ROS mediated anticandidal efficacy of 3-Bromopyruvate prevents vulvovaginal candidiasis in mice model

Abstract

Candidal infections, particularly vulvovaginal candidiasis (VVC), necessitate effective therapeutic interventions in clinical settings owing to their intricate clinical nature and elusive understanding of their etiological mechanisms. Given the challenges in developing effective antifungal therapies, the strategy of repurposing existing pharmaceuticals has emerged as a promising approach to combat drug-resistant fungi. In this regard, the current study investigates molecular insights on the anti-candidal efficacy of a well-proven anticancer small molecule -3-bromopyruvate (3BP) against three clinically significant VVC causing Candida species viz., C. albicans, C. tropicalis and C. glabrata. Furthermore, the study validates 3BP's therapeutic application by developing it as a vaginal cream for the treatment of VVC. 3BP exhibited phenomenal antifungal efficacy (killing >99%) with minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC) of 256 μg/mL against all tested Candida spp. Time killing kinetics experiment revealed 20 min as the minimum time required for 3BP at 2XMIC to achieve complete-killing (99.9%) in all Candida strains. Moreover, the ergosterol or sorbitol experiment explicated that the antifungal activity of 3BP does not stem from targeting the cell wall or the membrane component ergosterol. Instead, 3BP was observed to instigate a sequence of pre-apoptotic cascade events, such as phosphatidylserine (PS) externalization, nuclear condensation and ROS accumulations, as evidenced by PI, DAPI and DCFH-DA staining methods. Furthermore, 3BP demonstrated a remarkable efficacy in eradicating mature biofilms of Candida spp., achieving a maximum eradication level of 90%. Toxicity/safety profiling in both in vitro erythrocyte lysis and in vivo Galleria mellonella survival assay authenticated the non-toxic nature of 3BP up to 512 μg/mL. Finally, a vaginal cream formulated with 3BP was found to be effective in VVC-induced female mice model, as it significantly decreasing fungal load and protecting vaginal mucosa. Concomitantly, the present study serves as a clear demonstration of antifungal mechanistic action of anticancer drug -3BP, against Candida species. This finding holds significant potential for mitigating candidal infections, particularly VVC, within healthcare environments.

Fig 1

Determination of MIC of 3BP against C. albicans, C. tropicalis and C. glabrata through (a-c) spectrophotometrical and (d-f) spot method. After 24 hours of exposure to 3BP at concentrations ranging from 0–1024 g/mL, the growth OD600 was measured. VC denotes for vehicle control (ethanol at 10 μL).

Fig 2. Determination of 3BP’MFC on C. albicans, C. tropicalis and C. glabrata using spread plate method.

Fig 3. Time-kill kinetics displayed the inhibition of planktonic growth of all Candida strains treated with different concentration of 3BP (at MIC& 2XMIC) at various time intervals (30, 60, 90, 120,180, 210, 240, 270, 300, 330 & 360 min).

The graphs were plotted by considering (a-c) time versus growth OD and (g-i) time versus CFU. (d-f) Representative plate images showcasing growth pattern of Candida strains during treatment with 3BP at various time interval.

Fig 4. Analyzing cell membrane integrity in all tested Candida strains through PI staining (magnification: ×200, scale bar 50 μm).

a- C. albicans; b- C. tropicalis; c. C. glabrata.

Fig 5. Assessment of intracellular ROS accumulation in Candida cells upon manifestation with 3BP (at 1/2MIC & MIC) using DCFH-DA staining (magnification: ×200, scale bar 50 μm).

a- C. albicans; b- C. tropicalis; c. C. glabrata.

Fig 6. Investigation of nuclear membrane condensation through DAPI staining (magnification: ×200, scale bar 50 μm).

a- C. albicans; b- C. tropicalis; c. C. glabrata.

Fig 7. The influence of 3BP on mature biofilm production of Candida species using crystal violet staining method.

The “* “and “**” symbols represents the statistical significance of p < 0.05 and p < 0.01, respectively.

Fig 8

Assessment of 3BP cytotoxicity (a) Impact of 3BP on human erythrocyte cells. No haemolytic activity was observed. The graph was plotted with absorbance by using SDS (10%) as positive control, it seems to completely lysed the total erthryocytes. (b) Representative image showcasing the non-haemolytic activity of 3BP. (c) The survival graph of G. mellonella manifested with 3BP (at MIC & 2XMIC) for 5 days. (d) The representative image demonstrating the survival of G. mellonella at the end of the experiment (at 5^th day).

Fig 9. In vitro efficacy of 3BP loaded vaginal cream formulation.

Cream containing 2% miconazole served as positive control. (a) Optimization of 3BP various concentrations (0.25, 0.5 & 1%) against C. albicans growth (b) Antifungal efficacy of formulated vaginal creams against C. albicans under in vitro conditions. PC, NC, BP represented in the plates denotes the positive control (miconazole), negative control (cream formulation devoid of active agents), 3BP containing creams, respectively.

Fig 10

(a) In vivo efficacy checking of 3BP cream in VVC mice model. (A) Micrograph image demonstrates the appearance of vaginal tissue analyzed after hematoxylin-eosin staining. (A) infected control group (infection control), (B) non- infected animals (naive control), and (C) non- infected and treated with 3BP cream (toxicity control), (D) infected and treated with miconazole cream (vehicle control), infected and treated with 3BP cream (treatment control). Black colored arrows indicate the loss of membrane integrity upon C. albicans infection. Whereas red colored arrows denote intact vaginal membrane of mice vagina treated with both miconazole and 3BP containing cream. Impaired vaginal epithelium is designated in blue colored box in infected control group. (b) Fungal burden evaluated through CFU counting.