*3.2. Effects of Methylene Quinones against B. subtilis*

The time–kill curves of *B. subtilis* cultures showed a different behavior when celastrol and pristimerin were added at different growth stages (Figure 2). Addition of celastrol at 3 μg/mL in the lag phase (10<sup>5</sup> CFU/mL) had a bactericidal effect similar to ciprofloxacin with ≥3 Log10 in CFU reduction after 9 h of incubation (Figure 2A). Rifampicin and tetracycline also had a bactericidal behavior in the first hours of treatment, although the cultures recovered after 24 h of incubation. A bacteriostatic action and regrowth was observed with penicillin. Compared to celastrol, pristimerin at 10 μg/mL had a bacteriostatic effect, producing a minor reduction in the CFU counts (<3 Log10). However, when pristimerin was incorporated in the log phase of growth (107 CFU/mL), a bactericidal action was observed, reducing the bacterial population 4.2 Log10 in 2 h of treatment, with up to 99.9% dead after 24 h of incubation (Figure 2B). In addition, the drop in optical density values (OD550) revealed the bacteriolytic nature of this compound. Under these conditions of growth, celastrol had a bacteriostatic action, similar to that shown by ciprofloxacin, tetracycline, and rifampicin, with a clear regrowth of the culture after 24 h in the presence of rifampicin. Penicillin had a limited effect on the control of *B. subtilis* cells actively growing.

The action of the methylene quinones was also evaluated against different inoculum sizes of *B. subtilis* in lag-phase growth (Table 2). At the higher inoculum concentration (10<sup>7</sup> CFU/mL), celastrol showed a bacteriostatic activity during the exposition time and a lower reduction in CFU/mL compared to the log phase of growth (Figure 2B). A bacteriostatic effect was also observed at 106 and 105 CFU/mL and bactericide at 104 CFU/mL. Pristimerin showed a similar behavior to that obtained with celastrol. However, when this compound was added in the log phase of growth, a bacteriolytic effect was observed within 6 h of treatment (Figure 2B). These results indicate that both quinones show a stronger effect when *B. subtilis* is actively growing.

**Figure 2.** Time–kill curves of *B. subtilis* cultures expressed as Log10 of CFU counts after treatment with different antimicrobial substances (celastrol 3 μg/mL; pristimerin 10 μg/mL; ciprofloxacin 1.2 μg/mL; rifampicin 0.15 μg/mL; tetracycline 7.5 μg/mL; and penicillin 9 μg/mL) added in lag phase of growth (**A**) and log phase after three hours of preincubation (**B**). Cultures without drugs and with the maximum proportion of DMSO were used as controls. Error bars express SD with *n* = 3.

**Table 2.** Effect of celastrol at 3 μg/mL and pristimerin at 10 μg/mL on different inoculum sizes of *B. subtilis* at 3 and 6 h after treatment.


<sup>1</sup> Data are expressed as mean values ± standard deviations (*<sup>n</sup>* = 3).

#### *3.3. Mechanism of Action*

#### 3.3.1. Effects of Macromolecular Synthesis and Initial Uptake of Solutes

Initially, the incorporation of radiolabeled precursors into DNA, RNA, protein, and cell wall synthesis was measured. After addition of celastrol and pristimerin, the incorporation of all precursors into the macromolecular synthesis was blocked, but not simultaneously (Figure 3). Celastrol at 3 <sup>μ</sup>g/mL reduced by ≥70% the incorporation of [6-3H] thymidine and [5-3H] uridine within 5 and 10 min, respectively. The inhibitory effect of celastrol after 5 min was comparable to that observed with the specific inhibitor of the RNA synthesis, rifampicin. Pristimerin at 10 μg/mL needed at least 30 min to produce an inhibition of 57% and 47% of DNA and RNA synthesis, respectively (Figure 3A,B). Celastrol and pristimerin inhibited the incorporation of leucine into protein synthesis by >55% after 20 min, whereas tetracycline blocked this process (>70%) in 10 min. The incorporation of *N*-acetyl-D-[14C] glucosamine into peptidoglycan decreased rapidly within 2 min after the addition of celastrol and pristimerin, with inhibition values of 58% and 70%, respectively. However, this effect was not constant over time and the incorporation of the precursor gradually increased after 5 min. Initially, the inhibitory effect produced by penicillin at 30 μg/mL on cell wall synthesis was slower (32% in 2 min) than with celastrol and pristimerin, but the incorporation of the precursor gradually increased this up to 85% in 30 min (Figure 3D). Clofoctol, a cytoplasmic membrane disruptor [45], had a variable effect on the incorporation of precursors but blocked all biosynthetic processes (>70% of inhibition) after 30 min of evaluation.

**Figure 3.** Incorporation of precursor in the synthesis of DNA (**A**), RNA (**B**), protein (**C**), and cell wall (**D**) of *B. subtilis* cultures in the presence of triterpene methylene quinones (celastrol 3 μg/mL; pristimerin 10 μg/mL), specific inhibitors of each pathway (ciprofloxacin 1.25 μg/mL; rifampicin 0.2 μg/mL; tetracycline 10 μg/mL; and penicillin 30 μg/mL), and clofoctol (5 μg/mL). Data are expressed as percentage (%) of precursors' incorporation compared to controls without drugs but with the maximum proportion of DMSO. Error bars express SD with *n* = 3. Different letters above bars mean significant differences between treated cultures (*p* < 0.05, one-way ANOVA; Tukey's test).

The inhibition of all processes of macromolecular synthesis is more compatible with an indirect effect on biosynthetic pathways rather than a specific action on specific targets [46,47]. Thus, the inhibition produced by celastrol and pristimerin on macromolecular synthesis could be related with damage on the cytoplasmic membrane, as it happens with clofoctol. For this reason, we firstly determined the effect of both compounds on the uptake of glucose by *B. subtilis*, measured as total cell-associated counts after cell isolation from free labeled precursors in the incubation medium. As shown in Figure 4, celastrol at 3 μg/mL drastically reduced (>70%) the uptake of D-[1-14C]-glucose in only 2 min, whereas clofoctol needed at least 5 min to produce comparable reductions. Pristimerin weakly reduced the uptake of glucose and required up to 20 min to produce ≈50% inhibition.

**Figure 4.** Glucose uptake assay on *B. subtilis* cultures after the addition of the quinones celastrol (3 μg/mL) and pristimerin (10 μg/mL). Clofoctol (5 μg/mL), a known inhibitor of macromolecule uptake, was added as a positive control. Data are expressed as percentage (%) of precursor incorporation compared to controls without drugs but with the maximum proportion of DMSO. Error bars express SD with *n* = 3. Different letters above bars mean significant differences between treated cultures (*p* < 0.05, one-way ANOVA; Tukey's test).

Based on these results, we decided to investigate whether the uptake of radiolabeled precursors would also be blocked in the presence of terpenoids, as was the case with glucose. The uptake of precursors (thymidine, uridine, leucine, and N-acetyl glucosamine) was determined in the presence of celastrol, the triterpenoid that showed the greatest inhibitory effect on glucose uptake and macromolecular synthesis. Figure 5 shows how the addition of celastrol at 3 μg/mL to *B. subtilis* cultures rapidly inhibited the uptake of [6-3H] thymidine and [5-3H] uridine by >50% between 2 and 5 min. The specific inhibitors of DNA and RNA synthesis, ciprofloxacin and rifampicin, did not affect the uptake of these precursors. The uptake of [4,5-3H] leucine was inhibited by up to 43% after 20 min, while uptake of *N*-acetyl-D-[1-14C] glucosamine was weakly affected and gradually increased during the experimentation time (Figure 5C,D). The addition of tetracycline blocked 75% the uptake of leucine in 10 min, whereas the transport of *N*-acetyl-D-[1-14C] glucosamine into the cells was not affected in the presence of penicillin.

**Figure 5.** Incorporation (insoluble phase) and uptake (soluble phase) of DNA (**A**), RNA (**B**), protein (**C**), and cell wall (**D**) precursors on *B. subtilis* cultures in the presence of celastrol (3 μg/mL). Cultures in the same conditions but without celastrol and with the same proportion of DMSO were used as negative control. Error bars express SD with *n* = 3. Significant differences found in uptake (\*\*\*: *p* < 0.001. \*\*: *p* < 0.01. \*: *p* < 0.05) or incorporation (+++: *p* < 0.001. ++: *p* < 0.01) compared with control (one-way ANOVA; Tukey's test).

Our results indicate that both uptake and incorporation processes in *B. subtilis* were affected by celastrol. Inhibition of macromolecular synthesis implies a low demand for biosynthetic precursors. Under these conditions, it would be reasonable to expect a slowdown in the uptake of precursors into the cell if both processes are coupled. In contrast, a primary blockage in precursor transport into the cells would also lead to inhibition of biosynthetic pathways due to low precursor availability. However, the uptake of precursors does not necessarily have to be linked to their incorporation in macromolecular synthesis. As Chou and Pogell [48] have previously suggested, if transport is inhibited and the process is not tightly coupled to macromolecular synthesis, the initial accumulation of precursors into the acid-soluble pool should be much more inhibited than their incorporation during macromolecular synthesis. In fact, these authors described how panamycin, an antibiotic that affects membrane-associated cellular functions, inhibits the uptake of uridine, while incorporation into biosynthetic processes is minimally affected. Thus, an ideal scenario would be one in which all of the precursors taken up by cells are in acid-soluble form to determine to what extent a blockage in molecular transport is a key process in the mechanism of action of the antibacterial agent [48].

Therefore, an experiment was conducted to investigate the effect of celastrol on the uptake of radiolabeled thymidine in *B. subtilis* when DNA synthesis was blocked by ciprofloxacin (1.25 μg/mL during 30 min). As seen in Figure 6, total radioactivity in control cells increased gradually throughout the incubation time. As expected, the incorporation of precursor slowed down when the biosynthetic process was specifically inhibited by ciprofloxacin. In this case, 5 min after the addition of the radiolabeled precursor, only ≈29%

of available thymidine was incorporated into macromolecular synthesis. By contrast, in the absence of ciprofloxacin, ≈74% of the precursor present in the cells was incorporated in the same time (Figure 5A). This observation suggests that uptake and incorporation are independent processes and that the precursors can accumulate in the cytoplasm in the absence of incorporation. The addition of celastrol at 3 μg/mL not only blocked the uptake of thymidine radiolabeled precursor in the treated cells but also produced a slight leakage of accumulated [6-3H] thymidine from *B. subtilis*. This effect could be related to an action of celastrol on the integrity of the membrane, affecting its permeability and producing a release of the cytoplasmic content. Similar results have been observed on *S. aureus* cells treated with zeylasterone, another triterpenoid with antibacterial properties, for which an action on the cytoplasmic membrane has also been suggested [49]. Additionally, the arrest of uptake was accompanied by an immediate blockage of incorporation into macromolecular synthesis. The results obtained reinforce the idea that celastrol could first target the function of the cytoplasmic membrane by affecting the transport of solutes and other essential molecules into the cell.

**Figure 6.** Incorporation (insoluble phase) and uptake (soluble phase) of radiolabeled 3H-thymidine on *B. subtilis*. Bacterial culture were pretreated with ciprofloxacin (1.25 μg/mL), a specific inhibitors of DNA biosynthesis before the addition of the radiolabeled precursor (time 0). The triterpene celastrol (3 μg/mL), or the same proportion of DMSO used as control, was added at the time indicated by the arrow. Error bars express SD with *n* = 3. Significant differences found in uptake (\*\*\*: *p* < 0.001) or incorporation (+++: *p* < 0.001) compared with control (one-way ANOVA; Tukey's test).

#### 3.3.2. Effect of Celastrol on the Integrity and Functions of the Cytoplasmic Membrane

The cytoplasmic membrane is a delicate and metabolically active structure essential for the survival of microorganisms. It acts as a selective permeability barrier and prevents the loss of essential components of low molecular weight and nucleotide. Antibacterial agents targeting the cytoplasmic membrane affect its functions and cause a rapid release of low molecular weight compounds [50]. It has been described that the primary target site of the phenolic compounds in bacteria is the cytoplasmic membrane [51]. Damage to the cytoplasmic membrane impacts permeability barrier functions, which subsequently leads to a loss of structural integrity and a leakage of intracellular material. Thus, we also investigated the effect of celastrol on the cytoplasmic membrane of *B. subtilis* using (i) the BacLight test, (ii) detection of UV-absorbing material efflux, and (iii) determination of potassium leakage. Fluorescent dye and "LIVE/DEAD" BacLight Bacterial Viability Kits

have the capability of monitoring the viability of bacteria as a function of the cell membrane integrity [52,53]. Surprisingly, microscopic observations after the BacLight assay showed that the cells treated with celastrol maintained membrane integrity like the untreated cells. In contrast, cultures treated with clofoctol showed red fluorescence, an observation indicating membrane damage (Supplementary Materials Figure S1). When UV-absorbing material from *B. subtilis* cultures was monitored, concentrations up to 20 μg/mL of celastrol did not alter the UV spectrum in comparison to the untreated cultures (Supplementary Materials Figure S2). Clofoctol used as a control clearly induced the release of UV-absorbing nucleotides in treated cells. Similar results were previously observed with netzahualcoyone, a terpenoid that interacts with the cytoplasmic membrane but also does not release materials that absorb at 260/280 nm [21]. We also determined the potassium released by *B. subtilis* cells as the first index of membrane damage [54]. Exposure to celastrol for 5 min induced intracellular potassium release compared with untreated cells, although the effect was significantly weaker than that observed with clofoctol (Figure 7). These data suggest that celastrol could act on biological membranes, altering their functions and modifying cell permeability.

**Figure 7.** Potassium (K+) leakage (μg/mL) from *Bacillus subtilis* cells exposed to celastrol (3 μg/mL). Cultures with clofoctol (5 μg/mL) or the maximum proportion of DMSO were used as positive and negative controls, respectively. Error bars express SD with *n* = 3.

#### 3.3.3. Transmission Electron Microscopy

To determine whether celastrol induces noticeable cell membrane damage, transmission electron microscopy was performed on thin sections of *B. subtilis* treated with the terpenoid for 1 h. Compared to the untreated control, cells treated with celastrol exhibited abnormally long cells, variability in wall thickness, compact ribosomes underlying the plasma membrane, and mesosome-like structures arising from the septa (Figure 8).

**Figure 8.** TEM of *B. subtilis* exposed for 1 h to celastrol at 3 μg/mL (**B**,**C**) or pristimerin at 10 μg/mL (**D**,**E**). Untreated cells (**A**). CM, cytoplasmic membrane; PG, peptidoglycan layer; R, ribosomes; M, mesosome-like structures.

Despite the multiple effects produced by celastrol, the technique did not allow the observation of visible damage to the cell membrane. For comparison purposes, *B. subtilis* treated with pristimerin at 10 μg/mL, which showed a bacteriolytic effect during killing curves assays, was also observed. As with celastrol, the cells were extremely long, spindle-shaped, with thin cell walls and slightly electrodense cytoplasm with ribosomes associated to the inner face of the membrane (Figure 8D,E). These observations resemble those obtained by da Cruz et al. [42] on cultures of *S. aureus* treated with pristimerin, where the cells presented a disrupted membrane, as well as a loss of cell integrity.

### 3.3.4. Effect of Celastrol on Cellular Respiration

Another fundamental function of the cytoplasmic membrane is cellular respiration. The respiratory chain plays an important role in the energetic metabolism of a cell, the maintenance of intracellular redox balance, or the protection against oxidative stress [55]. The membranes harbor the electron transport chain, a well-known system with oxygen as the final electron acceptor in aerobic respiration processes.

Thus, the effect of celastrol on oxygen consumption was also evaluated on cell cultures of *B. subtilis* and *E. coli*, as well as in acellular preparations of these bacteria obtained by cell disruption. Table 3 summarizes the results of the effect of celastrol on glucose-dependent oxygen uptake in intact cells of *B. subtilis* and *E. coli*. Both celastrol and NaCN produced an immediate inhibition of oxygen consumption in *B. subtilis*, reaching 60% at 8 min after their addition compared to the untreated control. As expected, celastrol barely affected the oxygen uptake in *E. coli*, as Gram-negative bacteria are insensitive to the terpene quinone.

**Table 3.** Mean values ± standard deviations of means (*n* = 3) of oxygen consumption rates (μL O2/min) at different times in whole cells or membrane preparations of *B. subtilis and E. coli* without treatment or after treatments with celastrol at 3 μg/mL or sodium cyanide (NaCN) at 6.7 mM used as positive controls. The percentage (%) of inhibition in the oxygen consumption referring to the untreated cells (negative control) is shown in parentheses.


<sup>1</sup> For each assessment, values with different superscript letters within each given time point indicate statistically significant differences (ANOVA, Tukey's multiple comparison test, *p* < 0.05).

> The same evaluation was carried out on acellular preparations where the oxygen consumption was coupled to the oxidation of NADH. Unlike when whole cells were used, here, NADH oxidation only depended on the amount of dissolved oxygen and the function of the respiratory chain. In these conditions, the addition of celastrol and NaCN inhibited the oxygen consumption in *B. subtilis* and *E. coli* preparations. The different behavior shown by celastrol in intact and disrupted preparations of *E. coli* cells shows that the cell membranes of both Gram-positive and -negative bacteria are equally sensitive to its action. The outer membranes of Gram-negative bacteria act as a permeability barrier, holding celastrol physically distant from its target of action. These results indicate that celastrol has a direct effect on the electron transport chain, affecting the consumption of oxygen and, consequently, the oxidation of NADH associated with respiration processes. However, in the presence of celastrol, neither a spontaneous oxidation of NADH nor a reduction in NAD+ was observed.

> An additional experiment was carried out to verify previous results indicating an action of celastrol on the inhibition of enzymatic activity. Nagase et al. [56] reported that celastrol can inhibit topoisomerase II and trigger apoptosis in HL-60 cells. DNA gyrase is an important bacterial topoisomerase II that catalyzes ATP-dependent negative supercoiling

of bacterial DNA. The essential role of gyrase is to maintain the topological constitution of DNA and, hence, the survival of bacteria [57]. Eukaryotic cells lack the enzyme, which has led to the development of specific antimicrobials targeting the gyrase functions [58]. Our results confirmed the effect of celastrol at 50 μg/mL on the gyrase activity of supercoiling plasmid pBR322, as did ciprofloxacin, used as a positive control (Supplementary Materials Figure S3). Interestingly, pristimerin has shown a weaker antibacterial action compared to celastrol and did not affect the enzymatic activity of gyrase. This mechanism of action has been suggested for other triterpenoid compounds showing activity against topoisomerases I and II [59]. Some models have been proposed for the mode of action of triterpenoids on topoisomerases, which can be either by binding to the enzyme at the DNA binding site or by binding to the ATP binding site, conformationally blocking DNA binding to the enzyme [60]. Thus, the effect on DNA supercoiling produced by celastrol could be related with an action on the gyrase rather than a direct DNA binding effect.

#### **4. Conclusions**

The antimicrobial action of celastrol and pristimerin, two natural triterpene methylene quinones, was evaluated, and the mechanism of action against the spore-forming bacteria *Bacillus subtilis* was also approached. Celastrol showed a higher antimicrobial effect compared with pristimerin, being active against Gram-positive bacteria. The results obtained in this study indicate that celastrol interacts with the cytoplasmic membrane of *B. subtilis*, preventing the transport of solutes into the cells and affecting basic membrane functions such as respiration processes. At the structural level, the membrane was not widely affected, suggesting a mechanism of action for celastrol other than a simple effect on structural components and subsequent membrane disruption. Furthermore, celastrol can also interact with enzymatic processes different from those exclusively located on the cytoplasmic membrane, as observed here for topoisomerase II. Although further investigations are required to elucidate a more precise mechanism of action, our results indicate that celastrol can act on multiple targets.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2304-815 8/10/3/591/s1, Figure S1: Epifluorescence microscopy images of *B. subtilis* stained with propidium iodide and SYTO 9 after treatment with celastrol (3 μg/mL) for 30 and 60 min (A,B) or clofoctol (5 μg/mL) for 30 min (C). Control cells were treated with the maximum proportion of DMSO (D). Scale bars correspond to 10 μm. Figure S2: Release of 260 and 280 nm absorbing material from *B. subtilis* cells treated with celastrol at 3, 6, and 10 μg/mL and clofoctol at 5 μg/mL. Cells exposed to the maximum proportion of DMSO were used as control. Error bars express SD with *n* = 3. Figure S3: DNA supercoiling assays. Electrophoresis gel shows a control with a negatively supercoiled plasmid (line 1) with DMSO (line 2), and after its treatment with ciprofloxacin at 25 (line 3) and 50 μg/mL (line 4), celastrol at 50 μg/mL (line 5), and pristimerin at 50 μg/mL (line 6).

**Author Contributions:** Conceptualization, L.M. and L.d.L.G.; methodology, L.M. and L.d.L.G.; investigation, N.P.-M. and L.d.L.G.; data curation, L.M., N.P.-M. and L.d.L.G.; writing—original draft preparation, L.M. and L.d.L.G.; writing—review and editing, L.M. and L.d.L.G.; supervision, L.M. and L.d.L.G.; funding acquisition, L.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by RTI2018-094356-B-C21 Spanish Ministerio de Economía, Industria y Competitividad (MINECO) and co-funded by the European Regional Development Fund (FEDER) projects.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Thanks are due to the Universidad de La Laguna and Servicio de Microscopia de la Universidad de Las Palmas de Gran Canaria.

**Conflicts of Interest:** The authors declare no conflict of interest.
