying concentrations of auranofin at 37 °C for 48 h.Sodium selenite was added to give a final concentration of 5 µM (red open circle) or 50 µM (red filled circle).The OD 600 of each culture was recorded at 48 h.The experiment was performed twice.Data points represent the means of triplicate cultures while error bars represent standard deviations.