AAC Accepts, published online ahead of print on 6 October 2014 Antimicrob. Agents Chemother. doi:10.1128/AAC.03083-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

1

1

Quinacrine inhibits Candida albicans growth and filamentation at neutral pH

2 3

Vibhati V. Kulkarny1, Alba Chavez-Dozal1, 2, Hallie S. Rane1, Maximillian Jahng1, Stella M. Bernardo1,

4

Karlett J. Parra3 and Samuel A. Lee1, 2, #

5 6

Section of Infectious Diseases, New Mexico Veterans Healthcare System, Albuquerque, NM,

7

USA1;Division of Infectious Diseases, University of New Mexico Health Science Center, Albuquerque,

8

NM, USA2; Department of Biochemistry, University of New Mexico Health Science Center,

9

Albuquerque, NM, USA3

10 11

Running head: Quinacrine vs. C. albicans growth and filamentation

12 13

#

14

Diseases, New Mexico Veterans Healthcare System; 1501 San Pedro SE, Mail Code: 111-J, Albuquerque,

15

NM 87108; Phone: 505-265-1711; Fax: 505-256-2803; Email: [email protected]

To whom correspondence should be addressed: Samuel A. Lee, M.D., Ph.D., Section of Infectious

1

2

16

ABSTRACT

17

Candida albicans is a common cause of catheter-related bloodstream infections (CR-BSI), in part due

18

to its strong propensity to form biofilms. Drug repurposing is an approach that could identify agents able

19

to overcome antifungal drug resistance within biofilms. Quinacrine (QNC) is clinically active against the

20

eukaryotic protozoan parasites Plasmodium and Giardia. We sought to investigate the antifungal activity

21

of QNC against C. albicans biofilms. C. albicans biofilms were incubated with QNC at serially

22

increasing concentrations (4-2048µg/ml) and assessed using the XTT assay in a static microplate model.

23

Combinations of QNC and standard antifungals were assayed using biofilm checkerboard analyses. To

24

define a mechanism of action, QNC was assessed for inhibition of filamentation, effects on endocytosis,

25

and pH-dependent activity. High-dose QNC was effective for prevention and treatment of C. albicans

26

biofilms in vitro. QNC with fluconazole had no interaction, while the combination of QNC and either

27

caspofungin or amphotericin B demonstrated synergy. QNC was most active against planktonic growth

28

at alkaline pH. QNC dramatically inhibited filamentation. QNC accumulated within vacuoles as

29

expected, and caused defects in endocytosis. A tetracycline-regulated vma3 mutant lacking V-ATPase

30

function demonstrated increased susceptibility to QNC. These experiments indicate that QNC is active

31

against C. albicans growth in a pH-dependent manner. Although QNC activity is not biofilm-specific,

32

QNC is effective in the prevention and treatment of biofilms. QNC anti-biofilm activity likely occurs via

33

several independent mechanisms: vacuolar alkalinization, inhibition of endocytosis, and impaired

34

filamentation. Further investigation of QNC for treatment and prevention of biofilm-related Candida CR-

35

BSI is warranted.

2

3

36

INTRODUCTION

37

The ability of Candida albicans to form biofilms is a key attribute that enhances its ability to cause

38

opportunistic infections (1, 2). In sessile form, C. albicans undergoes pleiotropic phenotypic changes,

39

leading to protection from host immune defenses and increased resistance to antifungal therapy(3–6).

40

Moreover, biofilms serve as a protected nidus from which subsequent dissemination via the bloodstream

41

may occur. A wide range of adjunctive systemic therapies have been investigated for use in combination

42

with traditional antifungal therapy in order to improve efficacy against invasive or disseminated

43

candidiasis (3, 7, 8).

44

Quinacrine (QNC), a water-soluble acridone derivative, was widely used for prevention and treatment

45

of malaria during WWII and remains available as a highly active therapeutic agent against giardiasis.

46

QNC’s pharmacologic effects remain incompletely defined, but include inhibition of nucleic acid

47

synthesis through DNA intercalation, anti-prostaglandin and anti-platelet effects due to inhibition of

48

phospholipase A2 and anti-lipolytic activity, inhibition of platelet aggregation, and accumulation within

49

neutrophils (9–12).

50

QNC has activity against a number of protozoa, including Plasmodium, Giardia, and Leishmania

51

species. QNC accumulates within the vacuole of the malarial parasite, where it is thought to inhibit

52

hematin polymerization. Additionally, antifungal activity of QNC has been demonstrated in a limited

53

number of in vitro studies. High concentrations of QNC inhibit the growth of the yeast Saccharomyces

54

cerevisiae (13, 14), and QNC, which normally accumulates in vacuoles, has long been used as a marker

55

for the S. cerevisiae vacuole in cell and molecular biology (15). A study from 1975 showed that QNC, in

56

proline-rich medium, inhibited C. albicans mitochondrial synthesis and QNC concentrations >1mM

57

decreased filamentation (16). In Cryptococcus neoformans, QNC and to a lesser degree the closely-

58

related compound chloroquine have been shown to exhibit anti-cryptococcal activity; chloroquine was

59

shown to disrupt pH-dependent cellular processes after accumulation within vacuoles (17, 18).

60 61

We therefore sought to determine whether QNC was effective for the prevention and treatment of C. albicans biofilms, and to analyze the mechanistic effects of QNC with a focus on filamentation and 3

4

62

vacuolar function. We used a static microplate model of C. albicans biofilms to systematically determine

63

the optimal concentration of QNC needed to (i) inhibit mature, pre-formed C. albicans biofilms, (ii)

64

prevent C. albicans biofilm formation, (iii) determine activity when combined with standard antifungal

65

drugs, and (iv) identify potential mechanisms of action of QNC antifungal activity, with the overall

66

objective of defining the antifungal activity of QNC and its utility against C. albicans biofilms.

67 68

MATERIALS AND METHODS

69

Strains and Reagents. The clinically derived wild-type strain C. albicans SC5314 was used as a reference

70

isolate for detailed studies. C. albicans clinical isolates ATCC10231, ATCC14053, ATCC24433, and the

71

echinocandin-resistant clinical isolates 42379 and 53264 were also studied (4, 19). For biofilm formation,

72

strains were grown overnight at 30°C in yeast peptone dextrose (YPD) (Fisher Scientific). The harvested

73

cells were washed twice and re-suspended in phosphate-buffered saline (PBS) (Sigma-Aldrich). For

74

biofilm studies, a standardized cell suspension was made in RPMI-1640 (Mediatech) supplemented with

75

L-glutamine (Gibco) and buffered with 165mM morpholinepropanesulfonic acid (MOPS) (Sigma-

76

Aldrich) to pH 7.0, to attain a cell density of 1x106 cells/ml. Mechanistic studies of the vacuole utilized

77

strains impaired in vacuolar ATPase (V-ATPase) function that have previously studied by our groups: (i)

78

the vph1∆ null mutant, and its re-integrant, vph1∆ + VPH1, the stv1∆ null mutant, and its re-integrant,

79

stv1∆ + STV1,and DAY185 as a positive control (20), and (ii) the tetR-VMA3 strain, a conditional mutant

80

in which VMA3 has been placed under the control of a tetracycline-regulated promoter (21) and its

81

positive control THE1-CIp10, in which the URA3 gene has been integrated into the genome (22). The

82

vph1Δ and stv1Δ strains are each deficient in one of the two isoforms of the Voa subunit of V-ATPase,

83

VPH1 and STV1. Specifically, the vph1Δ strain is deficient in V-ATPase at the vacuolar membrane, and

84

the stv1Δ strain is deficient in V-ATPase at Golgi and pre-vacuolar compartments (23). In the tetR-

85

VMA3 strain, treatment with 20μg/ml doxycycline (DOX) abolishes all V-ATPase activity.

86

Biofilm Formation and Susceptibility Assays. The antifungal activity of quinacrine dihydrochloride

87

(QNC, FlukaBiochemika) against pre-formed, mature biofilms, for prevention of biofilm formation, and 4

5

88

pH dependence, was assessed using the XTT [i.e., 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-

89

tetrazolium-5-carboxanilide] (Sigma-Aldrich) reduction assay in a standard 96-well static microplate

90

model as described by Ramage and Ribot-Lopez (24). In detail, 100μL of the standardized cell

91

suspension was added to designated wells of 96-well microtiter plates and cell-free RPMI-1640 was

92

added to designated negative control wells; plates were incubated at 37°C for 24 hours to allow for

93

biofilm formation. Mature biofilms were washed gently three times with PBS and incubated again for 24

94

hours at 37°C with increasing concentrations (4 – 2048µg/ml in doubling serial dilutions in buffered

95

RPMI-1640) of QNC. Drug-free buffered RPMI-1640 was added to wells designated for positive

96

controls (i.e. no treatment). After QNC treatment, biofilms were again washed gently with PBS. The

97

XTT-reduction assay was used to determine the metabolic activity of the biofilms (24). Microtiter plates

98

were incubated at 37°C for 1.5 to 2 hours to allow the optimal formation of formazan. Color intensity of

99

XTT was measured at OD490 on a BioTek ELx808 microplate reader (BioTek Instruments).The antifungal

100

activity of each QNC treatment was expressed as a percentage relative to the metabolic activity of the

101

untreated biofilms. Each biofilm experiment was repeated three times independently, and results

102

presented are the mean of all three experiments.

103

To test the pH-dependence of QNC antifungal activity for prevention of biofilm formation, the

104

protocol above was followed, except cells were incubated in RPMI-1640 buffered to a specified pH;

105

acidic pH buffers are comprised of 50mM succinic acid/50mM Na2PO4 and alkaline pH buffers are

106

comprised of 50mM MES hydrate/50mM MOPS.

107

To test for interactions between QNC and known antifungals against mature biofilms, we used a

108

checkerboard assay (25).Two-fold dilutions of amphotericin B (AMB);caspofungin (CAS); and

109

fluconazole (FLC) were prepared according to CLSI guidelines (26). After formation of biofilms, serially

110

increasing concentrations of QNC and antifungal drugs were combined. After 24h of co-incubation, the

111

XTT assay was used to determine biofilm metabolic activity.

112

Growth Curves and Growth at varying pH. Growth was assessed in complete synthetic media (CSM) by

113

co-incubating strains with serially increasing concentrations of QNC and measuring OD600 at fixed 5

6

114

intervals in an automated Synergy-H1M Microplate Reader (Bio-Tek Instruments). Growth curves were

115

generated using Prism 6.0 (Graph-Pad Software, Inc.). Growth at pH 4.0-8.5 was also tested in liquid

116

CSM: C. albicans SC5314 was tested for growth at varying pH (4.0–8.5) using pH-buffered CSM ±QNC

117

[1024µg/ml]. C. albicans SC5314, V-ATPase-impaired strains, and their controls were tested for growth

118

at varying pH (4.0 – 8.5) using pH-buffered CSM with agar ± QNC[1024µg/ml], and with and without

119

DOX for strains THE1-CIp10 and tetR-VMA3. Cells from overnight cultures were washed and counted

120

as previously described (19). PBS was inoculated with cells from overnight cultures to a starting density

121

of 108 cells/ml. Next, a total of six 5-fold dilutions (1.0×108, 2.0×107, 4.0×106, 8.0×105, 1.6×105,

122

3.2×104, 6.4×103, and 1.3×103 cells/ml) were completed in 96-well plates, and cells were spotted onto

123

agar plates using a multiblot replicator (VP Scientific) and incubated at 37°C for 48 hours.

124

Filamentation Assays. Solid media tested were YPD with 10% fetal calf serum (FCS, Invitrogen);

125

Medium 199 supplemented with L-glutamine; and RPMI/L-glutamine. All filamentation media were

126

prepared ±QNC[1024µg/ml], and all filamentation media were prepared with 2% (w/v) agar. 3µl SC5314

127

cells from overnight cultures were spotted onto agar plates and incubated at 37°C for 120h. Liquid

128

RPMI-1640 ±QNC[1024µg/ml] was inoculated with cells from overnight culture to a starting density of

129

5×106 cells/ml and grown at 37°C, 200rpm for 2 to 24h. Cells were visualized via light microscopy at

130

selected time-points.

131

Vacuolar Morphology and Fluorescence Imaging. FM4-64 is a lipophilic dye that stains membranes and

132

is actively endocytosed to the vacuole; due to these properties, it has been used extensively as both a

133

vacuolar membrane stain and an endocytic marker. To stain vacuoles with FM4-64 [N-(3-

134

triethylammoniumpropyl)-4-(6-(4- (diethylamino)phenyl)hexatrienyl) pyridiniumdibromide, Life

135

Technologies], cells were first grown in YPD overnight. Next, cells were washed in 1X PBS, then

136

resuspended in fresh YPD with or without QNC [1024µg/ml] and grown for 4 hours. Cells were

137

resuspended to an OD600 of 2 to 4 in YPD with 40 µM FM4-64, incubated for 15 min at 30°C, and then

138

washed and resuspended in fresh YPD and incubated for 60 min at 30°C with images taken every 15

139

minutes via microscopy using the Texas Red filter. A Zeiss Imager M1 system was used for capturing 6

7

140

images using DIC and fluorescence microscopy, and rendering was completed using AxioVision 4.7

141

software (Zeiss). To quantify vacuolar morphology, images of at least 10 random fields were taken per

142

condition and the number of visible vacuolar vesicles in 100 cells per experiment was determined; then,

143

cells were grouped into one of three categories (1-2, 3-4 or ≥5 vacuoles/cell). Data from three

144

independent experiments with standard deviation are presented here as previously shown in Baars et al.

145

(27).

146

Statistical analyses. Metabolic activity of the prevention and treatment groups was compared to controls

147

using one- or two-way analyses of variance (ANOVA) and Dunnett’s multiple-comparison post-test.

148

Differences were considered significant at p 0.5-4.0 indicates ‘indifference’, and >4.0

158

indicates ‘antagonism.’

159 160

RESULTS

161

Quinacrine demonstrates in vitro activity against C. albicans biofilms. The antifungal effect of 4-

162

2048µg/ml QNC against mature C. albicans SC5314 biofilms was tested in a static microplate model (Fig

163

1A). QNC>128µg/ml caused a statistically significant reduction in C. albicans biofilm metabolic

164

activity. The concentration of QNC needed to reduce biofilm (sessile) metabolic activity by 50%

165

(sMIC50) was 256µg/ml and by 90% (sMIC90) was 1024µg/ml. Next, we assayed QNC’s ability to prevent 7

8

166

biofilm formation by co-incubating SC5314 with serially increasing concentrations of QNC (Figure 1B).

167

QNC>256µg/ml significantly decreased biofilm formation and, notably, at a concentration of 1024µg/ml

168

QNC, biofilms were 98.5% inhibited. The sMIC50 of QNC for biofilm prevention was 512µg/ml and the

169

sMIC90 was 1024µg/ml. A small but statistically significant difference was also seen at 8 and 16µg/ml

170

QNC. Thus, high-dose QNC was active for the prevention and treatment of C. albicans biofilms in vitro.

171

Light microscopy to visualize gross biofilm structure indicated that QNC treatment of pre-formed

172

biofilms resulted in a subtle reduction in overall biofilm density (data not shown). This modest difference

173

is consistent with prior studies in our laboratory, in which treatment with repurposed agents has had little

174

gross structural impact on mature biofilms (30, 31). In contrast, there was a dramatic reduction in biofilm

175

density when co-incubated with QNC in our model of biofilm prevention (Figure 1C). Further, stunted

176

hyphal cells were observed in biofilms co-incubated with high doses of QNC (Figure 1C). Next, QNC

177

activity against biofilms formed by C. albicans clinical isolates was tested (Supplemental Figure 1). The

178

clinical isolates ATCC10231, ATCC14053 and ATCC24433 were studied, as well as 42379 and 53264,

179

two clinical isolates with mutations in the fks1 gene conferring echinocandin resistance (4, 19). QNC had

180

activity against both mature biofilms and biofilm formation for all five clinical isolates.

181

Next, the interaction of QNC with standard antifungal agents (amphotericin, AMB; caspofungin, CAS;

182

or fluconazole, FLC) was tested in biofilm checkerboard assays (Table 1). The combination of QNC with

183

FLC against mature biofilms had no interaction (an ‘indifferent effect’). However, both AMB and CAS

184

had synergy with QNC against mature biofilms. Thus, AMB and CAS act synergistically with QNC

185

against mature, pre-formed C. albicans biofilms.

186

Quinacrine activity is pH-dependent. To test whether QNC effects on biofilm were due to growth

187

inhibition, we tested growth of QNC-treated planktonic cells in pH-buffered media as well as media that

188

had not been buffered to a specific pH (i.e., unbuffered media). Planktonic growth with QNC was

189

assessed for pH dependency in liquid CSM buffered to pH 4 – 8.5±QNC[1024µg/ml] for 30h. Cells grew

190

well at acidic pH, but no growth was detected in basic media (Figure 2A). Interestingly, in unbuffered

191

media, cells grown in the presence of QNC grew as well as untreated cells (Figure 2A). Additionally, 8

9

192

serial dilutions of C. albicans SC5314 planktonic cells spotted onto unbuffered agar media containing

193

QNC[16 – 2048µg/ml] showed no difference in colony growth (Figure 2B). However, when C. albicans

194

SC5314 cells were spotted onto solid media buffered to pH 4.0 to 8.5 ±QNC, no growth was seen on

195

alkaline media with QNC (Figure 2C). Therefore, we conclude that QNC affects planktonic growth at

196

alkaline pH, but has no effect on growth on unbuffered, media.

197

We next tested QNC anti-biofilm activity for pH-dependency. QNC significantly decreased biofilm

198

formation at pH 7.5 and 8.5, preventing 87.3% and 88.5% biofilm formation, respectively (Figure 3). In

199

contrast, QNC had only modest effects against C. albicans biofilm formation at more acidic pH (4.0-5.0)

200

(Figure 3). Therefore, the effects of QNC against biofilms are pH dependent, and likely due to impaired

201

growth at alkaline pH.

202

Quinacrine inhibits C. albicans filamentation. C. albicans biofilms are composed of complex

203

communities of both hyphal and yeast cells surrounded by an extracellular matrix; the presence of hyphae

204

is required for biofilm structural integrity. Because we observed less extensive filaments in biofilms

205

formed in the presence of high-dose QNC (Fig 1C), and because we observed that cells grown on agar

206

media with QNC showed a smooth rather than wrinkly appearance (data not shown), suggesting a defect

207

in filamentation, we next analyzed the effect of QNC on C. albicans hyphal formation. On unbuffered

208

solid media, filamentation was reduced by QNC in a dose-dependent manner under a variety of conditions

209

(Figure 4A). Hyphal formation in the presence of high-dose QNC was further studied via a 24h time-

210

course in liquid RPMI. After 2h incubation with QNC, germ tubes had emerged and QNC localized

211

primarily to the vacuole (data not shown). By 6h, filamentation was disrupted, with cells predominantly

212

found as pseudohyphae (Figure 4B). After 24h, cells remained in a pseudohyphal state. Notably, QNC

213

fluorescence still localized to vacuoles, but also displayed some cytosolic localization (Figure 4B). Thus,

214

QNC inhibits C. albicans filamentation while simultaneously localizing primarily to the vacuole.

215

Quinacrine inhibits active endocytosis and passive diffusion into the vacuole. Vacuolar morphology

216

differs greatly between yeast and filamentous growth forms. In the yeast form vacuoles are spherical,

217

whereas hyphal vacuoles are enlarged and ellipsoid. Upon induction of hyphal formation, vacuoles 9

10

218

evaginate and segregate into pseudohyphal cells, where they later enlarge and elongate (32). Under

219

hyphal-inducing conditions, cells treated with high dose QNC had vacuolar morphology resembling yeast

220

or pseudohyphae rather than hyphae. After 4h QNC treatment, vacuoles demonstrated evagination into

221

pseudohyphae (data not shown), but by 6h, vacuoles in QNC-treated cells remained spherical rather than

222

ellipsoid (Figure 4B). Due to these changes in the vacuole during morphogenic transitioning, we next

223

sought to assay both vacuolar morphology and functional aspects of vacuolar trafficking in the presence

224

of high-dose QNC.

225

We utilized FM4-64, a lipophilic dye that is actively endo- and exocytosed. FM4-64 initially stains

226

endocytic vesicle membranes and then accumulates at the vacuolar membrane before being actively

227

exocytosed back to the plasma membrane (PM) (33). To assay for defective endocytosis, we treated yeast

228

cells with QNC for 4h, then stained with FM4-64 and imaged at 15min intervals. At 15min, FM4-64

229

accumulated within discrete endocytic structures in the cell but did not reach the vacuolar membrane. At

230

30min, FM4-64 was again visible at the PM, indicating active exocytosis. At 1h, faint FM4-64 staining

231

of vacuolar membranes was visible, suggesting some degree of completed endocytosis to the vacuole

232

(Figure 5A). These data indicate an endocytic delay of approximately 45min.Actively metabolizing cells

233

contain multiple medium-sized vacuoles; in contrast, exposure to hypo-osmotic conditions causes the

234

vacuole to enlarge, resulting in a single vacuole occupying a large volume of the cell (34). We observed

235

that QNC treatment resulted in enlarged single vacuoles, compared to multiple smaller vacuoles observed

236

in controls. Quantification of this phenotype revealed that QNC treatment resulted in a significantly

237

larger portion of cells with one to two vacuoles, whereas cells containing three to four vacuoles were

238

more common in the controls (Figure 5B).

239

Quinacrine affects growth of C. albicans V-ATPase mutants. The vacuolar-ATPase (V-ATPase) mediates

240

acidification of the vacuole, and strains in S. cerevisiae and C. albicans bearing genetic deletions of V-

241

ATPase subunits are characterized by alkaline pH-conditional lethality (21, 35). Further, V-ATPase

242

deficient cells are viable on unbuffered media, but not on buffered media at alkaline pH, mirroring the

243

pH-dependency of QNC(21). Functional V-ATPase allows for active transport of protons across the 10

11

244

vacuolar membrane; accordingly, proper assembly of V-ATPase is required for vacuolar acidification

245

(21). We therefore tested whether QNC’s mechanism of action was related to V-ATPase activity using C.

246

albicans strains previously studied in our laboratory with partial (stv1Δ and vph1Δ strains) or total (tetR-

247

VMA3) loss of V-ATPase function (20, 21).Both vph1∆ and stv1∆ mutants were viable at all QNC

248

concentrations, as were the DAY185 control and the isogenic VPH1 and STV1 reintegrant strains (Figure

249

6A, 6B). Interestingly, upon repression of the VMA3 gene with DOX, the tetR-VMA3strain exhibited

250

decreased growth with increasing QNC concentrations (Figure 6C). Notably, minimal growth was seen at

251

QNC[1024µg/ml] and no growth was seen at QNC[2048µg/ml] (Figure 6C). These results suggest that

252

QNC may act in a parallel pH-dependent pathway to the V-ATPase protein complex.

253 254 255

DISCUSSION We systematically examined QNC’s activity against C. albicans biofilms, planktonic growth and

256

filamentation. Our findings indicate that high-dose QNC is effective for both prevention and treatment of

257

C. albicans biofilms. QNC≥128µg/ml caused a statistically significant decrease in the metabolic activity

258

of pre-formed, mature C. albicans SC5314 biofilms and QNC≥256µg/ml caused a statistically significant

259

decrease in biofilm formation. A concentration of QNC[2048µg/ml] was required to completely prevent

260

biofilm formation. Similar results were seen when QNC was utilized against clinical isolate biofilms.

261

We also studied QNC in combination with traditional antifungal drugs using biofilm checkerboard

262

analyses and found that QNC and CAS, as well as QNC and AMB display synergistic activity against C.

263

albicans biofilms. Recently, high doses of AMB have been shown to cause vacuolar membrane

264

fragmentation; this activity has been hypothesized to contribute to the fungicidal activity of AMB (36).

265

Our data indicate that a sub-lethal dose of AMB (0.25µg/ml) enhances the effectiveness of QNC in

266

synergistic fashion against mature biofilms. Further, we have shown that QNC localizes primarily to the

267

vacuole, in accordance with many previous studies showing QNC accumulation in vacuoles. These data

268

therefore suggest that the antifungal action of QNC occurs via a vacuole-specific mechanism. Quinacrine

269

inhibition of vacuolar function is probably related to its basic properties. Under acidic conditions the 11

12

270

weakly basic QNC passively diffuses across membranes and accumulates in acidic compartments. There,

271

QNC likely becomes protonated (i.e., by excess protons available under acidic environmental conditions,

272

or available in acidic compartments even under alkaline conditions). This protonation is likely required

273

for QNC fluorescence to occur (37). Thus, protonation of QNC in acidic vacuoles would interfere with

274

normal vacuolar function by alkalinizing the vacuole. Under acidic environmental conditions, however,

275

there would be enough excess protons available to quench quinacrine and thus avoid vacuolar

276

alkalinization.

277

We also discovered a pH-dependent phenotype in which biofilms co-incubated with QNC at acidic pH

278

displayed only slightly reduced levels of both filamentation and biofilm formation, whereas biofilms co-

279

incubated with QNC at alkaline pH had minimal adherent cells, no filamentation, and no visible biofilm.

280

The C. albicans response to host pH is well described, and is critical for survival within the host as well

281

as virulence (38). Consistent with this, C. albicans planktonic cells were unable to grow at alkaline pH in

282

the presence of QNC in liquid or on solid media. However, QNC-treated planktonic cells were able to

283

grow as well as untreated cells in unbuffered media. Cells grown on unbuffered media are able to

284

modulate environmental pH (39); thus, these results suggest that C. albicans cells are able to negate the

285

effects of QNC via manipulation of extracellular pH. In this model, if extracellular pH cannot be

286

modulated due to pH buffering, QNC affects both C. albicans growth and filamentation. Because the

287

physiological pH of the mammalian host environment is under tight homeostatic regulation, QNC activity

288

should be achievable in vivo. Further studies are required to test this hypothesis.

289

Filamentation is a major contributor to biofilm development and pathogenesis; we found that

290

filamentation on solid media was inhibited at QNC>256µg/ml in a dose-dependent manner. Additionally,

291

we found that QNC≥1024µg/ml completely inhibited filamentation in liquid culture. Therefore, high-

292

dose QNC inhibits C. albicans hyphal formation. A previous study found that, in the presence of proline,

293

a filament-inducer, QNC>1mM repressed hyphal induction in C. albicans (16). Exploiting QNC’s ability

294

to fluoresce, we found that QNC localized to vacuoles 2h after induction of filamentation, and

295

additionally, by 24h had dispersed throughout the cytosol. In C. neoformans, QNC uptake was 12

13

296

diminished at low pH, suggesting that QNC’s weakly basic properties drive its localization to the vacuole

297

(17, 40). We have shown that filamentation is impaired in the presence of QNC, in accordance with a

298

previously published study showing QNC decreased filamentation in the presence of proline (16). We

299

have tested filamentation in the presence of QNC under a wide variety of other filamentation-inducing

300

conditions, and found that QNC treatment led to reduced filamentation under every condition tested.

301

Therefore, QNC is a strong repressor of filamentation in C. albicans. A possible explanation is suggested

302

by QNC localization to the cytosol after 24h of incubation in filamentation-inducing liquid media. At

303

high extracellular pH, the pH gradient from the alkaline extracellular media to the neutral cytosol, then to

304

acidic organelles, could drive QNC into acidified intracellular compartments. We hypothesize that QNC,

305

a weak base, may partially alkalinize the acidic vacuole by taking up excess protons. After alkalinizing

306

the vacuole, QNC would migrate to the now relatively acidic cytosol, disrupting pH-dependent processes

307

in C. albicans including filamentation. However, at low extracellular pH, this gradient would not be

308

present, preventing QNC accumulation in internal organelles and thus allowing some degree of

309

filamentation to occur. Mechanistic studies of pH-dependent regulation of filamentation are currently

310

underway in our laboratories in order to further define these processes.

311

S. cerevisiae and C. albicans strains bearing genetic deletions of V-ATPase subunits develop a similar,

312

pH dependent phenotype to QNC-treated cells, characterized by alkaline pH-conditional lethality (21, 35).

313

Our results suggest that QNC inhibits acidification of the vacuole and acts in a similar or parallel manner

314

to V-ATPase pumps. To further clarify this possibility, we treated C. albicans V-ATPase mutants with

315

QNC. The V-ATPase complex consists of the V1 and Vo subcomplexes, in which V1 is responsible for

316

ATP hydrolysis and Vo is responsible for proton transport into the vacuole (35). We have previously

317

studied C. albicans V-ATPase subunits Voa, encoded byVPH1 (the vacuolar Voa subunit isoform) and

318

STV1 (the Golgi and endosomal Voa subunit isoform), and Voc, encoded by VMA3 (20, 21). Loss of

319

VMA3 suppresses all V-ATPase activity and more severely inhibits filamentation than loss of VPH1or

320

STV1in C. albicans (20), and this difference may implicate a mechanism whereby-ATPase complexes at

321

the vacuole as well as other organelles contribute to filamentation. These experiments demonstrate that, 13

14

322

similar to its effect on wild-type cells, QNC does not affect vph1Δ and stv1Δ planktonic growth. In

323

contrast, the tetR-VMA3 strain demonstrates increased susceptibility to QNC, with 2048µg/ml QNC

324

completely inhibiting planktonic growth. Because vph1Δ and stv1Δ mutants have partially impaired V-

325

ATPase function in specific organelles, but tetR-VMA3 has fully impaired V-ATPase, a drug specifically

326

targeting the V-ATPase would be expected to have a greater effect on growth of the vph1Δ and stv1Δ

327

mutants than the tetR-VMA3 strain, in which V-ATPase is already fully impaired. However, we observed

328

the opposite effect of QNC on these strains, suggesting that QNC’s antifungal activity does not work by

329

disrupting V-ATPase complexes, but rather by disrupting a pH-dependent pathway which functions in

330

parallel with the V-ATPase complex.

331

Oral quinacrine has established efficacy for the systemic treatment of malaria and other parasitic

332

diseases. In humans, QNC is highly and rapidly absorbed in the gastrointestinal tract after oral

333

administration; of note, QNC is highly water soluble and has an extremely high volume of distribution.

334

Thus, while serum concentrations of QNC are low, QNC achieves extremely high tissue concentrations,

335

particularly in the spleen, pancreas, lungs, bone marrow, skeletal muscles, and especially in the liver (41).

336

Concentrations in the liver may be 20,000 times that in the plasma (42). Due to accumulation into tissue,

337

QNC has a long half-life and is slowly excreted through urine and other bodily fluids (43). Thus,

338

quinacrine may achieve clinically relevant concentrations in tissues including the liver and spleen, which

339

have direct relevance to disseminated candidiasis, particularly in combination with AMB or CAS, where

340

we demonstrated in vitro synergy. Common adverse drug reactions of QNC include gastrointestinal

341

effects (e.g., nausea, diarrhea, cramps) and dermatologic effects including rash, dermatitis, and yellowish

342

skin discoloration (skin accumulation) (43, 44). Alternatively, high concentrations could be utilized as a

343

local antifungal lock strategy in catheter-related blood stream infections (7). Overall, these findings

344

demonstrate that, although QNC activity is not biofilm-specific, QNC inhibits C. albicans growth,

345

filamentation, and normal vacuolar function in a pH-dependent manner. Further investigation of the

346

clinical utility and efficacy of QNC against invasive candidiasis and other invasive systemic mycoses is

347

warranted. 14

15 348

ACKNOWLEDGEMENTS

349

We thank William Fonzi (Georgetown University) for providing strain SC5314.

350

This work was supported in part by Department of Veterans Affairs Merit Award 5I01BX001130 [to

351

S.A.L.]; and the Biomedical Research Institute of New Mexico [to S.A.L.]. A.A.C.D. was supported by

352

the National Institute of General Medical Sciences (NIGMS) through the Institutional Research and

353

Career Development Award and the Academic Science Education and Research Training Program

354

(IRACDA-ASERT).

355

15

16 356

REFERENCES

357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Ramage G, Saville SP, Thomas DP, López-Ribot JL. 2005. Candida albicans: an update. Eukaryot. Cell 4:633–638. Douglas LJ. 2003. Candida biofilms and their role in infection. Trends Microbiol.11:30–36. Bachmann SP, Ramage G, VandeWalle K, Patterson TF, Wickes BL, López-Ribot JL. 2003. Antifungal combinations against Candida albicans biofilms in vitro. Antimicrob. Agents Chemother.47:3657–3659. Ramage G, Mowat E, Jones B, Williams C, Lopez-Ribot J. 2009. Our current understanding of fungal biofilms. Crit. Rev. Microbiol. 35:340–355. Katragkou A, Chatzimoschou A, Simitsopoulou M, Dalakiouridou M, Diza-Mataftsi E, Tsantali C, Roilides E. 2008. Differential activities of newer antifungal agents against Candida albicans and Candida parapsilosis biofilms.Antimicrob. Agents Chemother.52:357–360. Tobudic S, Kratzer C, Lassnigg A, Graninger W, Presterl E. 2010. In vitro activity of antifungal combinations against Candida albicans biofilms. J. Antimicrob. Chemother.65:271–274. Walraven CJ, Lee SA. 2013. Antifungal Lock Therapy. Antimicrob. Agents Chemother.57:1–8. Lewis RE, Kontoyiannis DP, Darouiche RO, Raad II, Prince RA. 2002. Antifungal activity of amphotericin B, fluconazole, and voriconazole in an in vitro model of Candida catheter-related bloodstream infection. Antimicrob. Agents Chemother.46:3499–3505. Ehsanian R, Van Waes C, Feller SM. 2011. Beyond DNA binding - a review of the potential mechanisms mediating quinacrine’s therapeutic activities in parasitic infections, inflammation, and cancers. Cell Commun. Signal. CCS 9:13. Roy C, Gagné V, Fernandes MJG, Marceau F. 2013. High affinity capture and concentration of quinacrine in polymorphonuclear neutrophils via vacuolar ATPase-mediated ion trapping: comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs. Toxicol. Appl. Pharmacol. Wallace DJ, Gudsoorkar VS, Weisman MH, Venuturupalli SR. 2012. New insights into mechanisms of therapeutic effects of antimalarial agents in SLE. Nat. Rev. Rheumatol. 8:522–533. Koranda FC. 1981. Antimalarials. J. Am. Acad. Dermatol. 4:650–655. Finkelstein DB, Strausberg S. 1979. Metabolism of alpha-factor by a mating type cells of Saccharomyces cerevisiae. J. Biol. Chem. 254:796–803. Delling U, Raymond M, Schurr E. 1998. Identification of Saccharomyces cerevisiae genes conferring resistance to quinoline ring-containing antimalarial drugs. Antimicrob. Agents Chemother. 42:1034–1041. Weisman LS, Bacallao R, Wickner W. 1987. Multiple methods of visualizing the yeast vacuole permit evaluation of its morphology and inheritance during the cell cycle. J. Cell Biol. 105:1539– 1547. Land GA, McDonald WC, Stjernholm RL, Friedman L. 1975. Factors affecting filamentation in Candida albicans: changes in respiratory activity of Candida albicans during filamentation. Infect. Immun. 12:119–127. Harrison TS, Griffin GE, Levitz SM. 2000. Conditional lethality of the diprotic weak bases chloroquine and quinacrine against Cryptococcus neoformans. J. Infect. Dis. 182:283–289. Levitz SM, Harrison TS, Tabuni A, Liu X. 1997. Chloroquine induces human mononuclear phagocytes to inhibit and kill Cryptococcus neoformans by a mechanism independent of iron deprivation. J. Clin. Invest. 100:1640–1646. LaFleur MD, Kumamoto CA, Lewis K. 2006. Candida albicans biofilms produce antifungaltolerant persister cells. Antimicrob. Agents Chemother.50:3839–3846. Raines SM, Rane HS, Bernardo SM, Binder JL, Lee SA, Parra KJ. 2013. Deletion of vacuolar proton-translocating ATPase V(o)a isoforms clarifies the role of vacuolar pH as a determinant of virulence-associated traits in Candida albicans. J. Biol. Chem. 288:6190–6201. 16

17 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453

21. 22. 23. 24. 25. 26. 27. 28. 29.

30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.

Rane HS, Bernardo SM, Raines SM, Binder JL, Parra KJ, Lee SA. 2013. Candida albicans VMA3 is necessary for V-ATPase assembly and function and contributes to secretion and filamentation. Eukaryot. Cell 12:1369–1382. Bernardo SM, Khalique Z, Kot J, Jones JK, Lee SA. 2008. Candida albicans VPS1 contributes to protease secretion, filamentation, and biofilm formation. Fungal Genet. Biol. FG B 45:861–877. Patenaude C, Zhang Y, Cormack B, Köhler J, Rao R. 2013. Essential role for vacuolar acidification in Candida albicans virulence. J. Biol. Chem. 288:26256–26264. Ramage G, López-Ribot JL. 2005. Techniques for antifungal susceptibility testing of Candida albicans biofilms. Methods Mol. Med. 118:71–79. Pillai SK, Moellering RC, Eliopoulos GM. 2005. Antibiotics in Laboratory Medicine, 5th ed. Lippincott Williams & Wilkins. Clinical and Laboratory Standards Institute. 2008. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Third Informational Supplement M27-S3, 15th ed. Clinical and Laboratory Standards Institute, Wayne, PA. Baars TL, Petri S, Peters C, Mayer A. 2007. Role of the V-ATPase in regulation of the vacuolar fission-fusion equilibrium. Mol. Biol. Cell 18:3873–3882. Odds FC. 2003. Synergy, antagonism, and what the chequerboard puts between them. J. Antimicrob. Chemother.52:1. Holmes AR, Keniya MV, Ivnitski-Steele I, Monk BC, Lamping E, Sklar LA, Cannon RD. 2012. The monoamine oxidase A inhibitor clorgyline is a broad-spectrum inhibitor of fungal ABC and MFS transporter efflux pump activities which reverses the azole resistance of Candida albicans and Candida glabrata clinical isolates. Antimicrob. Agents Chemother.56:1508–1515. Ku TSN, Bernardo SM, Lee SA. 2011. In vitro assessment of the antifungal and paradoxical activity of different echinocandins against Candida tropicalis biofilms. J. Med. Microbiol. 60:1708– 1710. Ku TSN, Palanisamy SKA, Lee SA. 2010. Susceptibility of Candida albicans biofilms to azithromycin, tigecycline and vancomycin and the interaction between tigecycline and antifungals. Int. J. Antimicrob. Agents 36:441–446. Veses V, Gow NAR. 2008. Vacuolar dynamics during the morphogenetic transition in Candida albicans. FEMS Yeast Res. 8:1339–1348. Vida TA, Emr SD. 1995. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J. Cell Biol. 128:779–792. Li SC, Kane PM. 2009. The yeast lysosome-like vacuole: endpoint and crossroads. Biochim. Biophys. Acta BBA - Mol. Cell Res. 1793:650–663. Kane PM. 2006. The where, when, and how of organelle acidification by the yeast vacuolar H+ATPase. Microbiol. Mol. Biol. Rev. MMBR 70:177–191. Brajtburg J, Powderly WG, Kobayashi GS, Medoff G. 1990. Amphotericin B: current understanding of mechanisms of action. Antimicrob. Agents Chemother.34:183–188. Weisman LS, Bacallao R, Wickner W. 1987. Multiple methods of visualizing the yeast vacuole permit evaluation of its morphology and inheritance during the cell cycle. J. Cell Biol. 105:153947. Fonzi WA. 2002. Role of pH response in Candida albicans virulence. Mycoses 45:16–21. Vylkova S, Carman AJ, Danhof HA, Collette JR, Zhou H, Lorenz MC. 2011. The fungal pathogen Candida albicans autoinduces hyphal morphogenesis by raising extracellular pH. mBio 2:e00055–00011. O’Neill PM, Bray PG, Hawley SR, Ward SA, Park BK. 1998. 4-Aminoquinolines--past, present, and future: a chemical perspective. Pharmacol. Ther.77:29–58. Marshall EK, Dearborn EH. 1946. The relation of the plasma concentration of quinacrine to its antimalarial activity. J. Pharmacol. Exp. Ther. 88:142–153. 17

18 454 455 456 457 458 459 460

42. 43. 44.

US Pharmacopeial Convention Inc Staff. 1997. Drug Information for the Health Care Professional 17th edition. Convention, Inc, Rockville, MD. Quinacrine DRUGDEX® System. Thomson Healthcare, Greenwood Village, CO. Wallace DJ. 1989. The use of quinacrine (Atabrine) in rheumatic diseases: a reexamination. Semin. Arthritis Rheum. 18:282–296.

18

19 461 462

TABLES

463 464

TABLE 1.Biofilm checkerboard assays of varying QNC concentrations (4-2048µg/mL) in

465

combination with varying concentrations of Antifungals (Af) .Amphotericin B (AMB) concentrations

466

0.0156–1µg/ml; caspofungin (CAS) concentrations 0.0156–1µg/ml; fluconazole (FLC) concentrations 2–

467

128µg/ml; QNC concentrations 4-2048µg/ml, all in 2-fold dilutions, on C. albicans SC5314 mature,

468

preformed biofilms. Synergy or indifference was demonstrated quantitatively in terms of FIC (fractional

469

inhibitory concentration) and FIC index (FICI) definitions. Experiment

QNC x AMB

QNC x CAS

QNC x FLC

Agent

sMIC50 (mg/L) of each agent

FICI

Alone

Combination

(combination)

AMB

0.25

0.03125

0.375

QNC

256

64

CAS

0.125

0.03125

Outcome

Synergism

Synergism 0.3125

QNC

64

4

FLC

64

16

Indifference 1.25

QNC

128

128

470 471

19

20 472

FIGURE LEGENDS

473

Figure 1. Effect of varying quinacrine (QNC) concentrations on C. albicans SC5314 biofilm

474

formation and mature biofilms. (A) The in vitro effect of QNC against mature (pre-formed) C. albicans

475

SC5314 biofilms was assayed in a 96-well static microplate biofilm model. Biofilms were challenged

476

with buffered RPMI-1640 containing QNC at concentrations 4-2048µg/ml for 24 hours. MIC50 =

477

128µg/ml, MIC80= 512µg/ml (B) The effect of varying concentrations of QNC against formation of C.

478

albicans SC5314 biofilms was assessed in a 96-well static microplate model. Cells were co-incubated

479

with RPMI-1640 containing QNC at concentrations 4-2048 µg/ml for 24 hours. MIC50 = 256µg/ml,

480

MIC80= 512µg/ml. Bar graphs represent experiments performed three times independently (biological

481

replicates) each time in quadruplicate (technical replicates). Metabolic activity was expressed as a

482

percentage relative to the metabolic activity of the untreated biofilms. Statistical analysis was completed

483

using ANOVA and significant differences (p < 0.05) in reduction in biofilm metabolic activity with QNC

484

compared to the no-treatment control (cells containing only media and biofilms) are indicated by asterisks

485

(*). (C) Structural analyses of QNC activity against C. albicans SC5314 biofilm formation (prevention)

486

was performed using standard light microscopy, 40X power field. Biofilms were formed as described

487

above and biofilm morphology was assessed; representative images are shown. Visual differences in

488

biofilm architecture are apparent at five different QNC concentrations (128, 256, 512, 1024 and

489

2048µg/ml).

490 491

Figure 2. Effect of QNC on C. albicans SC5314 planktonic growth on unbuffered and pH-buffered

492

media. (A) Effect of QNC on growth in liquid media at varying pH. SC5314 cells from overnight

493

cultures were diluted to an OD600nm=0.05 in CSM either without a pH buffer (unbuffered) or buffered to

494

pH 4.0 - 8.5 as indicated, with or without QNC [1024µg/ml] added. Cells were grown at 30°C for 24h

495

with OD600nm readings taken at 15 min intervals. Growth of SC5314 ± 1024µg/ml QNC, as indicated by

496

OD600nm readings, is shown in a separate graph for each pH condition tested. Growth of SC5314 is 20

21 497

indicated by open black circles, and growth of SC5314 + 1024µg/ml QNC is indicated by closed red

498

circles. (B) Effect of varying QNC on growth on unbuffered CSM agar. Serial dilutions of overnight

499

cultures of SC5314 were spotted onto agar media containing varying concentrations of QNC and

500

incubated for 2 days at 30°C. The arrow to the right indicates decreasing cell densities (1.0×108, 2.0×107,

501

4.0×106, 8.0×105, 1.6×105, and 3.2×104 cells/ml from left to right) and decreasing concentrations of QNC

502

(0, 16, 32, 128, 256, 1024 and 2048 µg/ml) are noted on the bottom. (C) Effect of QNC on growth on

503

solid media at varying pH. Serial dilutions of SC5314 overnight cultures were spotted onto solid CSM

504

pH-buffered agar media containing QNC[1024µg/ml] and incubated for 48h at 30°C. The arrow to the

505

right indicates decreasing cell densities (1.0×108, 2.0×107, 4.0×106, 8.0×105, 1.6×105, and 3.2×104

506

cells/ml from left to right) and the pH of the media is noted on the bottom.

507 508

Figure 3. Effect of QNC [1024µg/ml] on C. albicans SC5314 biofilm formation in pH-buffered

509

RPMI-1640. The effect of varying concentrations of QNC against formation of C. albicans SC5314

510

biofilms was assessed in a 96-well static microplate model. Cells were co-incubated with pH-buffered

511

RPMI-1640 containing QNC[1024µg/ml] for 24 hours.

512 513

Figure 4. Effect of varying QNC concentrations on C. albicans SC5314 filamentation on solid media

514

and QNC[1024µg/ml] in liquid media. (A) Growth of SC5314 colonies on YPD+10% Fetal Calf Serum

515

(FCS) solid media, M199, and RPMI-1640, all with varying concentrations of QNC (No treatment, 16,

516

128, 256 and 1024µg/ml from left to right). (B) Filamentation of SC5314 in RPMI-1640 with and without

517

QNC [1024 µg/ml]. Filamentation was assessed hourly by microscopy. DIC and QNC images at 6 (top

518

panel) and 24 (bottom panel) hours are shown.

519 520

Figure 5. Effect of QNC[1024µg/ml] on active endocytosis to the vacuole and vacuolar morphology.

521

(A) SC5314 cells were incubated in the presence of QNC[1024 µg/ml] (yellow) for 4 hours, followed by

522

incubation with 40 µM FM4-64 (red), images were taken every 15 minutes to visualize active endocytosis 21

22 523

from the plasma membrane (PM) via vesicles to the vacuole and back to the PM. (B) To quantify

524

vacuolar morphology, photos of at least 10 random fields were taken per condition and the number of

525

visible vacuoles in 100 cells per experiment was determined. Cells were grouped into one of three

526

categories (1-2, 3-4 or 5 and greater vacuoles/cell) and then graphed as percentage of cells counted per

527

condition. Data from three independent experiments are presented with standard deviation.

528 529

Figure 6. Effect of increasing QNC concentrations on the growth of C. albicans V-ATPase-deficient

530

strains on solid media.(A) Serial dilution of DAY185, vph1Δ, and its reintegrant, vph1Δ+ VPH1:

531

overnight cultures were spotted onto solid YPD agar media containing QNC [No treatment, 256, 512,

532

1024, and 2048µg/ml] and incubated for 2 days at 37°C. (B) Serial dilution of DAY185, stv1Δ, and its

533

reintegrant, stv1Δ + STV1: overnight cultures were spotted onto solid YPD agar media containing QNC

534

[No treatment, 256, 512, 1024, and 2048µg/ml] and incubated for 2 days at 37°C. (C) Serial dilution of

535

strains THE1-CIp10 and tetR-VMA3: overnight cultures (after 24 hour pre-incubation with DOX) were

536

spotted onto solid YPD agar media, with and without DOX (indicated above as “+ DOX”), containing a

537

range of QNC concentrations (No treatment, 256, 512, 1024, and 2048µg/ml) and incubated for 2 days at

538

37°C.

22

RPMI

M199

FCS

A

0µg/ mL

24hr

6hr

B

Not r eat ment

QNC

+1024µg/ mLQNC