Molecular mechanisms of action of herbal antifungal alkaloid berberine, in Candida albicans

Abstract

Candida albicans causes superficial to systemic infections in immuno-compromised individuals. The concomitant use of fungistatic drugs and the lack of cidal drugs frequently result in strains that could withstand commonly used antifungals, and display multidrug resistance (MDR). In search of novel fungicidals, in this study, we have explored a plant alkaloid berberine (BER) for its antifungal potential. For this, we screened an in-house transcription factor (TF) mutant library of C. albicans strains towards their susceptibility to BER. Our screen of TF mutant strains identified a heat shock factor (HSF1), which has a central role in thermal adaptation, to be most responsive to BER treatment. Interestingly, HSF1 mutant was not only highly susceptible to BER but also displayed collateral susceptibility towards drugs targeting cell wall (CW) and ergosterol biosynthesis. Notably, BER treatment alone could affect the CW integrity as was evident from the growth retardation of MAP kinase and calcineurin pathway null mutant strains and transmission electron microscopy. However, unlike BER, HSF1 effect on CW appeared to be independent of MAP kinase and Calcineurin pathway genes. Additionally, unlike hsf1 null strain, BER treatment of Candida cells resulted in dysfunctional mitochondria, which was evident from its slow growth in non-fermentative carbon source and poor labeling with mitochondrial membrane potential sensitive probe. This phenotype was reinforced with an enhanced ROS levels coinciding with the up-regulated oxidative stress genes in BER-treated cells. Together, our study not only describes the molecular mechanism of BER fungicidal activity but also unravels a new role of evolutionary conserved HSF1, in MDR of Candida.

Figure 1. Antifungal potential of BER (a) Growth curve of WT C. albicans cells at 100, 150 and 200 µg/ml, (b) serial dilution assays in solid (left panel) and liquid medium for testing BER susceptibility of C. albicans and non albicans species.

(c) Serial dilution assays of CDR1 (Gu5) and MDR1 (F5) overexpressing and (d) their deletions strains in presence of BER.

Figure 2. TF mutant library screening (a) Serial dilution assays of TF mutant strains in the presence of BER, (b) end point comparative RTPCR of HSF1 (gene deleted in JMR044) in WT strain (DAY286) in presence and absence of BER.

Figure 3. HSF1 conditional mutant is susceptible to various antifungal drugs (a) susceptibility WT, HSF1 conditional mutant and HSF1 heterozygous for BER (b) different classes of antifungal drugs; FLC, CAS, TRB, AMB, and their combination with BER, (c) CW perturbing agents; CFW, CR, SDS (d) TEM images of WT, HSF1 conditional mutant and HSF1 heterozygous in presence of BER.

Figure 4. Effect of BER on CW integrity mutants (a) serial dilution assay of calcineurin and MAP kinase pathway and HSP90 gene deleted to evaluate BER MIC₅₀, (b) end point comparative RTPCR of genes involved in CW integrity in WT C. albicans cells in presence and absence of BER, (c) and in HSF1 conditional mutant lane indicates 1: WT, 2: HSF1 TET/hsf1, 3: HSF1/hsf1, 4,5,6,: +Doxy, 7,8,9 :+BER, 10, 11, 12: +Doxy+Ber.

Figure 5. BER treatment results in dysfunctional mitochondria (a) growth of C. albicans cells in non-fermentable carbon source (glycerol) in presence of BER (b) MTR labeling of the active mitochondria by FACS in C. albicans WT cells in presence and absence of BER, bar graph representing number of events gated (c) MTR labeling were also done in WT, HSF1 conditional mutant and HSF1 heterozygous strains in presence and absence of BER.

Figure 6. Determination of endogenous ROS generation by BER and induction of apoptosis (a) (upper panel) bar graph representing relative fluorescent units when cells were treated with DCFDA in presence and absence of BER, AA is added to revert the ROS production, (lower panel) fluorescent microscopy images of WT C. albicans cells labeled with DCFDA, (b) Cytometric determination FITC Annexin V labeling in WT cells treated with BER.

Figure 7. Model depicting pathways affected by BER treatment in C. albicans.