# Molecular Mechanisms of Action of Herbal Antifungal Alkaloid Berberine, in Candida albicans

Sanjiveeni Dhamgaye, Frédéric Devaux, Patrick Vandeputte, Nitesh Kumar Khandelwal, Dominique Sanglard, Gauranga Mukhopadhyay, Rajendra Prasad

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Sanjiveeni Dhamgaye, Frédéric Devaux, Patrick Vandeputte, Nitesh Kumar Khandelwal, Dominique Sanglard, et al. Molecular Mechanisms of Action of Herbal Antifungal Alkaloid Berberine, in Candida albicans. PLoS ONE, 2014, 9 (8), pp.e104554. 10.1371/journal.pone.0104554. hal-01345117

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# Molecular Mechanisms of Action of Herbal Antifungal Alkaloid Berberine, in *Candida albicans*

Sanjiveeni Dhamgaye¹,², Frédéric Devaux³, Patrick Vandeputte⁴, Nitesh Kumar Khandelwal¹, Dominique Sanglard⁴, Gauranga Mukhopadhyay², Rajendra Prasad¹*

1 School of Life Sciences, Jawaharlal Nehru University, New Delhi, India, 2 Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India, 3 Sorbonne Universités, UPMC Univ Paris 06, UMR 7238, Laboratoire de genomique des microorganisms, Paris, France, 4 Institute of Microbiology, University of Lausanne and University Hospital Center, Lausanne, Switzerland

## 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.

**Citation:** Dhamgaye S, Devaux F, Vandeputte P, Khandelwal NK, Sanglard D, et al. (2014) Molecular Mechanisms of Action of Herbal Antifungal Alkaloid Berberine, in *Candida albicans*. PLoS ONE 9(8): e104554. doi:10.1371/journal.pone.0104554

**Editor:** Neeraj Chauhan, New Jersey Medical School, Rutgers University, United States of America

**Received** April 2, 2014; **Accepted** July 11, 2014; **Published** August 8, 2014

**Copyright:** © 2014 Dhamgaye et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability:** The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

**Funding:** Dept. of Biotechnology, Govt. of India (BT/01/CEIB/10/III/02) and (BT/PR 14879/BRB 10/885/2010, Swiss-Indo collaboration grant ISJRP 122917. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Competing Interests:** The authors have declared that no competing interests exist.

* Email: rp47jnu@gmail.com

## Introduction

*Candida albicans* is a part of normal commensal flora of human body, which becomes one of the most resilient fungal pathogen under low host immunity [1], [2]. Available antifungals predominantly include azoles, echinocandins, polyenes and allylamines [3]. Each of these classes of antifungals have a distinct mode of action, for example azoles target heme protein, cytochrome P450 lanosterol 14 α demethylase thereby impeding conversion of lanosterol to fecosterol and subsequently blocking ergosterol biosynthesis [4]. Echinocandins interfere with CW synthesis by inhibiting β1-3 glucan synthase [5]. Polyenes have affinity to bind membrane sterols that results in the formation of aqueous pores ensuing the leakage of crucial cellular components and subsequent cell death [6]. Allylamines are relatively newer class of antifungal that also inhibit ergosterol biosynthesis but by specifically targeting squalene epoxidase [7].

Notwithstanding the fact that some pathogenic Candida species are intrinsically resistant to antifungals, prolonged use of drugs also leads to an emergence of multidrug resistant (MDR) strains posing additional clinical challenges [8]. Because of the limited arsenal of

antifungals, there is constant need to find new antifungals with novel targets. In this context, herbal antifungals have acquired significance owing to their natural origin [9–11]. Besides herbal compounds there are innumerable non-herbal chemical compounds with antifungal potentials. For example, gentian violet has been used to treat recurrent vulvovaginal candidosis [12]. Idarubicin, a DNA topoisomerase inhibitor is effective against *Aspergillus niger*, *Cryptococcus neoformans* and *C. glabrata* [13], caffeine exhibits antifungal potential by interfering with MAPK signaling and CW integrity pathway [14]. Recently, our group has shown that a bacteriological stain malachite green (MG) exerts antifungal effect by shifting the metabolic flux of Candida cells towards fermentation, increasing generation of ROS and leading to cell necrosis [15].

Isoquinoline alkaloid berberine (BER), which is bright yellow colored, is widely used for dyeing wool, leather and wood. BER is shown to elicit anticancerous, antidiabetic, anti atherosclerotic and neuroprotective properties. Additionally, BER also exhibits antibacterial, and has also shown anti-inflammatory potentials [16], [17]. Although there are instances where BER is shown to
<figure><img src="image_2.png" /><figcaption>**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.
doi:10.1371/journal.pone.0104554.g001</figcaption></figure>

elicit antifungal activity against *C. albicans* and *C. glabrata* [18],
how it exerts its effect is not understood.

In the present study, we attempted to elucidate the antifungal
effect of BER on *C. albicans* and non-*albicans* species. For this, we
screened the transcriptional factor (TF) mutants in house library of
*C. albicans* and identified a mutant of heat shock factor (*HSF1*)
that was hypersusceptible to BER [19]. *HSF1* activates transcrip-
tion via heat shock element (HSE) in response to thermal
adaptation. Our study provides evidence that the treatment of
Candida cells with BER compromises CW integrity via the
calcineurin pathway leading to cell death. The study also assigns a
new role to *HSF1* in combating MDR.

**Material and Methods**

Strains and Media

All the media components were purchased from HiMedia
(Mumbai, India). BER, 2', 7' -dichlorofluorescein diacetate
(DCFDA), and glycerol were purchased from Sigma chemicals
Co. (St. Louis, MO). Ascorbic acid (AA) was purchased from SRL
(Mumbai, India). Ranbaxy, India generously provided FLC.
AMB, CAS, CR, CFW, SDS, TRB were purchased from Sigma
chemicals Co. (St. Louis, MO) Superscript-II Reverse transcriptase
enzyme, oligo dt(18)primer and random primer from Invitrogen,
USA. dATP, dGTP, dTTP and dCTP were bought from Sigma.
All the yeast strains were grown in yeast-extract peptone dextrose
(YPD) agar and incubated at 30°C. All the strains were stored as
15% glycerol stocks in -80°C storage which were freshly revived

on YPD before beginning of the experiment. Details of all the
strains used in the study is described in table S1 in File S1.

Growth curve and serial dilution assays

Freshly revived *C. albicans* strains were inoculated at 0.1 OD₆₀₀ in YPD in four different tubes. BER was added at 100, 150, 200 µg/ml in three of the tubes while no BER was added to the control tube. Cells were allowed to grow at 30°C at 200 rpm. 1 ml of culture is withdrawn from each tube at interval of 4 hours and absorbance is taken at 600 nm. Serial dilution assays were done in both liquid and solid YPD medium for testing the drug susceptibility essentially by following the guidelines provided by CLSI (Clinical and Laboratory Standards Institute) [20],[21]. Concentration of each drug used in the study is provided in parenthesis BER (200 µg/ml, 300 µg/ml depending on the WT strain used), doxycyclin (DOX; 0.6 µg/ml), caspofugin (CAS; .05 µg/ml), amphotericin B (AMB; 4 µg/ml), calcofluor white (CFW; 60 µg/ml), congo red (CR; 150 µg/ml), SDS (0.05%), ascorbic acid (AA; 0.3 µg/ml).

Screening of TF mutant library for BER susceptibility

We have employed an in-house TF mutant library containing 240 homozygous mutant strains individually deleted for genes with DBD (DNA binding domain) by UAU transposition (~70%) or by URA blaster technique or by PCR product recombination PCR [15]. The TF mutants were subjected to the screening for BER susceptibility using serial dilution assays as described above. This was done thrice and the TF mutant strain that was reproducibly susceptible to BER was tested in the final screen.
<figure><img src="image_3.png" /><figcaption>**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.
doi:10.1371/journal.pone.0104554.g002</figcaption></figure>

## End point comparative reverse transcription PCR (RTPCR)

RNA was extracted using RNeasy mini kit Qiagen essentially by following their protocol. RNA is quantitated by NanoDrop 2000 UV-Vis spectrophotometer from Thermo Scientific. 1 µg/ml of DNase treated RNA was taken further for cDNA preparation using RevertAid H Minus Reverse Transcriptase kit from Thermo Scientific using protocol provided by the manufacturer. 1 µl of the cDNA product was used for amplification using Taq DNA polymerase from New England Biolabs in a total reaction volume of 50 µl. Primers used for the amplification of target genes are listed in table S2 in File S1.

## Transmission electron microscopy (TEM)

TEM was performed to examine the effect of BER on the cell wall of *HSF1tet/hsf1Δ* strain in comparison with the WT as described elsewhere [22]. Cells were grown to mid-exponential phase and treated with DOX, BER or BER +DOX. The cells were washed with PBS buffer to remove the media and then cells were chemically fixed using 2% glutaraldehyde at 4°C for 2 hours followed by 0.5% osmium tetroxide. The cells were finally embedded in epon resin. The samples were visualized by TEM microscope by Jeol JEM-2100F, Tokyo Japan.

## BER transport

BER transport inside the cells was measured exploiting its fluorescence. BER has excitation wavelength of 405 nm and emission is at 520 nm [23]. Late stage cells were washed with PBS twice and the cells were resuspended in PBS to 0.1 OD₆₀₀. Cells were incubated for 1 hour and then washed cells were resuspended in PBS with 2% glucose with BER (MIC₅₀ concentration).

Fluorescence detection in cells was carried out with a Fluorescence Activated Cell Sorter (FACS) caliber flow cytometer (Becton-Dickinson Immunocytometry Systems, San Jose, CA) at FL-1. Ten thousand events were counted and fluorescence intensity was compared between untreated cells and BER-treated cells using Cell Quest software.

## Measurement of ROS (Reactive Oxygen Species) generation

Endogenous ROS generation was measured using DCFDA (2',7'-Dichlorofluorescin diacetate) which is an oxidant sensitive fluorescent probe [24]. The method employed is previously described in details [15]. Briefly, wild type cells were inoculated in YPD broth at 0.1 OD₆₀₀ and allowed to incubate at 30°C until the OD₆₀₀ reaches 0.8. BER (MIC₅₀) was added to the cells and same volume of mock in the control then allowed to grow further for 80 min. We used antioxidant AA for reversal of generation of ROS. Cells were pretreated with 5 mM AA 60–90 min and then incubated in presence of BER for 80 mins after washing the cells, 10 µM DCFDA was added. Cells were then kept at 30°C for 30 min. Fluorescence was measured (Excitation and emission at 488 nm and 540 nm respectively) and quantified by FACS as mentioned above.

## Evaluation of induction of apoptosis by BER treatment

Cells undergoing apoptosis was determined by the externalization of PS (phosphatidylserine), an apoptotic marker, by using FITC (Fluorescein isothiocyanate) labeled Annexin V kit (FITC Annexin V Apoptosis Detection Kit I, BD Biosciences). Exponentially growing cells were treated with BER (at MIC₅₀; 200 µg/ml).
<figure><img src="image_4.png" /><figcaption>**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.*
doi:10.1371/journal.pone.0104554.g003</figcaption></figure>

Cells were then treated with zymolyase to obtain spheroplasts, which were incubated with FITC Annexin V and PI (propidium iodide). The fluorescent signal was assessed using a Fluorescence Activated Cell Sorter (FACS) caliber flow cytometer (Becton-Dickinson Immunocytometry Systems, San Jose, CA). We used 488 nm excitation and a 515 nm band pass filter for FITC detection and a filter >560 nm for PI detection. A total of 10,000 events were counted at the flow rate. Data analysis was performed using Cell Quest software. Cells labeled with FITC indicate onset of apoptosis while those with PI label predominantly show the presence of necrotic cells (AnnV/PI) [15].

## Determination of active mitochondria using MitoTracker

Exponentially growing cells were treated with BER (MIC₅₀), for 80 min washed and resuspended in YPD and treated with 200 nM of MitoTracker Deep Red FM (MTR-FM) [25]. The cells were allowed to incubate for 30–45 min at 30°C with 200 rpm. The cells were washed and resuspended in 400 µL of PBS fluorescence is measured by FACS analysis as described above. MTR-FM has excitation and emission spectra of 644 nm and 665 nm respectively.

## Results

### Antifungal potential of BER does not depend on major efflux pump proteins

For an in depth analysis of mechanism of action of antifungal potentials of an herbal alkaloid BER, we have examined the growth pattern of *C. albicans* and observed that BER maximally

retarded cell growth at 200 µg/ml (Figure 1(a)). In serial dilution assays, BER at 200 µg/ml was also effective against all the tested non-*albicans* species with variable susceptibilities (Figure 1(b)). For instance, *C. utilis*, *C. kefyr* and *C. krusei* were relatively hypersusceptible to BER and their MIC₅₀ ranged between 25 µg/ml and >50 µg/ml, while *C. glabrata* and *C. tropicalis* were the least susceptible species with MIC₅₀ of 200 µg/ml in contrast to *C. albicans*. Notably, BER has reported to be non-toxic to human cells [26].

One of the predominant mechanisms to cope up with the antifungal stress is an overexpression of drug efflux pump encoding genes such as *CDR1* and *MDR1* belonging to the ABC (ATP Binding Cassette) and MFS (Major Facilitators) super families of transporters, respectively [27]. We assessed the implication of these pump proteins in withstanding BER stress. For this, we used azole-susceptible (AS) and azole-resistant (AR) clinical isolates overexpressing these pump encoding genes [28], [29], and examined the effect of BER. As depicted in Figure 1(c) and 1(d), the susceptibility to BER in *CDR1* or *MDR1* overexpressing strains was only modestly affected as compared with susceptible strains. The possibility that the observed modest increase in susceptibility of clinical resistant isolates towards tested drugs might arise due to acquired mutations in other genes, that can also not be ruled out. To check the specific roles of these transporters, we have used strains that were individually disrupted for these genes but the susceptibility in these nulls (DSY449 and SFluM4A) remained unaffected in comparison to the respective WT strains. Together, these results imply that efflux pumps do not impact BER activity (Figure 1 (c) and 1(d)).
<figure><img src="image_5.png" /><figcaption>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.</figcaption></figure>

doi:10.1371/journal.pone.0104554.g004

HSF1 levels affect susceptibility to BER and collateral
susceptibilities to other drugs

To understand the mechanism behind susceptibility to BER, we
carried out a screen of an in-house library of TF mutant strains of
*C. albicans* [15]. There are ~275 genes that contains DBD in *C.
albicans* these genes were deleted individually as explained in
materials and methods section. All the strains that appear
reproducibly (in triplicate) susceptible were subjected to a final
screen as shown in Figure 2(a). Among all the TFs, *HSF1*, a heat
shock factor mutant (JMR044) was most susceptible to BER
treatment (MIC₅₀ 25 µg/ml) in comparison with WT strain
(DAY286) (MIC₅₀ 100 µg/ml) (figure S1 in File S1). End point
comparative RTPCR revealed a slight increase in the expression
of *HSF1* after BER treatment in *C. albicans*, thus implying a
direct effect of BER on its expression (Figure 2(b)).

HSF1 is an essential gene and it could be likely that this TF mutant that was generated by UAU transposition could still exhibit HSF1 activity due to the chosen disruption method [19], [30]. To reconfirm HSF1 impact on MDR, we employed a conditional HSF1 mutant (hsf1Δ/tet-HSF1) in which HSF1 expression is under control of the TET promoter [19]. We tested the susceptibility of the HSF1 conditional mutant in presence of TET analogue, DOX, which shuts off the expression of HSF1. The results mirrored the BER susceptibility phenotype that was observed with the mutant strain JMR044 of TF library screen (Figure 3(a)). We also verified whether conditional mutant hsf1Δ/ tet-HSF1 was collaterally affecting susceptibility to other drugs. FLC, AMB, TRB, and CAS (caspofungin). Additionally, we have also evaluated the effect of combination of BER and these drugs on conditional mutant hsf1Δ/tet-HSF1. Interestingly, the conditional HSF1 mutant was highly susceptible towards different classes of antifungal drugs including FLC, AMB, TRB, and CAS (caspofungin) (Figure 3(b)). Our susceptibility assays revealed that hsf1Δ/tet-HSF1 not only displayed enhanced susceptibility to BER but also to drugs acting on membrane ergosterol (TRB, FLC, AMB) or inhibit CW synthesis (CAS) (Figure 3(b)) [31], [32]. However, combination of BER and above mentioned drugs did

not seem to enhance the susceptibility of *HSF1* conditional
mutant (Figure 3(b)). This highlights that the effects of BER and
*HSF1* leading to drug susceptibility are manifested by indepen-
dent circuitry. The impact of *HSF1* on drug susceptibilities
appeared to be independent of known attributes of MDR [33],
[34]. For example, the expression of *CDR1*, *CDR2*, *MDR1*,
*ERG11*, *TAC1*, *UPC2* did not change in the *HSF1* conditional
mutant in presence and absence of BER (figure S2 in File S1).

Susceptibility of *hsf1Δ/tet-HSF1* towards ergosterol and CW synthesis inhibitors led us to evaluate specific effect of CW perturbing agents such as SDS (sodium dodecyl sulphate), CR (congo red) and CFW (calcofluor white) [35] on this mutant. We performed serial dilution assays in the presence of these CW perturbing agents. As evident from Figure 3(c), *hsf1Δ/tet-HSF1* was susceptible to SDS (0.05%), CR (150 µg/ml) and CFW (60 µg/ml). The impact of *HSF1* on CW was reinforced by TEM of *hsf1Δ/tet-HSF1* strains. One could visualize in TEM pictures, the damage to the CW of *hsf1Δ/tet-HSF1* strains. When *hsf1Δ/tet-HSF1* strains were treated with BER, the impact appeared to be more aggravated as was evident from the rupturing of the CW and shrinkage of the cytoplasm of *hsf1Δ/HSF1* cells after BER treatment (Figure 3(d)). Interestingly, the damage to CW as revealed by TEM picture analyses following BER treatment was not as severe as compared to untreated *hsf1Δ/HSF1* cells (Figure 3(d)).

BER affects CW integrity independent of HSF1

Taking clue from *hsf1Δ/tet-HSF1*, which was susceptible to CW
inhibitors, we explored whether the genes involved in the CW
integrity pathway were affected in BER-treated cells [36]. By
employing liquid assay according to the CLSI protocol, we
compared BER MICs in mutant strains for the genes involved in
heat shock (*HSP90*), calcineurin (*CNA1*, *CNA2*, *CRZ1*), and
MAP kinase pathways (*BCK1*, *MKC1*). As evident from the assay,
calcineurin (*crz1Δ/crz1Δ*, *cna1Δ/cna1Δ*) and MAP kinase path-
ways (*bck1Δ/bck1Δ*) mutants were more susceptible to BER
treatment with MIC₅₀ of 25 µg/ml as compared to wild type
<figure><img src="image_6.png" /><figcaption>**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.</figcaption></figure>

doi:10.1371/journal.pone.0104554.g005

(100 µg/ml) (Figure 4(a)). End point comparative RTPCR confirmed that the expression of genes involved in these pathways (including *CRZ1*, *CNA1* and *BCK1*) was increased upon BER treatment (Figure 4(b)). Notably, depletion of *HSF1* levels did not arrest the increased expression of these genes upon BER treatment (Figure 4(c)) in combination of DOX indicating BER effect on these genes is independent of *HSF1*.

## BER treatment results in mitochondrial dysfunction

To assess functional mitochondria, we compared the growth of BER treated cells in non-fermentative and in fermentative carbon sources and compared it with the growth of untreated cells. We observed that, even at non-lethal BER concentration (below MIC₅₀; 75 µg/ml), cells were unable to grow when non-fermentable glycerol was provided as sole carbon source (Figure 5(a)). We addressed the mitochondrial activity by employing MTR-FM probe [25]. MTR-FM probe is known to passively diffuse across yeast cell membrane and to accumulate in active

mitochondria, which can be visualized by enhanced fluorescence. Using flow cytometry depicted in Figure 5(b) BER-treated cells showed poor fluorescence intensity as compared to untreated cells. Together, poor growth in non-fermentable carbon source and decrease in MTR-FM fluorescence reinforced dysfunctional mitochondria following BER treatment. Notably, *HSF1* conditional mutant did not show any impact on mitochondrial functional status (Figure 5 (c))

## Determination of generation of ROS and apoptosis on BER treatment

Mitochondrial dysfunction on BER treatment led us to determine the status of ROS in presence of BER. To measure the changes in ROS generation we exploited a fluorescent molecule chloromethyl-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA), which can readily enter into the cells and is sensitive to redox changes [24]. It is deacetylated by endogenous esterase into dichlorofluorescein, which fluoresces on oxidation by
<figure><img src="image_7.png" /><figcaption>**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.
doi:10.1371/journal.pone.0104554.g006</figcaption></figure>

ROS. Wild type cells treated with a MIC₅₀ concentration of BER (100 µg/ml) were allowed to incubate in presence of 10 µM of CM-H₂DCFDA. As shown in Figure 6, fluorescence increased after BER treatment indicating generation ROS. The increase in ROS following BER treatment could be partially reversed by the addition of antioxidant such as AA [37]. This implied that BER

treatment results in production of ROS, which could contribute to
the antifungal effect exerted by BER.

To estimate whether generation of ROS leads to apoptosis on
BER treatment, we checked for externalization of phosphatidyl
serine (PS), a marker for apoptotic cells. Annexin V (Annexin V
Apoptosis Detection Kit I, BD Biosciences) binds to PS which is in
turn linked to FITC hence can be detected through Flow

<figure><img src="image_8.png" /><figcaption>**Figure 7.** Model depicting pathways affected by BER treatment in *C. albicans*.
doi:10.1371/journal.pone.0104554.g007</figcaption></figure>
cytometry as explained in material and methods. However, there
was no significant change in the population of cells showing FITC
labeling on treatment with BER at MIC₈₀ as seen in Figure 6(b).
For example, our data show that the percentage of gated cells
labeled with Annexin V-FITC did not change significantly as it
ranged between 0.67% in WT strain to 0.86% in cells treated with
BER. This excluded the possibility that BER at MIC₈₀ does not
trigger apoptosis in *C. albicans* cells.

Discussion

In this study, we show that the plant alkaloid BER displays
potential anti-Candida activity which has been earlier reported to
be non-toxic to humans [26] Our data supports that *HSF1*, a
transcription regulator that is an essential gene involved in thermal
adaptation of Candida cells, impacts BER susceptibility of *C. albicans* cells. *HSF1* protects cells from thermal assault by
activating the expression of HSPs (Heat Shock Proteins) that act
as chaperones to rescue proteins from misfolding under duress
[19]. Our observation suggests that *HSF1*, which not only impacts
BER susceptibility, also influences susceptibility of Candida cells to
different drugs with distinct targets. Thus *HSF1* mutant is also
observed to be collaterally susceptible to other drugs (Figure 3(b)).
However, the impact of BER and *HSF1* on drug susceptibility is
manifested by independent pathways in Candida cells. Our
conclusions are based upon the following supporting observations.
The treatment of TF mutant strains of Candida cells with BER
revealed that *HSF1* mutant was most susceptible to this antifungal
compound. BER treatment affects the calcineurin pathway and
CW integrity leading to dysfunctional mitochondria and cell
death. *HSF1* levels also impact CW integrity, which is apparent
from the susceptibility of *HSF1* conditional mutants towards CW
inhibitors and from disrupted CW visualized under TEM images.
However, unlike BER treatment, the impact of *HSF1* on CW
integrity was independent of calcineurin stress pathway. For
example, neither *HSF1* levels could affect the expression of
calcineurin pathway genes nor observed dysfunctional mitochon-
dria and enhanced ROS levels following BER treatment were
associated with *HSF1* phenotype. Although, *HSF1* levels poten-
tiate the antifungal activity of BER but its influence in enhancing
drug susceptibility adopts different regulatory circuitry as shown in
Figure 7. Together, our results show that BER displays its
antifungal potential through its ability to impair mitochondrial
function, generation of ROS, targeting CW integrity pathway and
also affecting heat shock transcription factor *HSF1*.

BER has been touted as potential anticancerous agent as it
generates ROS and induces apoptosis in human prostrate cancer
cells [38], however, our Annexin V labeling data rules out any
induction of apoptosis in *C. albicans* following BER treatment
(Figure 6(b)). Implication of BER in dysfunction of mitochondria
in HSC-3 oral cancer cells has also been studied [39]. Another
report shows that it is accumulated inside mitochondria, which
results in the arrest of cell proliferation, mitochondrial fragmen-
tation, along with depolarization and oxidative stress in K1735-
M2 mouse melanoma cells [40]. Considering evidences from other
systems, the possibility of other pathways, which may also govern
BER susceptibility, could not be excluded.

Several major mechanisms of MDR have been identified.
However, recent studies reveal newer regulatory pathways that
govern the development of MDR in Candida cells. For instance,
the 'two component system' composed of a membrane-bound,
sensor histidine kinase (HK) protein, a cytoplasmic response
regulator (RR) and an intermediate histidine phosphotransfer
(HPt) protein. The two-component signal transduction pathways

which help to adapt yeast cells to environmental stress is also
shown to impact drug susceptibility of Candida cells [41]. The
depletion of cellular iron pools leads to decrease resistance to
several drugs involving ergosterol metabolism and calcineurin
dependent core stress response pathways [42]. The role of
mitochondria in MDR of *C. glabrata* is well documented [43].
Recently, electron transport defective null mutants of *goal1* and
*ndh51* of *C. albicans* were shown to display increased susceptibility
towards FLC [44]. Overwhelming emerging evidences also suggest
an intricate relationship between membrane lipids and MDR
wherein any compositional imbalance considerably influences
drug susceptibility of Candida cells [45], [46]. Similarly, *CZF1*, a
TF hitherto known to be responsible for white/opaque switching,
was identified from whole genome RNA-Seq as another mediator
the acquisition of MDR [47].

In the perspective of uncovering unconventional drugs that possess antifungal property, we had earlier shown that a chemical dye malachite green (MG), which is commonly used in fish industry as an antifungal, antiparasitic and antibacterial agent, could effectively kill *C. albicans* and non-albicans species [15]. MG owes its antifungal effect by shifting the metabolic flux towards fermentation, ROS generation leading to cell necrosis and is independent of known MDR mechanisms [15]. UPC2 and STP2 aid in tolerating MG stress of Candida cells [15]. In another study, natural polyphenol, curcumin (CUR) was shown to be cidal to *C. albicans* as well as to non-*albicans* species and display synergism when combined with known antifungals. CUR treatment induces ROS generation and an early apoptosis in *C. albicans* cells [11].

Together, the fungicidal effects of natural and synthetic
compounds not only provide a platform to improve therapeutic
strategies but also unravel novel regulatory circuitry that impacts
MDR in Candida. This also indicates the flexibility of *C. albicans*
cells to tolerate stress due to a variety of drugs with different targets
by adopting different regulatory routes. Our results highlight that
the development of tolerance towards antifungal drugs is a
multifactorial phenomena, and the combat against MDR still
remains a challenge.

**Supporting Information**

**File S1 Supporting Information.** Figure S1 in File S1. Serial dilution assay showing MIC₅₀ concentration for WT (BWP17) and *HSF1* mutant JMR044. Figure S2 in File S1. End point comparative RTPCR of genes involved in MDR in presence and absence of BER lane indicates 1: WT, 2: HSF1 TET/hsf1, 3: HSF1/hsf1, 4,5,6,: +Doxy, 7,8,9 :+BER, 10, 11, 12: +Doxy+Ber. Table S1 in File S1. List of strains used in the study. Table S2 in File S1. List of primers used in the study. (DOC)

Acknowledgments

SD likes to acknowledge UGC Dr. D.S. Kothari Post-doctoral fellowship, Advanced Instrument Research Facility, Jawaharlal Nehru University, New Delhi, India. SD is thankful to Council of Scientific and Industrial Research, Government of India, for awarding Senior Research Fellowship. We are thankful to Prof. Alistair Brown (Aberdeen Fungal Group, University of Aberdeen) for providing *HSF1* conditional mutant, heterozygote and WT strains.

Author Contributions

Conceived and designed the experiments: RP FD SD DS. Performed the
experiments: SD PV NKK. Analyzed the data: SD GM RP FD.
Contributed reagents/materials/analysis tools: RP DS FD. Contributed
to the writing of the manuscript: SD RP FD GM.
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