Celastrol boosts fluconazole efficacy against vaginal candidiasis: in vitro and in vivo evidence

Abstract

Candida albicans is a commensal fungus that naturally inhabits the vagina. However, overgrowth of C. albicans can result in vulvovaginal candidiasis (VVC), one of the most prevalent fungal infections affecting women. The rapid emergence of azole resistance in C. albicans, in addition to the limited available antifungal agents, complicates the treatment and emphasizes the urgent need for novel therapeutic options. Efflux-mediated azole resistance is a common resistance mechanism in fluconazole (FLZ)-resistant C. albicans. Combination therapy using natural compounds is a potential approach that can restore fluconazole's antifungal activity in azole-resistant isolates via efflux pump inhibition. This study aimed to evaluate the ability of celastrol, a natural triterpene, to retrieve FLZ antifungal activity against azole-resistant C. albicans in vitro and in vivo. Celastrol did not exhibit antifungal activity against the tested clinical isolates; however, the sub-MIC of celastrol inhibited rhodamine 6G (R6G) efflux and increased R6G accumulation inside celastrol-treated C. albicans cells. Synergy was spotted between celastrol and FLZ via a checkerboard assay. Quantification of m-RNA levels of efflux-mediated azole resistance genes within azole-resistant C. albicans demonstrated CDR1 overexpression. Upon celastrol treatment, a significant decline in ABC transporters transcript levels were detected. Moreover, molecular docking demonstrated that celastrol is a potential ABC efflux transporters blocker that successfully fits into target binding pockets. A negligible hemolytic effect of celastrol against human erythrocytes was observed. In the in vivo model of VVC, the combination of FLZ and celastrol in vaginal gel revealed a drastic reduction in the fungal burden with apparently normal vaginal tissue. Celastrol promising in vitro and in vivo findings strengthen its future use for the treatment of azole-resistant C. albicans.

Keywords: Candida albicans; Azole-resistance; Celastrol; Efflux pumps; Vaginal candidiasis.

Fig. 1

The activity of ABC efflux pump. A Viable count of celastrol-treated and untreated C. albicans cells indicated no significant difference in CFU/mL confirming non-fungicidal activity of celastrol at sub-MIC (500 µg/mL). B The R6G efflux was quantified in the presence or absence of celastrol at sub-MIC by measuring the supernatant absorbance at an excitation wavelength of 529 nm and an emission wavelength of 553 nm. Furthermore, R6G intracellular accumulation was assayed, C Fluorescence microscopic images of R6G stained azole-resistant C. albicans under treatment with celastrol. D Fluorescence intensity measured by Image J software and corrected total cell fluorescence (CTCF) was calculated

Fig. 2

A Celastrol enhanced fluconazole activity in azole-resistant C. albicans isolates as revealed by heatmap, the color bar indicates relative OD growth. B Celastrol had negligible in vitro hemolytic activity. Cytotoxic activity of celastrol was tested against human RBCs, Triton X-100 served as a positive control

Fig. 3

A Fold change in expression levels of CDR1, CDR2 and MDR1 in azole-resistant C. albicans clinical isolate (CA29). Expression levels of each gene were calculated using the 2^−ΔΔCt method using ACT1 as a housekeeping gene and azole-sensitive C. albicans as a calibrator. Grey and Patterned bars represent azole mediated efflux pump genes in calibrator and CA29, respectively. B Downregulation of efflux genes CDR1, CDR2 and MDR1 by celastrol. Celastrol remarkably reduced the expression of the three efflux genes. P values were calculated using one-way ANOVA test

Fig. 4

The interactions of celastrol with the crystal structure of; CDR1-AF-Q5ANA3-F1-model_v1 represented by 2D A and 3D structure B, CDR2-AF-P78595-F1-model_v4 represented by 2D (C) and 3D structure (D), MDR1-AF-Q5ABU7-F1-model_v1 represented by 2D (E) and 3D structure (F)

Fig. 4

The interactions of celastrol with the crystal structure of; CDR1-AF-Q5ANA3-F1-model_v1 represented by 2D A and 3D structure B, CDR2-AF-P78595-F1-model_v4 represented by 2D (C) and 3D structure (D), MDR1-AF-Q5ABU7-F1-model_v1 represented by 2D (E) and 3D structure (F)

Fig. 5

Celastrol decreased the vaginal fungal burden and improved FLZ in vivo activity. A Graphical illustration of vulvovaginal candidiasis model. B Vulvovaginal images in mice model (Upper panel). Fungal cultures of mice vaginal lavage (Lower panel). C Colony count of C. albicans in vaginal lavage fluid of mice after vaginal gel application. The amount of C. albicans in VVC was quantified by using a viable count technique and expressed as CFU/mL. Histopathological examination of vaginal tissue; D 2 days post infection with C. albicans showing presence of hyphae contact to the mucosal surface (arrowhead) with congestion of submucosal blood vessels (thick arrow) and perivascular inflammatory cellular infiltration (thin arrow), E Mice infected with C. albicans and treated with vaginal gel containing combination of FLZ and celastrol showing absence of both hyphae and endocytosed hyphae with apparently normal mucosa and submucosa, F Control-untreated mice post infection with C. albicans showing presence of hyphae in contact to the mucosal surface (arrow head) with presence of few endocytosed hyphae in the mucosa (arrows) and hyperkeratosis of stratified epithelium was detected, G Mice infected with C. albicans and treated with FLZ containing gel showing presence of hyphae contact to the mucosal surface (arrows head) with cystic dilation of some glands (arrows), H Mice infected with C. albicans and treated with celastrol containing vaginal gel showing diffuse severe congestion of submucosal blood vessels (arrows head), I Mice applying gel only (vehicle-control) showing apparently normal mucosal and submucosal tissue with no evidence to fungal infection. The vaginal tissue was stained with hematoxylin and eosin stain (scale bar = 100 μm)

Fig. 5

Celastrol decreased the vaginal fungal burden and improved FLZ in vivo activity. A Graphical illustration of vulvovaginal candidiasis model. B Vulvovaginal images in mice model (Upper panel). Fungal cultures of mice vaginal lavage (Lower panel). C Colony count of C. albicans in vaginal lavage fluid of mice after vaginal gel application. The amount of C. albicans in VVC was quantified by using a viable count technique and expressed as CFU/mL. Histopathological examination of vaginal tissue; D 2 days post infection with C. albicans showing presence of hyphae contact to the mucosal surface (arrowhead) with congestion of submucosal blood vessels (thick arrow) and perivascular inflammatory cellular infiltration (thin arrow), E Mice infected with C. albicans and treated with vaginal gel containing combination of FLZ and celastrol showing absence of both hyphae and endocytosed hyphae with apparently normal mucosa and submucosa, F Control-untreated mice post infection with C. albicans showing presence of hyphae in contact to the mucosal surface (arrow head) with presence of few endocytosed hyphae in the mucosa (arrows) and hyperkeratosis of stratified epithelium was detected, G Mice infected with C. albicans and treated with FLZ containing gel showing presence of hyphae contact to the mucosal surface (arrows head) with cystic dilation of some glands (arrows), H Mice infected with C. albicans and treated with celastrol containing vaginal gel showing diffuse severe congestion of submucosal blood vessels (arrows head), I Mice applying gel only (vehicle-control) showing apparently normal mucosal and submucosal tissue with no evidence to fungal infection. The vaginal tissue was stained with hematoxylin and eosin stain (scale bar = 100 μm)