Mechanism and bioinformatics analysis of the effect of berberine-enhanced fluconazole against drug-resistant Candida albicans

Abstract

Biofilms produced by Candida albicans present a challenge in treatment with antifungal drug. Enhancing the sensitivity to fluconazole (FLC) is a reasonable method for treating FLC-resistant species. Moreover, several lines of evidence have demonstrated that berberine (BBR) can have antimicrobial effects. The aim of this study was to clarify the underlying mechanism of these effects. We conducted a comparative study of the inhibition of FLC-resistant strain growth by FLC treatment alone, BBR treatment alone, and the synergistic effect of combined FLC and BBR treatment. Twenty-four isolated strains showed distinct biofilm formation capabilities. The antifungal effect of combined FLC and BBR treatment in terms of the growth and biofilm formation of Candida albicans species was determined via checkerboard, time-kill, and fluorescence microscopy assays. The synergistic effect of BBR and FLC downregulated the expression of the efflux pump genes CDR1 and MDR, the hyphal gene HWP1, and the adhesion gene ALS3; however, the gene expression of the transcriptional repressor TUP1 was upregulated following treatment with this drug combination. Furthermore, the addition of BBR led to a marked reduction in cell surface hydrophobicity. To identify resistance-related genes and virulence factors through genome-wide sequencing analysis, we investigated the inhibition of related resistance gene expression by the combination of BBR and FLC, as well as the associated signaling pathways and metabolic pathways. The KEGG metabolic map showed that the metabolic genes in this strain are mainly involved in amino acid and carbon metabolism. The metabolic pathway map showed that several ergosterol (ERG) genes were involved in the synthesis of cell membrane sterols, which may be related to drug resistance. In this study, BBR + FLC combination treatment upregulated the expression of the ERG1, ERG3, ERG4, ERG5, ERG24, and ERG25 genes and downregulated the expression of the ERG6 and ERG9 genes compared with fluconazole treatment alone (p < 0.05).

Keywords: Candida albicans; Berberine; Biofilm; Ergosterol; Fluconazole.

Fig. 1

Molecular structure of BBR

Fig. 2

Strains with distinct biofilm formation capacities. C. albicans biofilms were isolated and cultured in 96-well plates for 48 h to produce a mature biofilm. The morphology of the biofilm was assessed via crystal violet staining and observed under a microscope. (a) A characteristic image of C. albicans with low biofilm formation (LBF). (b) A characteristic image of C. albicans with intermediate biofilm formation (IBF). (c) A characteristic image of strains with high biofilm formation (HBF). (d) Twenty-four strains of clinical Candida albicans strains were classified as LBF, IBF, or HBF strains

Fig. 3

MH-HB agar plate assay evaluating the activity of FLC against the SC5314 reference strain and C. albicans isolates (resistant strains) with the agar plate containing 25 µg FLC. (a), (b) Images of the strains were taken after 24 h of incubation at 37 ℃. The diameter of the inhibition zone of the SC5314 reference strain was significantly larger than that of the C. albicans isolates

Fig. 4

Concentration growth curves of C. albicans with different concentrations of FLC and drug combinations; these results demonstrated the antifungal effects of different concentrations

Fig. 5

A combination of FLC and BBR inhibited the growth of the SC5314 reference strain. (a) untreated group, (b) FLC 16 µg/ml treated group, (c) BBR 4 µg/ml treated group, and (d) combination of 16 µg/ml FLC and 4 µg/ml BBR. The strains were incubated at 37℃ for 6 h

Fig. 6

A combination of FLC and BBR inhibited the growth of C. albicans isolates. (a) untreated group, (b) FLC 16 µg/ml treated group, (c) BBR 4 µg/ml treated group, and (d) combination of 16 µg/ml FLC and 4 µg/ml BBR. The samples were incubated at 37℃ for 6 h

Fig. 7

Time-kill curves of C. albicans isolates with the different treatments—control, FLC alone, BBR alone, and combination—using inoculums of 5 × 10⁴ CFU/ml. Aliquots were acquired at serial time points, and colonies were counted on agar plates after 48 h of incubation

Fig. 8

C. albicans isolates were cultured with 16 µg/ml FLC and 4 µg/ml BBR for 36 h. The inhibition of C. albicans growth was evaluated by fluorescence microscopy. BBR significantly increased FLC-induced inhibition of the growth of biofilm-producing C. albicans isolates after 36 h of incubation, and a combination of 16 µg/mL FLC and 4 µg/mL BBR dramatically inhibited cell growth and biofilm formation compared with the control treatment (p < 0.05)

Fig. 9

Comparison of cell hydrophobicity in the different treatment groups. The agents synergistically decreased the CSH of C. albicans isolates. C. albicans isolates were cultured in the presence of 32 µg/ml FLC or 8 µg/ml BBR alone or a combination of BBR and FLC. The BBR and FLC combination led to a significant decrease in the CSH of the C. albicans isolates, although BBR and FLC alone also moderately reduced the CSH when compared to the untreated control, BBR alone, and FLC alone groups (p < 0.05)

Fig. 10

The mRNA expression levels of CDR1, MDR, ALS3, ERG11, TUP1, and HWP1 in the different drug treatment groups were determined via qRT‒PCR, *p < 0.05

Fig. 11

Significantly enriched gene ontology categories with the classification of differentially expressed genes. The results are summarized in the following three categories: molecular function, cellular component, and biological process

Fig. 12

KEGG pathway enrichment analysis results in C. albicans isolates; multiple metabolic pathways were highly enriched. The X-axis indicates the name of the pathway; the Y-axis denotes the number of genes

Fig. 13

A phylogenomic tree was constructed based on selected proteins. Phylogenetic relationships among 13 species are shown. The numbers indicate the branch lengths

Fig. 14

The yellow box shows forward genome chaining; the blue box shows reverse genome chaining. The width of the fill color in the box indicates the similarity of the alignment, with a full fill indicating a 100% similarity

Fig. 15

The steroid biosynthesis and transformation pathway proceeds from terpenoid backbone biosynthesis to the end products in C. albicans. The pathway shows the common ergosterol synthesis process with enzymes and key genes. The metabolites in red indicate the genes that had upregulated expression in the steroid biosynthesis pathway

Fig. 16

Gene expression in C. albicans after BBR and FLC treatment. C. albicans isolates were treated with 32 µg/ml FLC and 8 µg/ml BBR, 32 µg/ml FLC alone, or 8 µg/ml BBR alone; the control group was not treated. Three independent experiments were performed, with 8 replicates in each group