Abstract
The emergence of resistant Candida albicans has made clinical fluconazole (FLC) treatment difficult. Improving sensitivity to FLC is an effective way to treat resistant isolates. Berberine hydrochloride (BBH) is a commonly used traditional Chinese medicine with antimicrobial effects, especially in resistant isolates. We investigated the molecular mechanisms underlying BBH and FLC synergism on biofilm-positive FLC-resistant C. albicans inhibition. Checkerboard microdilution assays and time-kill assays showed a strong synergistic effect between BBH and FLC in resistant C. albicans isolates, causing a significant 32–512-fold reduction in minimum inhibitory concentrations. BBH combined with FLC inhibited intracellular FLC efflux due to key efflux pump gene CDR1 downregulation, whereas FLC alone induced high CDR1 transcription in resistant strains. Further, BBH + FLC inhibited yeast adhesion, morphological hyphae transformation, and biofilm formation by downregulating the hyphal-specific genes ALS3, HWP1, and ECE1. BBH caused cytoplasmic Ca2+ influx, while FLC alone did not induce high intracellular Ca2+ levels. The vacuolar calcium channel gene YVC1 was upregulated, while the vacuolar calcium pump gene PMC1 was downregulated in the BBH + FLC and BBH alone groups. However, vacuolar calcium gene expression after FLC treatment was opposite in biofilm-positive FLC-resistant C. albicans, which might explain why BBH induces Ca2+ influx. These results demonstrate that BBH + FLC exerts synergistic effects to increase FLC sensitivity by regulating multiple targets in FLC-resistant C. albicans. These findings further show that traditional Chinese medicines have multi-target antimicrobial effects that may inhibit drug-resistant strains. This study also found that the vacuolar calcium regulation genes YVC1 and PMC1 are key BBH + FLC targets which increase cytoplasmic Ca2+ in resistant isolates, which might be critical for reversing biofilm-positive FLC-resistant C. albicans.
Highlights
MATERIALS AND METHODSCandida is a common pathogen of nosocomial bloodstream infections, causing high-mortality invasive candidiasis
Previous studies revealed that ergosterol synthesis inhibition and apoptosis induced by endogenous reactive oxygen species (ROS) augmentation contribute to the synergistic effect of berberine plus FLC against C. albicans (Xu et al, 2009; Xu et al, 2017; Yang et al, 2018)
Combined use could increase C. albicans sensitivity to FLC and berberine hydrochloride (BBH), causing decreased FLC minimum inhibitory concentrations (MICs) from ≥512 to 1 μg/mL and reduced BBH MIC from 64 to 2–4 μg/mL. These results demonstrate that the FLC MIC is decreased by 256–512-fold with minute BBH addition
Summary
Candida is a common pathogen of nosocomial bloodstream infections, causing high-mortality invasive candidiasis. Previous studies revealed that ergosterol synthesis inhibition and apoptosis induced by endogenous ROS augmentation contribute to the synergistic effect of berberine plus FLC against C. albicans (Xu et al, 2009; Xu et al, 2017; Yang et al, 2018) This combination could downregulate efflux pump genes CDR1 and CDR2 overexpression (Zhu et al, 2014). Berberine hydrochloride (BBH) combined with FLC was tested to explore the molecular mechanism underlying the synergistic effect on efflux pump activity, biofilm formation, and intracellular calcium homeostasis. To evaluate the combined BBH and FLC effect on resistant C. albicans drug efflux, Rh6G assays were performed as previously described, with some modifications (Xu et al, 2019).
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