mechanism of the synergistic effect of amiodarone and … · in vivo studies were carried out in a...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2010, p. 1753–1761 Vol. 54, No. 50066-4804/10/$12.00 doi:10.1128/AAC.01728-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Mechanism of the Synergistic Effect of Amiodarone and Fluconazolein Candida albicans�†

Soledad Gamarra,1 Elousa Maria F. Rocha,1 Yong-Qiang Zhang,2 Steven Park,1Rajini Rao,2 and David S. Perlin1*

Public Health Research Institute, New Jersey Medical School-UMDNJ, Newark, New Jersey 07103-3535,1 andThe Johns Hopkins University School of Medicine, Baltimore, Maryland 212052

Received 8 December 2009/Returned for modification 29 January 2010/Accepted 20 February 2010

The antiarrhythmic drug amiodarone has been found to have fungicidal activity. In Saccharomyces cerevisiae,its antifungal activity is mediated by calcium overload stress, which leads to a rapid nuclear accumulation ofthe calcineurin-regulated transcription factor CRZ1. In addition, low doses of amiodarone have been reportedto be synergistic with fluconazole in fluconazole-resistant Candida albicans. To establish its mechanism oftoxicity in C. albicans, we used expression profiling of key pathway genes to examine cellular responses toamiodarone alone and in combination with fluconazole. Gene expression profiling of 59 genes was done in fiveC. albicans strains (three fluconazole-susceptible strains and two fluconazole-resistant strains) after amioda-rone and/or fluconazole exposure. Of the 59 genes, 27 analyzed showed a significant change (>2-fold) inexpression levels after amiodarone exposure. The up- or downregulated genes included genes involved in Ca2�

homeostasis, cell wall synthesis, vacuolar/lysosomal transport, diverse pathway regulation, stress response,and pseudohyphal morphogenesis. As expected, fluconazole induces an increase in ergosterol pathway genesexpression levels. The combination treatment significantly dampened the transcriptional response to eitherdrug, suggesting that synergism was due to an inhibition of compensatory response pathways. This dampeningresulted in a decrease in total ergosterol levels and decreased pseudohyphal formation, a finding consistentwith decreased virulence in a murine candidiasis model.

Candida albicans is the most frequently observed opportu-nistic human fungal pathogen causing mucosal and systemicinfections in individuals with compromised immune defenses(44). Antifungal therapy is limited by the paucity of chemicalclasses, toxicity, drug resistance, moderate response rates, andsubstantial interpatient variation in serum drug levels. Thus,candidiasis remains a challenging opportunistic infection withhigh mortality, despite current available treatment. There is apressing need for alternative treatments with new drug classesrepresenting novel drug targets. One promising new antifungalclass is represented by amiodarone (AMD), a drug now inclinical use as an antiarrhythmic. AMD has shown fungicidalactivity against yeasts and a range of clinically important fungi,including C. albicans, Cryptococcus neoformans, Fusariumoxysporum, and Aspergillus nidulans (9, 53). In addition, lowdoses of AMD have been reported to be synergistic with dif-ferent azoles in itraconazole-resistant A. fumigatus strains (1)and also in the protozoans Trypanosoma cruzi (4) and Leish-mania mexicana (49).

In Saccharomyces cerevisiae, it is known that AMD affectscalcium homeostasis (10), leading to an immediate influx ofCa2� and a rapid activation of the calcineurin pathway, includ-ing nuclear accumulation of the calcineurin-regulated Crz1p.Transcriptional profiling also revealed an apparent disruption

of nutrient sensing/signaling within minutes based on the up-regulation of genes involved in utilization of alternative carbonand nitrogen sources and in mobilizing energy reserves in aCa2�-independent fashion (53). Genes involved in all stages ofthe cell cycle were downregulated by AMD, and a prominentcalcineurin-dependent delay in G2/M transition was observed(18, 53). Collectively, these data suggest that AMD influencesa set of cellular pathways distinct from existing antifungaldrugs.

Less is known about the mechanism of action of AMD in C.albicans. As a first approximation, it can be inferred that AMDacts in the same way as in S. cerevisae, inducing calcium stressand modifying the calcineurin pathway regulation altering thetolerance to antifungal agents, cation homeostasis, and viru-lence (47). However, the direct and indirect components of thecalcineurin pathway still remain to be elucidated in this yeastspecies. Moreover, the mechanism of regulation differs be-tween fungal species (21), which makes it critical to evaluateAMD action in pathogenic fungi.

Recently, it was observed that synergy occurred with thecombination fluconazole (FLC) and AMD against FLC-resis-tant C. albicans (17). This unexpected result raised the possi-bility of a novel pathway, perhaps influenced by changes inmembrane composition, which contributes to the observed syn-ergy. To evaluate the underlying basis of AMD toxicity in C.albicans, expression profiling of key pathway genes, some iden-tified previously in S. cerevisiae, was used to examine cellularresponses to AMD and AMD-FLC treatment. These observa-tions were confirmed and extended by analysis of total ergos-terol content, in vitro hyphal growth in liquid media and amurine candidiasis model.

* Corresponding author. Mailing address: Public Health ResearchInstitute, New Jersey Medical School-UMDNJ, Newark, NJ 07103-3535. Phone: (973) 854-3200. Fax: (973) 854-3101. E-mail: [email protected].

† Supplemental material for this article may be found at http://aac.asm.org/.

� Published ahead of print on 1 March 2010.

1753

MATERIALS AND METHODS

Strains and compounds. Seven C. albicans strains were used throughout thepresent study, including five FLC-susceptible strains (clinical strains 1002 and610 and control strains ATCC 36082, ATCC 90028, and SC5314), and twoFLC-resistant strains (Table 1). Strain 3795 is an FLC-resistant isolate harboringtwo ERG11 mutations (T376C and A428G) and is isogenic with the FLC-susceptible strain 1002 (42). The other FLC-resistant strain (strain 611) is iso-genic with the FLC-susceptible strain 610 and showed CDR1 overexpression(kindly provided by Richard D. Cannon) (20). Control strains ATCC 90028 andSC5314 were used only for MIC and synergism evaluation, ergosterol quantifi-cation, and filamentous forms inhibition studies. Antifungal agents utilized wereFLC (Pfizer, New York, NY) and AMD (Sigma-Aldrich, St. Louis, MO). AMDwas dissolved in 100% dimethyl sulfoxide (DMSO; Sigma).

Antifungal susceptibility testing and drug interaction evaluation. AMD activ-ity varies depending on the pH (37). Thus, the working pH was established inorder to obtain the biggest AMD effect. Individual MICs and minimum fungi-cidal concentrations (MFCs) were obtained for all of the strains according to theCLSI document M27-A3 (6) using RPMI 1640 medium buffered with morpho-linepropanesulfonic acid at different pH (ranging from pH 4 to 8) (33, 53).AMD-FLC in vitro drug interactions were evaluated with a two-dimensional,two-agent broth microdilution checkerboard technique using the fractional in-hibitory concentration (�FIC) (1, 39).

Establishing the optimal culture conditions for expression profiling. ATCC36082 strain was grown in YPD agar (1% yeast extract, 2% Bacto peptone, 2%dextrose, 1.5% agar) for 16 h at 37°C. Then, one colony was transferred to YPDbroth and incubated for 16 h at 37°C at 250 rpm. Subsequently, the culture washarvested and washed two times with sterile distilled water. From this pellet, a107-CFU/ml inoculum was obtained and was grown at 37°C at 250 rpm in RPMI1640 (pH 5 to 8) in the presence of different drug concentrations (AMD from 1to 50 �M and FLC from one-half MIC to full MIC) and different exposure times(5, 10, 15, 30, 45, 60, and 90 min). DMSO was added to the control cultures inthe same proportion as in the test cultures. All of the genes studied here wereevaluated for each condition. Finally, the condition with the maximum differ-ences in gene expression was selected.

RNA isolation and expression profiling. A 107-CFU/ml inoculum was obtainedas described above. Cells were grown in RPMI 1640 pH 5 in the presence of 25�M AMD for 10 min and/or one-half MIC of FLC for 1 h. Total RNA wasextracted by a hot phenol-chloroform extraction and ethanol precipitation pro-tocol (7). Gene expression profiles were performed using a one-step Sybr greenquantitative reverse transcription-PCR kit (Stratagene, La Jolla, CA) on a Strat-agene Mx3005P multiplex quantitative PCR system. C. albicans URA3 (GenBankaccession no. XP_721787.1) was used for normalization. The relative expressionwas evaluated using the method described by Pfaffl (43). Differential expressionwas analyzed for 59 C. albicans genes in the following functional categories: Ca2�

pumps/channels/transporters (n � 6), calmodulin-calcineurin pathway (n � 4),cell wall (n � 7), alkaline pH and cation overload response (n � 2), GTPaseactivity (n � 2), transcription factor activity (n � 6), cell cycle (n � 3), heat shockprotein (n � 1), oxidation of fatty acids (n � 2), ergosterol biosynthesis (n � 7),morphogenesis and hyphal formation (n � 5), Tor signaling pathway (n � 3),GDP-mannose transporter (n � 1), amino acid transport and metabolism (n �

2), ammonium transmembrane transporters (n � 2), and glucose metabolismand transporters (n � 6) (see the supplemental material).

Ergosterol extraction and quantification. Sterols were extracted by the alco-holic KOH method and ergosterol content was established as a percentage of thedry weight of the cells by the method described by Arthington-Skaggs et al., withmodifications (3). Inocula of 107 CFU/ml were obtained as described above andinoculated into 100 ml of RPMI 1640 (pH 5) alone or RPMI 1640 (pH 5) plusAMD, FLC, or AMD-FLC. Then, they were incubated for 16 h at 37°C at 250rpm. Finally, the cells were divided in two fractions and weighed. The firstfraction was used for moisture determination by drying the cells at 50°C untilachieving a constant weight. The second fraction was used to measure theergosterol content of the samples by scanning spectrophotometrically between240 and 300 nm with a Nano-Drop spectrophotometer (NanoDrop Technolo-gies, Inc., Wilmington, DE) (3).

In vivo study. In vivo studies were carried out in a murine candidiasis model.Female BALB/c mice (CRL) were challenged with the C. albicans strains 1002,3795, 610, and 611 administered by tail vein injection. At 3 h postinfection, micewere given single or in combination treatments of AMD (Amiodarone-HClinjection; Bioniche) and FLC (LKT Laboratories, Inc.) via the intraperitonealroute. AMD treatment doses ranged between 5.0 to 25 mg/kg, while FLCdoses ranged from 0.1 to 1 mg/kg in mice infected with FLC-susceptiblestrains and from 5.0 to 40 mg/kg for animals infected with FLC-resistantstrains. The treatments were given once daily for 3 days after the first dose.On day 4 postinfection, mice were euthanized, kidneys were harvested andenumerated for C. albicans burdens (41). Combinatorial drug efficacy wasassessed by C. albicans kidney burdens reduction in the treated mice relativeto the untreated controls.

Pseudohyphae and true hyphae growth in liquid media. C. albicans cells wereincubated at 37°C in RPMI 1640 (pH 5) for 16 h in the absence or in the presenceof different concentrations of AMD (0.5 to 25 �M), FLC (one-half MIC), andAMD-FLC (25 �M and one-half MIC for FLC). All cells were viewed by lightmicroscopy at �400 to assess pseudohyphal and true hyphal formation.

Statistical analysis. In vitro susceptibility, synergism, and expression profilingexperiments were repeated at least three times on different days. Arithmeticmeans and standard deviations were used to statistically analyze the continuousvariables (�FIC, expression profiling data, ergosterol percentage, and kidneyburdens). Geometric means were used to statistically compare MIC results. Inorder to approximate a normal distribution, the MICs were transformed to log2

values to establish susceptibility differences between strains. Both on-scale andoff-scale results were included in the analysis. The off-scale MICs were con-verted to the next concentration up or down. The significance of the differ-ences in MICs, �FICs, ergosterol content, and kidney burdens was deter-mined by using the Student t test (unpaired, unequal variance). Significantdifferences in MIC values and in vivo studies were determined by using theStudent t test (unpaired, unequal variance) and by the Wilcoxon rank sumtest, respectively; a P value of �0.05 was considered significant. Statisticalanalysis was done with the Statistical Package for the Social Sciences (version13.0; SPSS, Inc., Chicago, IL).

TABLE 1. Antifungal susceptibility testing and drug interaction evaluation of all the strains used in this studya

Strainb Amino acidsubstitution in Erg11p

Geneoverexpression

MICc

FLC-AMD interactionStandard MIC Combination MIC

FLC AMD FLC AMD �FIC Interpretationd

36082 None None 1.00 25.0 0.50 1.56 0.563 IN5314 None None 0.50 25.0 0.20 6.25 0.625 IN90028 None None 1.00 25.0 0.50 1.56 0.563 IN1002* None None 0.50 25.0 0.06 12.5 0.625 IN3795* F136L/K134R None �128 25.0 0.50 6.25 0.250 SYN610† None None 0.25 25.0 0.12 18.75 1.250 IN611† None CDR1 64.0 25.0 8.00 6.25 0.375 SYN

a MICs and �FIC values were obtained for all strains according to CLSI document M27-A3 using RPMI 1640 medium buffered at pH 5.b �, matched clinical isolates (41); †, matched clinical isolates (20).c MICs represent geometric means of at least three MICs determined on different days. Values are expressed in �g/ml for FLC and in �M for AMD. “Combination”

refers to the MIC of each drug in combination with the other. These MIC combinations together with the standard MICs were used to obtain the �FIC values displayedin the table.

d As reported by F. C. Odds (39).

1754 GAMARRA ET AL. ANTIMICROB. AGENTS CHEMOTHER.

RESULTS

Antifungal susceptibilities and synergism. The MIC andMFC values for AMD against C. albicans showed significantvariation as a function of pH (P � 0.01) with the lowest valuesobserved at a pH of �5 for all strains examined. MFC valueswere the same as the MIC values demonstrating that AMD hasfungicidal properties against C. albicans. AMD was ineffectiveagainst C. albicans at higher pH values (MIC � 800 �M).Conversely, the FLC MICs obtained using RPMI 1640 at pH 7(CLSI document M27A3 condition) (6) showed no statisticallysignificant differences compared to those obtained at pH 5(P � 0.54). These observations were congruent with previousstudies (9, 18, 33, 37). Thus, RPMI 1640 growth medium at pH5 was adopted for all of the experiments with AMD and FLC,both individually and in AMD-FLC combination. Under theseconditions, all strains showed the same AMD MIC (25 �M),whereas the FLC MICs ranged from 0.25 to �128 �g/ml (Ta-ble 1). The FLC-susceptible strains tested displayed an AMD-FLC �FIC index that was interpreted as synergistic indiffer-ence (range, 0.563 to 1.250). On the other hand, AMD-FLCshowed a synergistic effect with FLC-resistant strains (�FIC �0.500) (Table 1).

Transcriptional changes underlying AMD-FLC antifungalaction against C. albicans. Transcriptional profiling was used toassess changes in gene expression associated with the AMD,FLC, and AMD plus FLC treatment in C. albicans. Many ofthe target genes of interest were identified from prior tran-scriptional microarray studies performed on S. cerevisiae (53).Optimal expression profiling growth conditions were estab-lished using early time points (5, 10, 15, and 30 min) anddifferent AMD and FLC concentrations (1, 10, 25, and 50�g/ml) using a control strain ATCC 36082. For AMD, thebiggest fold increases in expression were observed at 25 �M(MIC) (Fig. 1A) and when the cells were exposed to AMD for10 min (Fig. 1B). These data suggest that for several key genes,the transcriptional response reached its peak shortly after drugexposure and diminished over time. For FLC, the biggest dif-ferences in expression were seen when strain ATCC 36082 wasexposed to 0.5 �g of FLC/ml (one-half MIC). When differenttime points were evaluated, the highest expression differenceswere observed at 1 h of FLC exposure (not shown). On thebasis of these results, the condition established for analyzingthe AMD-FLC combination influence in expression profiling

was 1-h FLC (one-half MIC) exposure, followed by 10 min ofgrowth in the presence of 25 �M AMD.

After drug exposure with 25 �M AMD for 10 min, 22 of 59genes analyzed showed a significant change in expression levels(expression fold change � 2). These changes were bigger forFLC-resistant clinical strains (Table 2). The upregulated genesincluded genes involved in cell wall organization, Ca2� over-load stress response, transcription factors involved in stressresponse, and calmodulin-calcineurin pathway. Of these cate-gories, the Ca2� homeostasis pathway genes, RIM101 andTAC1 stood out. We examined six Ca2� transporter genes,including plasma membrane Ca2� channels (CCH1 and MID1)(15, 29), ATP-driven Ca2� pumps in vacuolar and Golgi mem-branes (PMC1 and PMR1) (11, 16, 28, 47), a vacuolar Ca2�/H�

vacuolar exchanger (VCX1) (11), and a voltage-dependent,Ca2� selective vacuolar ion conductance channel (YVC1) (40).Of these, CCH1, MID1, PMC1, and PMR1 were upregulated inresponse to AMD treatment, showing that the AMD mecha-nism of action is likely linked to Ca2� overload, as observed inS. cerevisiae (53). Furthermore, of four genes examined in thecalmodulin-calcineurin pathway (CMD1, CNA1, CNB1, andCRZ1), CMD1 and CRZ1 were strongly activated in responseto AMD, supporting the notion that the AMD mechanism oftoxicity is mediated by Ca2� overload, as in S. cerevisiae (18,53). AMD altered the expression of other genes involved incell wall synthesis and regulation, diverse pathway regulation(transcription factors), GTPase activity, pH and cation regula-tion, vesicle transport, and cycle cell regulation (Table 2). Asexpected, FLC exposure induced expression of genes for er-gosterol biosynthesis pathway (ERG1, ERG11, ERG3, andERG6) in the FLC-susceptible strains. On the other hand,ERG2, ERG7, and ERG24 and all of the other categories didnot show expression changes after FLC treatment. Unexpect-edly, the expression of ergosterol synthesis pathway genes wassignificantly reduced (P � 0.01) by the combination FLC-AMD treatment in the FLC-susceptible strain studies (Table2). Moreover, all genes overexpressed under AMD treatmentshowed reduced expression when FLC plus AMD was used(Table 2). In general, this reduction was statistically significantand greater (P � 0.01) in the FLC-resistant strains, helping toexplain the synergistic in vitro effect of FLC-AMD observed inFLC-resistant strains.

FIG. 1. Changes in gene expression (in fold induction) in response to different AMD concentrations and different exposure times at fixed AMDconcentration using a control strain ATCC 36082. A 107-CFU/ml inocula was grown at 37°C at 250 rpm in RPMI 1640 in the presence of differentdrug concentrations (AMD from 1 to 50 �M and FLC from one-half MIC to full MIC) (A) and different exposure times (B), as indicated. Geneexpression profiles were performed using a one-step Sybr green quantitative reverse transcription-PCR. URA3 was used for normalization.

VOL. 54, 2010 AMD AND FLC SYNERGIZE AGAINST C. ALBICANS 1755

AMD and FLC reduce the total ergosterol content in asynergistic manner. All clinical strains and the control strainATCC 90028 showed 2- to 4-fold more ergosterol than thecontrol strains ATCC 36082 and SC5314 (Table 3). These lasttwo strains showed a massive filamentous form production inthe growth condition used, suggesting that ergosterol content islower in filamentous forms than in planktonic cells. Indepen-dent of the FLC susceptibility and filamentous form produc-tion, all of the strains showed statistically significant reductionsin ergosterol content in the presence of FLC (P � 0.05) (Table3). As expected, FLC-susceptible strains showed a greater re-duction in ergosterol content than FLC-resistant strains afterFLC treatment (4- to 1.6-fold and 1.4 to 2.0- fold, respectively).On the other hand, treatment with AMD alone did not affect

the ergosterol content, both in FLC-susceptible and -resistantstrains, suggesting that AMD does not directly influence theergosterol pathway. Despite this lack of effect, and in accor-dance with the �FIC values, the ergosterol content decreasedmore in the FLC-resistant than in FLC-susceptible strainswhen AMD was used in combination with FLC (Table 3).

In vivo AMD and FLC synergy studies. A murine model ofdisseminated candidiasis was used to assess the effect of AMD-FLC combination against FLC-susceptible and -resistant iso-lates. To observe potential synergy, drug concentrations forAMD and FLC were selected in order to have a minimal effecton kidney burdens. Figure 2 shows that treatment with AMDalone at 25 or 5.0 mg/kg/day for 5 days did not affect kidneyburden (P � 0.10). Similarly, FLC at 0.1 mg/kg/day failed to

TABLE 2. Genes up- and downregulated in C. albicans in response to FLC, AMD, and both drugs in combination

Gene Category

Up- and downregulation (fold) inductiona in strain:

36082 (WT) 1002 (WT) 3795 �erg11(F136L/K134R) 610 (WT) 611 (overexpression

CDR1)

F A F�A F A F�A F A F�A F A F�A F A F�A

MID1 Ca2� pumps, channels,and transporters

0.8 1.3 0.7 0.8 2.4 1.7 0.9 4.2 1.9 0.9 7.2 4.6 1.1 4.7 3.1

CCH1 Ca2� pumps, channels,and transporters

1.3 7.6 1.8 1.1 28 15.4 1.1 53 25.2 0.7 9.2 4.6 1.1 9.3 3.9

PMC1 Ca2� pumps, channels,and transporters

1.5 28 2.3 1.2 65 20.9 1.0 121.6 28.6 0.9 35.9 7.9 0.9 25 7.4

CRZ1 Calmodulin-calcineurinpathway

1.2 45.2 2.6 2.4 13.1 4.0 1.1 26.1 3.9 0.8 5.6 3.1 2.0 11.6 1.7

CMD1 Calmodulin-calcineurinpathway

1.3 0.9 1.3 0.8 2.7 1.8 0.8 3.3 2.6 0.8 3.5 2.0 1.2 7.1 1.6

FKS2 Cell wall 1.1 2.6 0.6 1.0 2.3 1.2 1.0 4.6 2.2 0.7 1.2 2.0 1.3 0.6 0.7TUS1 Cell wall 1.9 4.5 2.0 1.0 4.8 3.0 1.3 18.9 8.5 0.8 2.0 1.7 1.4 3.1 3.1SKN1 Cell wall 1.5 3.7 0.7 0.7 2.4 1.0 1.2 2.3 1.4 1.0 5.4 3.2 1.6 6.0 3.0WSC2 Cell wall 1.3 3.8 0.6 0.7 2.5 1.9 1.0 2.7 1.5 0.9 6.6 3.3 0.8 3.9 2.8RIM101 Alkaline pH and cation

overload response1.0 20.8 2.7 1.1 28.4 6.8 1.1 31.8 8.1 1.2 13.7 10.6 1.0 16.7 6.8

GYP7 GTPase activity, vesicletransport

1.0 5.1 1.7 0.9 28.4 13.2 1.2 72.8 25.0 0.9 22.5 11.0 1.0 22.8 11.3

TAC1b Transcription factoractivity

1.3 35.9 6.2 1.3 44.2 23.8 1.2 43.3 36.7 0.8 36.0 18.1 1.2 5.4 3.6

ZCF39 Transcription factoractivity

1.3 4.3 1.4 1.2 28.3 11.4 1.3 53.4 17.4 1.1 14.6 8.3 1.0 11.2 5.1

RLM1c Transcription factoractivity

1.2 0.5 0.8 1.1 3.0 0.7 1.2 4.9 1.3 1.1 3.2 1.8 1.2 3.2 1.2

CLB2 Cell cycle 0.9 0.6 0.3 1.1 0.4 0.4 1.1 0.5 0.5 1.0 0.5 0.5 0.9 0.4 0.7ACE2 Cell cycle 1.0 1.8 0.3 1.0 2.2 2.2 1.2 3.2 3.0 1.0 3.6 3.4 1.1 3.2 1.9CLN1 Cell cycle 1.1 1.3 0.5 1.1 0.0 0.4 0.8 0.0 0.7 1.3 1.1 1.5 1.1 0.3 1.4HSP90 Heat shock protein.

molecular chaperone0.7 0.3 1.0 1.2 0.3 2.5 1.1 0.5 2.8 0.8 0.6 2.1 1.2 0.8 1.5

FOX2 Oxidation of fatty acids 1.1 4.4 1.3 1.1 13.7 7.9 1.2 32.0 18.8 0.8 6.9 17.2 1.2 2.9 7.1ERG1 Ergosterol biosynthesis 2.2 0.9 1.6 1.6 0.5 0.6 1.2 0.8 1.2 1.4 1.8 1.3 1.2 0.6 1.1ERG11 Ergosterol biosynthesis 2.4 1.3 1.6 2.7 0.7 1.1 1.2 0.7 1.5 1.3 0.5 1.3 1.7 0.3 1.1ERG3 Ergosterol biosynthesis 1.8 1.2 0.9 2.8 1.1 1.2 2.1 1.8 1.3 2.0 1.2 1.0 1.8 0.6 0.8ERG6 Ergosterol biosynthesis 2.4 0.8 1.0 3.3 0.5 1.1 1.8 0.6 1.9 0.9 0.8 0.9 1.4 0.7 0.8NRG1 Morphogenesis and

hyphal formation1.0 2.6 0.7 1.4 3.7 0.8 1.2 2.8 1.4 0.8 3.3 1.6 1.4 3.7 2.6

TUP1 Morphogenesis andhyphal formation

0.8 1.4 0.8 0.6 0.7 0.7 1.3 3.2 2.5 1.0 1.0 1.7 1.1 2.1 2.7

RFG1 Morphogenesis andhyphal formation

0.8 0.4 0.5 0.9 0.1 0.9 1.1 0.2 0.3 0.7 0.4 0.6 1.4 0.4 0.6

UME6 Morphogenesis andhyphal formation

0.7 0.0 0.5 0.7 0.0 0.1 0.6 0.0 0.2 0.9 0.5 0.7 1.1 0.1 0.2

a The data represent the arithmetic mean of three repetitions. Numbers in boldface represent upregulated genes (�2-fold). Numbers in italics represent down-regulated genes (�2-fold). F, fluconazole; A, amiodarone.

b Related to azole resistance by transcriptional activation of the CDR genes (8).c Related to stress response (37).


reduce organ burdens when the mice were inoculated with theFLC-susceptible strain 1002 (P � 1.00). On the other hand,when the animals were inoculated with strain 610, the treat-ment with FLC at 0.1 mg/kg/day produced a 2-fold reduction inkidney burdens (P � 0.008). FLC at 1 mg/kg/day showed 2.5-

and 3-log reduction for strains 1002 and 610, respectively.However, when animals were treated with a combination ofincreasing FLC concentrations (0.5 to 1 mg/kg/day) in thepresence of fixed amounts of AMD at 5 or 25 mg/kg/day, therewas an FLC dose-dependent reduction in organ burden (Fig.

TABLE 3. Sterol quantification expressed as a percentage of the cell dry weighta

Groupb Strain

Mean % concn SD P

NT FLC AMD FLC-AMD FLC vsNT

AMD vsNT

FLC-AMDvs NT

FLC�AMDvs FLC

FLC-AMDvs AMD

I 36082 0.45 0.05 0.11 0.01 0.60 0.01 0.70 0.05 0.0096 0.32 0.071 0.0001 0.05I 5314 0.70 0.05 0.40 0.03 0.80 0.01 0.70 0.01 0.0089 0.73 0.82 0.015 1.00II 90028 1.30 0.02 0.60 0.03 1.20 0.02 0.50 0.03 0.0001 0.27 0.0001 0.58 0.0003III 1002 1.25 0.01 0.30 0.00 1.15 0.00 0.50 0.00 0.0001 0.05 0.0001 0.0001 0.0001III 3795 1.05 0.05 0.75 0.01 0.85 0.05 0.40 0.02 0.017 0.15 0.0008 0.0016 0.005IV 610 1.75 0.05 1.1 0.02 1.45 0.00 0.8 0.02 0.0005 0.017 0.0001 0.0044 0.0001IV 611 1.45 0.00 0.7 0.05 1.35 0.00 0.6 0.02 0.0001 0.05 0.0001 0.58 0.0001

a NT, no treatment.b Groups I and II, control laboratory strains; group III, clinical isogenic strains; group IV, clinical isogenic strains; group I, massive filamentous form production;

groups II, III, and IV, regular pseudohypha and true hypha production.

FIG. 2. Efficacy of AMD and FLC combination in a murine candidiasis model. Female BALB/c mice (CRL) were challenged with theFLC-susceptible C. albicans strain 1002 (isogenic with 3795) (A), the FLC-resistant C. albicans strain 3795 (with two ERG11 mutations T376C andA428G) (B), the FLC-susceptible C. albicans strain 610 (isogenic with 611) (C), and the FLC-resistant C. albicans strain 611 (with overexpressionCDR) (D). At 3 h postinfection, mice were given single or combination treatments of AMD (5.0 to 25 mg/kg) and/or FLC (0.1 to 1 mg/kg in miceinfected with FLC-susceptible strains and 5.0 to 40 mg/kg in mice infected with the FLC-resistant strains) intraperitoneally. The treatments weregiven once daily for 3 days after the first dose. On day 4 postinfection, mice were euthanized, kidneys were harvested and enumerated for C.albicans burdens. White boxes represent kidney burdens in log CFU/g for each mouse; black boxes represent average kidney burden per group.Tx, treatment.


2) (P � 0.008). This drug synergy was saturated with AMD at5 mg/kg/day (P � 1 comparing AMD at 25 mg/kg/day and FLCat 1 mg/kg/day to AMD at 5 mg/kg/day and to FLC at 1mg/kg/day). To explore additional synergy with AMD and FLCin an azole-resistant background, animals were infected withFLC-resistant C. albicans strains (Table 1). In these strains,due to the strong FLC resistance phenotype, FLC alone had noeffect on reducing microbial burdens (P � 0.05). AMD aloneat 25 mg/kg/day had no significant affect on microbial burden(P � 0.05). The combination of increasing FLC concentration(5.00 to 40 mg/kg/day) in the presence of AMD at 5 or 25mg/kg/day showed strong synergy (P � 0.05). When the micewere inoculated with strain 3795, which contains the erg11mutations F136L and K134R, the treatment with FLC at 40mg/kg/day, showed a �4-log reduction in kidney burden whencombined with AMD at 25 mg/kg/day relative to the no-drugcontrol (P � 0.016). A more pronounced reduction in burdenwas observed when FLC (40 mg/kg/day) was used in combina-tion with AMD at 5 mg/kg/day (P � 0.008) (Fig. 2). Moreover,in three mice, this combination treatment led to total kidneysterilization. This led to the suggestion that changes in thelanosterol demethylase due to mutation may be important forvirulence. When FLC-resistant strain 611, which overexpressesCDR1, was used for infections, the FLC-AMD combinationtreatment (20 and 5 mg/kg/day) showed a smaller decrease infungal burden (P � 0.008).

AMD inhibits filamentous form development in C. albicans.The inhibition of filamentous form production was observed inall strains used throughout the present study independent ofgenotype. Reducing filamentous form production may helpaccount for reduced virulence after drug combination therapy.In all strains, AMD started showing filamentous form inhibi-tion at 1 �M and reached its maximum when AMD was addedat a final concentration of 10 �M (Fig. 3). The effect wasapparent in multiple strains but was more pronounced inazole-resistant strains (Fig. 3). The molecular mechanism in-volved in this phenotype could be linked with changes in theexpression of four genes related with hyphal and pseudohyphalproduction. The gene UME6, an inductor of hyphal develop-ment (52), showed no expression in all of the strains studied inthe presence of AMD (Table 2). Moreover, three well-charac-terized transcription factors, i.e., Tup1p, Nrg1p, and Rfg1p,acting as negative regulators of hyphal and pseudohyphal mor-phogenesis were upregulated in the presence of AMD (25).

DISCUSSION

AMD mechanism of action in Candida albicans. AMD is anantiarrhythmic drug observed to possess antifungal propertiesagainst a wide range of fungi in vitro (9). The mechanism ofAMD action has been linked to rapid opening of plasma mem-brane calcium channels (10, 18, 37), reduced membrane fluid-

FIG. 3. Evaluation of pseudohyphal growth in the presence of AMD. C. albicans grown in liquid media incubated at 37°C in RPMI 1640 (pH5) for 16 h in the absence or in the presence of different concentrations of AMD (0.5 to 25 �M), FLC (one-half MIC), and AMD-FLC (25 �Mand one-half MIC for FLC). (A) The effect of increasing amounts of AMD alone is shown on pseudohyphal development as viewed by lightmicroscopy at �400. (B to D) The percentages of pseudohyphal production were calculated from visual scoring and are shown for FLC-susceptibleC. albicans strain 1002 (isogenic with 3795) and FLC-resistant C. albicans strain 3795 (with two ERG11 mutations T376C and A428G) (B);FLC-susceptible C. albicans strain 610 (isogenic with 611) (C); and FLC-resistant C. albicans strain 611 (with overexpression CDR) and ATCC36082, ATCC 90028, and SC5314 (D).


ity (2, 46), and disruption of the cell cycle (53) in C. neoformansand S. cerevisiae. Recently, it was reported that the fungicidaleffect of AMD in S. cerevisiae is a consequence of calciumstress involving the calcineurin-responsive transcription factorCrz1p (51, 53). The transcriptional profile of key pathwaygenes in the present study (Table 2) suggests that the antifun-gal effect of AMD against C. albicans is also most likely me-diated by Ca2� stress, alteration of cell wall organization, dis-ruption of nutrient sensing/signaling, and perturbation of thetranscription regulation.

We analyzed six genes encoding Ca2� pumps, channels, andtransporters and a transcription factor (RIM101) involved inthe cation overload response (27). This group of genes includesa high-affinity plasma membrane Ca2� channels (CCH1 andMID1) (15, 29), a vacuolar calcium P-type ATPase essential forCa2� homeostasis (PMC1) (16, 28, 47), a Golgi apparatusCa2� pump (PMR1) (11, 28), a vacuolar Ca2�/H� vacuolarexchanger (VCX1) (11), and a voltage-dependent, Ca2� selec-tive vacuolar ion conductance channel (YVC1) (40). CCH1,MID1, PMC1, and RIM101 were overexpressed, strongly sug-gesting that AMD activity is linked with Ca2� and pH stress.This proposed mechanism of AMD action in C. albicans wasconfirmed by the induction of CRZ1 expression in response tothis drug. It was demonstrated that when Ca2� enters the cellusing Cch1p channels, the calcineurin signaling pathway isactivated and Crz1p is part of this pathway (34).

The implication of Ca2� in AMD mechanism of action wasalso evident when the GYP7 gene expression profiling wasstudied. This gene encodes a putative activator of the vacuolarYpt7p GTPase inducing an acceleration of its GTPase activity(50). This last protein interacts physically with Ccz1p (for cal-cium-caffeine-zinc sensitivity protein 1) in the presence of aCa2� overload (24, 30). Moreover, this interaction is essentialfor autophagy in S. cerevisiae (35), leading to a possible expla-nation of the fungicidal action of AMD against different fungalspecies. Our data also showed that AMD induces the overex-pression of two genes encoding putative transcription factorregulators with Cys-His zinc finger motifs, TAC1 and ZCF39.Tac1p regulates the main ABC transporter genes (CDR1 andCDR2) in C. albicans and is important for resistance to azoledrugs (8, 12). On the other hand, ZCF39 was linked to fila-mentation and biofilm formation (38). Collectively, the over-expression of these transcription factors by AMD could beexplained as a cell response to the stress induced by the drug.

The effect of AMD on the C. albicans cell wall was studiedby analyzing the expression of seven genes important for cellwall biosynthesis and the cell integrity signaling pathway (seethe supplemental material). TUS1 (a Rho1p exchange factormodulator) and FKS2 (a glucan synthase subunit linked withthe calcineurin pathway) showed modest but significant up-regulation (Table 2). Hence, AMD may alter cell wall regula-tion and other differential pathways since Rho1p is a ubiqui-tous regulatory protein contributing to cell wall integrity, cellpolarity, cytoskeleton reorganization, and protein kinase Cregulation (13, 14, 31, 45, 48).

Mechanism of AMD-FLC synergy in C. albicans. The syner-gistic effect of the combination AMD and azole drugs wasdemonstrated in vitro against azole-resistant C. albicans and A.fumigatus (1, 17). This effect was confirmed by using clonalFLC-susceptible and -resistant C. albicans clinical strains.

These data show that ERG11 mutations strongly reduce theAMD-FLC �FIC values from indifferent to synergistic. How-ever, to examine this further, we explored potential drug syn-ergy in a murine candidiasis model. Drug ranges for bothAMD and FLC were selected to have a minimal affect onmicrobial burdens when used alone. However, in combination,there was a pronounced (�2-log) reduction in kidney CFU fora wild-type susceptible strain. However, the affects were muchmore prominent for a FLC-resistant strain, which showed a�4-log reduction in the presence of both drugs (Fig. 2). Thisapparent discrepancy between in vivo and in vitro data may beexplained by the antifilamentous form activity of AMD, whichaffects virulence. The expression profiling data presented herehelps to explain the molecular mechanism of filamentous forminhibition by demonstrating an AMD-dependent upregulationof repressors of genes involved in this cellular response. Thisfact may increase mouse survival since filamentous form andUme6p are well-known C. albicans virulence factors (5, 52).

In order to understand the strongest synergistic activity ofthe FLC-AMD combination in FLC-resistant strains, expres-sion profiling was undertaken using five strains exposed to FLCand AMD alone and in combination. FLC treatment inducesthe hyperexpression of the ergosterol pathway genes as a com-pensatory response to reestablish the plasma membrane ergos-terol levels. On the other hand, AMD treatment causes thehyperexpression of vacuole Ca2� pumps and calcineurin path-way genes in order to overcome the Ca2� overload. Thesestress responses induced by individual drug treatments weretotally or partially inhibited when FLC was used in combina-tion with AMD independently of the FLC susceptibility. How-ever, the combination treatment produced a bigger dampeningin the transcriptional response (P � 0.01) in the FLC-resistantstrains than in the FLC-susceptible isolates.

The partial or total inhibition of the stress responses inducedby the combination treatment shows a possible connectionbetween ergosterol synthesis and Ca2� homeostasis pathways.This notion was described previously for mammal cells whereketoconazole and other azole derivatives were shown to have adirect effect on the inflammatory response by inhibiting thecalcineurin pathway via calmodulin (19). Moreover, some au-thors have hypothesized a possible mechanism of triazole-AMD synergism as the AMD inhibition of the pre-lanosterolenzyme Erg7p and Erg1p in the protozoans Trypanosoma cruziand Leishmania mexicana, respectively (4, 49), and the possibleimplication of Ca2� homeostasis disruptors in the inhibition ofS. cerevisiae sterol isomerases (ERG2) (36). However, none ofthese observations was reproduced in our experiments with C.albicans (Table 2). In this yeast, the synergistic effect of AMD-FLC against FLC-resistant isolates could be explained by analteration in membrane fluidity and enzyme function producedby the reduction of plasma and organelle membrane ergosterolin the erg11 mutant. A recent report of Maresova et al. (32)suggests that AMD is a membrane active drug that elicits atransient hyperpolarization of the membrane, followed by adepolarization resulting in the influx of Ca2� and H� and lossof cell viability. Since ergosterol is the central sterol in fungalcell membranes, it is expected that changes in the ergosterolbiosynthetic pathways could have downstream effects on mem-brane composition resulting in altered membrane fluidity andenzyme function. Ergosterol depletion of organelle mem-


branes impacts vacuolar H�-ATPase function, which greatlyalter cellular stress responses (Y.-Q. Zhang and R. Rao, un-published data). Such changes may account for reported syn-ergy observed between AMD and FLC with triazole-resistantstrains, especially those with amino acid changes in Erg11pdifferent than Y132H (22, 23, 26). Our ergosterol quantifica-tion data suggest that another mechanism may also have toexist to explain the reduction in total ergosterol when AMD-FLC combination treatment was compared to FLC aloneagainst FLC-resistant strains. The somewhat lower but signif-icant synergistic behavior of AMD-FLC treatment against thestrain harboring CDR overexpression can be explained by themodulation of efflux pumps induced AMD, as suggested pre-viously for azole-resistant A. fumigatus (1).

Overall, these data support a role for AMD and relatedmembrane active agents as a novel adjunct for existing anti-fungal therapy. The in vitro, in vivo, and expression profilingdata presented here demonstrate that pathways regulated byAMD could be considered as possible new antifungal targetsdiffering significantly from existing classes of antifungal drugs.

ACKNOWLEDGMENTS

This study was supported by NIH grants AI069397 to D.S.P. andAI065983 to R.R.

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mechanism of the synergistic effect of amiodarone and … · in vivo studies were carried out in a...

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