Clotrimazole as a Potent Agent for Treating the Oomycete Fish Pathogen Saprolegnia parasitica...

Clotrimazole to treat Saprolegnia

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Clotrimazole as a potent Agent for Treating the Oomycete 1

Fish Pathogen Saprolegnia parasitica through Inhibition of 2

Sterol 14α-Demethylase (CYP51) 3

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Andrew G.S. Warrilow,a Claire M. Hull,a Nicola J. Rolley,a Josie E. Parker,a 5

W. David Nes,b Stephen N. Smith,a Diane E. Kelly,a# and Steven L. Kelly a# 6

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a Centre for Cytochrome P450 Biodiversity, Institute of Life Science, College of 8

Medicine, Swansea University, Swansea, Wales, SA2 8PP, United Kingdom, 9

b Center for Chemical Biology, Department of Chemistry and Biochemistry, 10

Texas Tech University, Lubbock, Texas 79409-1061. 11

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Running title: Clotrimazole to treat Saprolegnia 14

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# To whom correspondence should be addressed: 17

Phone: +44 (0)1792 292207 Fax: +44 (0)1792 503430 18

Email: [email protected] or [email protected] 19

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AEM Accepts, published online ahead of print on 1 August 2014Appl. Environ. Microbiol. doi:10.1128/AEM.01195-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.


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A candidate CYP51 gene encoding sterol 14α-demethylase from the fish 21

oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based 22

on conserved CYP51 residues among CYPs in the genome. It was 23

heterologously expressed in Escherichia coli, purified and characterized. 24

Lanosterol, eburicol and obtusifoliol bound to purified SpCYP51 with 25

similar binding affinities (Ks 3 to 5 μM). Eight pharmaceutical and six 26

agricultural azole antifungal agents bound tightly to SpCYP51 with 27

posaconazole displaying the highest apparent affinity (Kd ≤3 nM) and 28

prothioconazole-desthio the lowest (Kd ~51 nM). The efficaciousness of 29

azole antifungals as SpCYP51 inhibitors was confirmed by IC50 values of 30

0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole 31

antifungal agents were less effective at inhibiting S. parasitica, S. diclina 32

and S. ferax growth. Epoxiconazole, fluconazole, itraconazole and 33

posaconazole failed to inhibit Saprolegnia growth (MIC100 >256 µg ml-1). The 34

remaining azoles only inhibited Saprolegnia growth at elevated 35

concentrations (MIC100 16 to 64 µg ml-1) with the exception of clotrimazole, 36

which was equally potent as malachite green (MIC100 ~1 µg ml-1). Sterol 37

profiles of azole treated Saprolegnia species confirmed that endogenous 38

CYP51 enzymes were being inhibited with the accumulation of lanosterol in 39

the sterol fraction. The effectiveness of clotrimazole against SpCYP51 40

activity (IC50 = ~1 µM) and inhibiting the growth of Saprolegnia species in 41

vitro (MIC100 ~1 to 2 µg ml-1) suggests that clotrimazole could be used 42

against Saprolegnia infections including as a preventative measure by 43


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pretreatment of fish eggs and for fresh water farmed fish as well as in 44

leisure activities. 45

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Keywords: Oomycete; Saprolegnia; CYP51; sterol; clotrimazole. 47

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Worldwide growth of aquaculture (at ~6% per annum from 2006 to 2011) is 50

reflective of increased consumption of fish, especially in China (1). Aquaculture 51

now accounts for more than 40% of the total fish production for human 52

consumption with freshwater aquaculture contributing ~70% in 2011 compared to 53

~30% from marine aquaculture (1). Diseased fish through bacterial and 54

oomycete (water mould) infections is the largest cause of economic loss in 55

aquaculture (2). Saprolegnia species are responsible for most oomycete 56

infections in farmed-fish (3) with S. parasitica being endemic to all fresh water 57

environments around the world. Losses in worldwide salmon aquaculture through 58

disease run into tens of millions of pounds per annum (4). S. parasitica has also 59

been implicated in the worldwide decline in wild salmon populations (5) and S. 60

ferax is thought to be partially responsible for declining numbers of amphibians in 61

natural ecosystems (6, 7). 62

Saprolegnia is an opportunistic pathogen that is saprotrophic and 63

necrotrophic (3), although some S. parasitica strains are very virulent and can 64

cause primary infections (8). Saprolegnia has a fairly wide range of temperature 65

tolerance (3 to 33°C) (9) with sudden changes in water temperature making fish 66

vulnerable to infection by Saprolegnia (3, 10). Saprolegnia infection can be 67

difficult to eradicate, especially in freshwater hatcheries and fish farms, in part 68

due to the ability of Saprolegnia species to form biofilm communities on its own 69

and with other microorganisms (11) which are more resistant to treatment with 70

antibiotic compounds and act as reservoirs of infection. Infections can affect both 71

eggs and fish. Salmonid eggs are particularly vulnerable to Saprolegnia infection 72


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during the several months spent on freshwater riverbeds prior to hatching (12). 73

Initial infections of ova result in dead eggs from which a mycelial mat spreads 74

with time to engulf neighboring live eggs (12) resulting in further losses and the 75

potential infection of hatchlings. Czeczuga et al (13) found Saprolegnia species 76

were present on the external surfaces of all sea trout eggs sampled from the 77

rivers and freshwater hatcheries of Poland. On fish, Saprolegnia invades 78

epidermal tissues, often beginning on the head or fins and eventually spreads 79

over the whole body surface (10) and is able to cause cellular necrosis as well as 80

dermal and epidermal damage (14). However, Saprolegnia infections do not 81

appear to be tissue specific. If untreated, Saprolegnia infection leads to death by 82

osmoregulatory failure (9, 14, 15). Up until 2002 Saprolegnia infections were well 83

controlled using the organic dye malachite green which is considered the most 84

effective chemical against Saprolegnia (3, 16). However, the use of malachite 85

green in aquaculture was banned by the USA and several other countries (17) 86

due to concerns the dye is a potential carcinogen (18) and a mutagen (3). Since 87

2002 infections caused by Saprolegnia species have re-emerged, with 88

Saprolgenia parasitica in particular being an economically important pathogen of 89

farmed fish such as salmon, trout and catfish (19). Improving water quality and 90

reducing stress and handling can significantly reduce the incidence and severity 91

of Saprolegnia infection in catfish (20). 92

Presently there are few licensed chemicals for controlling Saprolegnia 93

infection in salmonid eggs and no chemicals that give sufficient protection against 94

Saprolegnia infection after hatching (21). S. parasitica is inhibited by low 95


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concentrations of sodium chloride (22) with the sodium chloride helping to 96

counteract osmotic stress in affected fish with damaged skin. Sodium chloride at 97

high concentrations such as in sea water (~29 g/l) and salt water (~15 g/l) is 98

lethal to Saprolegnia (17, 23) and is effective for controlling S. parasitica (10). 99

However, use of sodium chloride in freshwater environments is not practical. 100

Ozone treatment of water has also been used to reduce S. parasitica infection 101

(21) although ozone can not be used to cure infected fish. Formalin has been 102

used to treat eggs and the first stages of larval development (3, 18, 24, 25). 103

However formaldehyde is harmful to the environment and personal health. 104

Hydrogen peroxide is a promising chemical for the treatment of Saprolegnia (17, 105

18, 26) with minimal impact to the environment and has been used as a 106

treatment of catfish eggs (20). Cupric sulphate and diquat (0.125 ppm) have 107

been used as prophylactic treatments to inhibit zoospores (20). Recently 108

alternate chemical approaches have been tried to control S. parasitica infection 109

with varying success including chemically modified chitosans (27), peracetic acid 110

(28), saprolmycins (29), oridamycins (30), pyceze (2-bromo-2-nitropropane-1,3-111

diol) (12) and essential oils and ethanol extracts of medicinal plants (31). In 112

addition, chitin synthases responsible for tip growth in Saprolegnia have been 113

demonstrated as potential targets for anti-oomycete drugs (32). Effective 114

strategies and chemicals still need to be developed to control or eradicate 115

Saprolegnia infections in aquaculture, especially in fresh-water environments, 116

with at least the same efficacy as malachite green. 117


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Previously oomycetes, such as Phytophthora species, were considered 118

sterol auxotrophs utilizing sterols from the surrounding environment or the host 119

organism (33) and lacked functional CYP51 genes (34). This explained the 120

ineffectiveness of azole antifungal agents against many oomycete species as the 121

azole target enzyme CYP51 was absent. However, the recent discovery of a 122

sterol metabolism pathway in the oomycete Aphanomyces euteiches (a legume 123

root pathogen) (35), including a CYP51 gene, led us to investigate whether such 124

a pathway was also present in S. parasitica. Previous investigations into the 125

sterol profile of Saprolegnia were limited to S. ferax (36, 37). 126

In this study we identified the CYP51 gene from the genome sequence of 127

S. parisitica, cloned, expressed and purified the recombinant CYP51 protein in an 128

active state. We characterized the effectiveness of fifteen azole antifungal agents 129

used in the clinic (pharmazoles) and in agriculture (agriazoles) at inhibiting S. 130

parasitica CYP51 activity in vitro and growth of Saprolegnia species. Finally we 131

discuss the potential of using azole antifungal agents to control Saprolegnia 132

infections in freshwater aquaculture. 133

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MATERIALS AND METHODS 136

Construction of the pCWori+:SpCYP51 expression vector. The S. parasitica 137

CYP51 gene (BROAD Institute accession number SPRG_09493.2 - 138

http://www.broadinstitute.org/annotation/genome/Saprolegnia_parasitica/MultiHo139

me.html) was identified by performing a BLASTP search against the S. parasitica 140

genome database using the amino acid sequence of Candida albicans CYP51 141

(UniProtKB accession number P10613). The SpCYP51 gene was synthesized by 142

Eurofins MWG Operon (Ebersberg, Germany) incorporating an NdeI restriction 143

site at the 5' end and a HindIII restriction site at the 3' end of the gene cloned into 144

pUC57 plasmid. In addition the first eight amino acids were changed to 145

'MALLLAVF' (38) and a six-histidine extension (CATCACCATCACCATCAC) was 146

inserted immediately before the stop codon to facilitate protein purification by 147

Ni2+-NTA agarose affinity chromatography. The SpCYP51 gene was excised by 148

NdeI / HindIII restriction digestion followed by cloning into the pCWori+ 149

expression vector using Roche T4 DNA ligase. Gene integrity was confirmed by 150

DNA sequencing. 151

Heterologous expression in E. coli and isolation of recombinant 152

SpCYP51 protein. The pCWori+:SpCYP51 construct was transformed into 153

competent DH5α E. coli cells and transformants selected using 0.1 mg ml-1 154

ampicillin. Growth and expression conditions were identical to those previously 155

reported (39). Protein isolation was according to the method of Arase et al (40) 156

except that 2% sodium cholate was used in the sonication buffer. The solubilized 157

SpCYP51 protein was purified by Ni2+-NTA agarose affinity chromatography as 158


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previously described (41) using 1% L-histidine in 0.1 M Tris-HCl (pH 8.1) and 159

25% glycerol to elute SpCYP51, followed by dialysis against 5 liters of 25 mM 160

Tris-HCl (pH 8.1) and 10% glycerol. Ni2+-NTA agarose purified SpCYP51 was 161

used for all subsequent spectral and IC50 determinations. Protein purity was 162

assessed by SDS polyacrylamide gel electrophoresis followed by staining with 163

Coomassie Brilliant Blue R-250. 164

Determination of cytochrome P450 protein concentrations. 165

Cytochrome P450 concentration was determined by reduced carbon monoxide 166

difference spectra (42), with carbon monoxide passed through the cytochrome 167

P450 solution prior to addition of sodium dithionite to the sample cuvette, using 168

an extinction coefficient of 91 mM-1 cm-1 (43) for the absorbance difference 169

between 447 and 490 nm. Absolute spectra (700 to 300 nm) were determined 170

using 2 μM SpCYP51 in 0.1 M Tris-HCl (pH 8.1) and 25% glycerol as previously 171

described (41). Confirmation that isolated SpCYP51 was active was 172

demonstrated by measuring the 14α-demethylation of lanosterol, eburicol and 173

obtusifoliol using the CYP51 reconstitution assay detailed below. 174

CYP51 reconstitution assay system. IC50 determinations were 175

performed using the CYP51 reconstitution assay system (500 μl final reaction 176

volume) previously described (44) containing 0.5 μM SpCYP51, 1 μM Aspergillus 177

fumigatus cytochrome P450 reductase (AfCPR1 - UniProtKB accession number 178

Q4WM67), 50 μM eburicol, 50 μM dilaurylphosphatidylcholine, 4% (wt/vol) (2-179

hydroxypropyl)-β-cyclodextrin (HPCD), 0.4 mg ml-1 isocitrate dehydrogenase, 25 180

mM trisodium isocitrate, 50 mM NaCl, 5 mM MgCl2 and 40 mM MOPS (pH ~7.2). 181


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Azole antifungal agents were added in 2.5 μl dimethylformamide followed by 10 182

min incubation at 30°C prior to assay initiation with 4 mM β-NADPH-Na4. 183

Samples were then shaken for 30 min at 30°C. In addition, trial SpCYP51 assays 184

were also performed using 50 μM lanosterol and 50 μM obtusifoliol as substrates. 185

Sterol metabolites were recovered by extraction with ethyl acetate followed by 186

derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide and tetramethylsilane 187

in pyridine prior to analysis by gas chromatography mass spectrometry (45). 188

Eburicol was chosen as the substrate for the CYP51-azole IC50 determinations 189

as the TMS-derivatized 14-demethylated product could be readily separated from 190

TMS-derivatized eburicol during gas chromatography. IC50 in this study is defined 191

as the inhibitor concentration required causing 50% inhibition of CYP51 activity 192

under the stated assay conditions. 193

Sterol binding properties of SpCYP51. Stock 2.5 mM solutions of 194

lanosterol (the CYP51 substrate in animals and most yeasts), eburicol (the 195

CYP51 substrate in higher fungi) and obtusifoliol (the CYP51 substrate in plants) 196

were prepared in 40% (wt/vol) HPCD. Sterol was progressively titrated against 4 197

μM SpCYP51 protein in a quartz semi-micro cuvette (light path 4.5 mm) with 198

equivalent amounts of 40% (wt/vol) HPCD added to the reference cuvette also 199

containing 4 μM SpCYP51. The absorbance difference spectrum between 500 200

and 350 nm was determined after each incremental addition of sterol (up to 75 201

μM). Sterol saturation curves were constructed from ΔA385-421 derived from the 202

difference spectra. The substrate binding constants (Ks) were determined by non-203


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linear regression (Levenberg-Marquardt algorithm) using the Michaelis-Menten 204

equation. Each binding determination was performed in triplicate. 205

Azole binding studies. Binding azole antifungal agents to 2 μM 206

SpCYP51 were performed as previously described (46) using quartz split-207

cuvettes with a 4.5 mm light path. Stock 1, 0.5, 0.2 and 0.1 mg ml-1 solutions of 208

the pharmaceutical azole antifungals (pharmazoles) clotrimazole, econazole, 209

fluconazole, itraconazole, ketoconazole, miconazole, posaconazole and 210

voriconazole and the agricultural azole antifungals (agriazoles) epoxiconazole, 211

prochloraz, propiconazole, prothioconazole, prothioconazole-desthio, 212

tebuconazole and triadimenol were prepared in dimethylformamide. Azole 213

antifungals were progressively titrated against 2 μM SpCYP51 in 0.1 M Tris-HCl 214

(pH 8.1) and 25% glycerol at 22°C with equivalent volumes of dimethylformamide 215

also being added to the SpCYP51-containing compartment of the reference 216

cuvette. The absorbance difference spectra between 500 and 350 nm were 217

determined after each incremental addition of azole with binding saturation 218

curves constructed from ΔA428-412 against azole concentration. The dissociation 219

constant of the enzyme-azole complex (Kd) for each azole was determined by 220

non-linear regression (Levenberg-Marquardt algorithm) using a rearrangement of 221

the Morrison equation for tight ligand binding (47, 48). Kd values determined 222

using the Morrison equation are accurate to ~1/100th of the protein concentration 223

(49). Each binding determination was performed in triplicate. The chemical 224

structures of the azole antifungals used in this study are shown in Fig. 1. 225


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MIC studies with Saprolegnia species. Saprolegnia parasitica Coker 226

(CBS 223 63), Saprolegnia diclina Humphrey (CBS 326 35) and Saprolegnia 227

ferax Gruith (CBS 173 42) were retrieved from the CBS-KNAW Fungal 228

Biodiversity Centre (Netherlands) and maintained on 90 mm diameter Yeast Mold 229

agar plates (ATCC YM Medium 200) containing yeast extract (3%), malt extract 230

(3%), glucose (1%), peptone (0.5%) and agar (2%). Agar plates were routinely 231

inoculated with a single 5 mm plug of mycelium cut from stock cultures exhibiting 232

active radial growth. All strains were grown for 3 days at 20°C (± 2°C) prior to 233

use. Azole susceptibility assays were performed in 24-well (flat bottom, 16 mm 234

diameter round well) culture plates (IWAKI: 3820-024) using an adaptation of the 235

Clinical and Laboratory Standards Institute (CLSI) M27-A2 broth dilution method. 236

Briefly, all azoles and malachite green were dissolved in dimethylsulfoxide to a 237

stock concentration of 25.6 mg ml-1. Dilutions were then made with 238

dimethylsulfoxide to achieve 100-times stock solutions (1 ml volumes) of 25.6, 239

12.8, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05 and 0.025 mg ml-1. These stocks were 240

initially diluted 10-fold using YM medium prior to addition of 100 µl directly into 241

culture plate wells containing 900 µl YM to achieve final azole concentrations of 242

256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25 µg ml-1 and control wells containing 243

1% (vol/vol) dimethylsulfoxide. Culture wells were inoculated centrally with a 244

single 3 mm plug of mycelium excised from YM agar plates and incubated for 3 245

days at 20°C (± 2°C). Minimum inhibitory concentrations (MIC) were performed in 246

duplicate and scored manually. MIC100 is defined here as the lowest antifungal 247

concentration at which growth remained completely inhibited after 72 h at 20°C. 248


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Sterol composition of Saprolegnia species. Mycelial cakes were 249

removed from the plate wells of treated (compound concentration immediately 250

below MIC100) and untreated Saprolegnia species and then washed thrice with 251

sterile water. Nonsaponifiable lipids were extracted as previously reported (50) 252

and were derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide and 253

tetramethylsilane in pyridine prior to analysis by gas chromatography mass 254

spectrometry (45). Sterol composition was calculated using peak areas from the 255

gas chromatograms and the mass fragmentation patterns were used to confirm 256

sterol identity. 257

Data analysis. Curve-fitting of ligand binding data were performed using 258

the computer program ProFit 6.1.12 (QuantumSoft, Zurich, Switzerland). Spectral 259

determinations were made using quartz semi-micro cuvettes with a Hitachi U-260

3310 UV/VIS spectrophotometer (San Jose, California). 261

The N-terminal membrane anchor region of SpCYP51 was predicted using 262

the TargetP version 1.1 (http://www.cbs.dtu.dk/services/TargetP/) software. 263

Subcellular location was predicted using WoLF PSORT (http://wolfpsort.org/) 264

software. Phylogenetic analyses were performed by comparing the SpCYP51 265

amino acid sequence (SPRG_09493.2) against selected fungal, plant and animal 266

CYP51 proteins from the UniProtKB database 267

(http://www.uniprot.org/help/uniprotkb) using NCBI BLAST2 268

(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=269

blastn&BLAST_PROG_DEF=megaBlast&BLAST_SPEC=blast2seq) and 270

ClustalX version 1.81 (http://www.clustal.org/) sequence alignment software with 271


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a phylogenetic tree produced from the ClustalX generated Phylip-dnd file using 272

TreeviewX (http://www.darwin.zoology.gla.ac.uk/~rpage/treeviewx/) software. 273

The eukaryotic CYP51 sequences used for phylogenetic comparison are 274

detailed in the supplementary table. BLASTP searches of the Broad Institute S. 275

parasitica genome database 276

(http://www.broadinstitute.org/annotation/genome/Saprolegnia_parasitica/MultiHo277

me.html) were performed using sterol biosynthesis protein homologs from A. 278

euteiches (35), Aspergillus fumigatus (Δ5 sterol desaturase Q4WDL3, Δ3 sterol 279

dehydrogenase Q4X017) and Homo sapiens (Δ8 sterol isomerase Q15125). 280

Several sequences for ∆3 sterol keto reductase (fungal and mammalian) were 281

used. All S. parasitica protein sequences so identified were then used as query 282

sequences at NCBI BLASTP 283

(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastS284

earch&LINK_LOC=blasthome) to elucidate the most likely sterol biosynthesis 285

candidate. 286

Chemicals. All chemicals, including azole antifungals except 287

voriconazole, were obtained from Sigma Chemical Company (Poole, UK). 288

Voriconazole was supplied by Discovery Fine Chemicals (Bournemouth, UK). 289

Growth media, sodium ampicillin, IPTG and 5-aminolevulenic acid were obtained 290

from Foremedium Ltd (Hunstanton, UK). Ni2+-NTA agarose affinity 291

chromatography matrix was obtained from Qiagen (Crawley, UK). 292

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RESULTS 295

Analysis of SpCYP51 protein sequence. The SpCYP51 sequence we identified 296

agreed with that published (SPRG_09493.2) by Jiang et al (51) during the course 297

of our work. TargetP predicted the SpCYP51 N-terminal membrane anchor to 298

consist of the first 36 amino acid residues whilst WoLF PSORT predicted 299

SpCYP51 would be located in the endoplasmic reticulum. This agrees with the 300

observation that eukaryotic CYP51 proteins are associated with the membranes 301

of the endoplasmic reticulum (52). Alignment of SpCYP51 against other 302

eukaryotic CYP51 proteins confirmed that all 23 conserved amino acid residues 303

previously identified for CYP51 proteins were present (53). The resultant 304

phylogenetic tree (Fig. 2) showed SpCYP51 clustered with the CYP51 of fellow 305

oomycete A. euteiches, sharing 79% sequence identity. S. parasitica CYP51 was 306

more closely related to plant and animal CYP51 proteins than to fungal CYP51 307

proteins. For example, SpCYP51 shared 48% sequence identity with the marine 308

diatom T. psuedonana CYP51 and 45% identity with the thermo-acidophilic 309

unicellular red-alga G. sulphuraria CYP51. SpCYP51 shared 41-44% sequence 310

identity with higher plant CYP51 enzymes (44% with cucumber CYP51) and 36-311

38% identity with animal CYP51 proteins (37% identity with human CYP51) in 312

contrast to just 30-35% sequence identity with the 'true' fungal CYP51 proteins 313

(34% identity with C. albicans CYP51 and 31% identity with A. fumigatus 314

CYP51A). 315

Expression and purification of SpCYP51. The yield of SpCYP51 was 316

~120 (±20) nmoles per liter E. coli culture as determined by absolute 317


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spectroscopy (41) after purification by Ni2+-NTA agarose chromatography. This 318

yield was ~10-fold greater than that calculated by carbon monoxide difference 319

spectroscopy under reductive conditions (42) of the crude sodium cholate cell 320

extract due to the reduced CO-SpCYP51 adduct formed being unstable. SDS 321

polyacrylamide gel electrophoresis confirmed the purity of the Ni2+-NTA agarose 322

eluted SpCYP51 protein to be greater than 95% when assessed by Coomassie 323

Brilliant Blue R-250 staining intensity, with an apparent molecular weight of 324

~52,000 ±2,000 which was close to the predicted value of 55,487 including the 325

six-histidine C-terminal extension. 326

Spectral properties of recombinant SpCYP51 protein. The absolute 327

spectrum of SpCYP51 (Fig. 3A) was typical for a ferric cytochrome P450 enzyme 328

predominantly (~80%) in the low-spin state (41, 54) with α, β, Soret (γ) and δ 329

spectral bands at 565, 535, 417 and 360 nm, respectively. Reduced carbon 330

monoxide difference spectra (Fig. 3B) produced the characteristic red-shifted 331

Soret peak at 445-7 nm typical of ferrous cytochrome P450 enzymes complexed 332

with CO (42, 43). However, the reduced CO-adduct at 447 nm was unstable with 333

the absorbance peak quickly dissipating (t0.5 = 3.2 ±0.2 min) along with an 334

accompanying increase in the 'inactive' P-420 complex absorbance at 421 nm. 335

Binding studies with sterols. All three 14α-methylsterols produced 336

strong type I binding spectra with SpCYP51 (Fig. 4A) which was typical of the 337

interaction of substrates with cytochromes P450 (54) with a peak at ~385 nm and 338

trough at ~420 nm. Sterol saturation curves were constructed (Fig. 4B) to derive 339

substrate binding constants (Ks). SpCYP51 had similar affinity for all three sterols 340


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at 2.8 ±0.4, 3.3 ±0.5 and 4.6 ±0.2 μM for lanosterol, eburicol and obtusifoliol, 341

respectively. All three sterols were 14α-demethylated in the CYP51 reconstitution 342

assay using purified SpCYP51 protein and AfCPR1 as the redox partner with 343

turnover numbers of 0.63, 1.04 and 2.55 min-1 for lanosterol, eburicol and 344

obtusifoliol, respectively. This confirmed the putative CYP51 was indeed as 345

predicted and that SpCYP51 had been isolated in a fully functional form. 346

Azole antifungal agent binding studies. Tight type II binding spectra 347

were observed between 2 μM SpCYP51 and all fifteen azole antifungal agents 348

(Fig. 5 and 6), with the exception of prothioconazole, yielding a peak at ~428 nm 349

and a trough at ~412 nm. Type II binding spectra are caused by the triazole N-4 350

nitrogen (Fig. 1 - voriconazole) or the imidazole N-3 nitrogen (Fig. 1 - econazole) 351

coordinating as the sixth ligand with the heme iron (55) to form the low-spin 352

CYP51-azole complex resulting in a 'red-shift' of the heme Soret peak. No 353

reproducible binding spectra between prothioconazole and 2 μM SpCYP51 could 354

be obtained (data not shown) probably because of steric hindrance from the 355

sulfur atom preventing the direct coordination of the triazole N-4 nitrogen atom 356

with the heme ferric ion (Fig. 1). Tight binding is normally observed for azole 357

antifungals against fungal CYP51 proteins where the Kd for a ligand is similar or 358

lower than the concentration of the enzyme present (49). The imidazole-based 359

pharmazoles gave approximately 30% more intense (greater ΔAmax) difference 360

spectra with SpCYP51 than the triazole-based pharmazoles (Fig. 5), whereas the 361

agriazoles (all triazoles with the exception of the imidazole prochloraz) gave type 362

II difference spectra (Fig. 6) of similar intensity to the triazole-based 363


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pharmazoles. Azole-SpCYP51 saturation curves (Fig. 7) indicated that azole 364

binding affinities were similar with Kd values typically between 3 and 16 nM 365

(Table 1) with the exception of clotrimazole, fluconazole and prothioconazole-366

desthio which bound slightly less tightly with Kd values of 25, 40 and 51 nM, 367

respectively. These marginally higher Kd values may result from the relative 368

compactness of these three azoles preventing additional stabilizing interactions 369

with amino acid side-chains that line the SpCYP51 heme binding pocket. This 370

would need to be verified by in silico ligand docking experiments. Therefore 371

SpCYP51-catalyzed sterol demethylation should be strongly inhibited by azole 372

antifungal agents. 373

IC50 determinations for azole antifungal agents on SpCYP51 activity. 374

IC50 determinations using 0.5 μM SpCYP51 with the pharmazoles fluconazole, 375

itraconazole, ketoconazole and clotrimazole (Fig. 8A) and the agriazoles 376

epoxiconazole, propiconazole, prochloraz and tebuconazole (Fig. 8B) confirmed 377

that all eight azoles severely inhibited SpCYP51 activity using eburicol as 378

substrate and AfCPR1 as redox partner. The binding of epoxiconazole, 379

propiconazole and prochloraz to SpCYP51 was tight with similar IC50 values of 380

0.17, 0.20 and 0.15 μM, respectively, being observed along with no residual 381

CYP51 activity in the presence of 2 μM azole. The IC50 value for a tight binding 382

azole that can not be displaced by substrate would be half the CYP51 383

concentration (~0.25 μM) as was observed for epoxiconazole, propiconazole or 384

prochloraz. The binding of tebuconazole, itraconazole, clotrimazole, fluconazole 385

and ketoconazole appeared slightly less tight with IC50 values of 0.47, 0.84, 0.95, 386


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1.35 and 2.27 μM, respectively, with residual SpCYP51 activities of 27%, 19%, 387

27%, 30% and 38% observed at 4 μM azole, suggesting these five azoles were 388

displaced from the SpCYP51 heme by substrate during catalysis. 389

MIC studies with Saprolegnia species. The MIC results obtained with 390

most of the azole antifungals (Fig. 9 and Table 2) were surprising as all three 391

Saprolegnia species were insensitive to epoxiconazole, fluconazole, itraconazole 392

and posaconazole (MIC100 >256 µg ml-1) whilst the remaining azoles, except 393

clotrimazole, gave MIC100 values between 8 and 64 µg ml-1 in comparison to 1 µg 394

ml-1 for malachite green. Hu et al (56) also determined the MIC for malachite 395

green to be 1 μg ml-1 for Saprolegnia supporting the validity of our MIC 396

methodology. Clotrimazole though proved to be as effective as malachite green 397

against Saprolegnia species with an MIC100 value of 1 to 2 µg ml-1. Clotrimazole 398

proved to be effective despite having an apparent eight-fold lower affinity for 399

SpCYP51 than posaconazole (Kd ~25 nM compared to ≤3 nM) and an IC50 value 400

6-fold higher than prochloraz. The next effective azoles were econazole and 401

miconazole (MIC100 8 μg ml-1) suggesting imidazoles were generally more 402

effective at controlling Saprolegnia infection then triazoles. 403

Sterol composition of Saprolegnia species. The sterol composition of 404

untreated and prochloraz-treated (16 µg ml-1) S. diclina, S. ferax and S. parasitica 405

were determined after 72 h at 20°C (Fig. 10 and Table 3). Untreated S. diclina, S. 406

ferax and S. parasitica contained four main sterols desmosterol, 24-methylene 407

cholesterol, fucosterol (the Saprolgenia equivalent to ergosterol in fungi) and 408

cholesterol with traces of lanosterol also detectable. S. diclina and S. ferax had 409


20

similar sterol profiles, whilst S. parasitica had a ten-fold lower abundance of 410

fucosterol and a two- to three-fold higher abundance of desmosterol. 411

The large build-up of lanosterol in the prochloraz-treated samples (Fig. 10 412

and Table 3) indicated prochloraz uptake in Saprolegnia and inhibition of CYP51 413

activity preventing the 14α-demethylation of lanosterol, which leads to the 414

depletion of the other major Saprolegnia sterols. Prochloraz-treated S. parasitica 415

and S. diclina had similar sterol profiles with lanosterol constituting 90 to 97% of 416

the total sterol and the depletion of cholesterol, desmosterol, 24-methylene 417

cholesterol and fucosterol. However, the sterol profile of prochloraz-treated S. 418

ferax differed in that fucosterol content remained constant whilst cholesterol, 419

desmosterol and 24-methylene cholesterol content were depleted and lanosterol 420

accumulation was less at only 58% of the sterol content. Further investigations 421

are required to explain the differences in sterol composition amongst Saprolegnia 422

species in response to azole treatment. Clotrimazole-treated (0.5 μg ml-1) S. 423

parasitica also resulted in a large accumulation of lanosterol (92% of total sterol) 424

and the depletion of the other sterols (Table 3). Therefore clotrimazole was 425

effective at inhibiting CYP51 activity in vivo at a 32-fold lower concentration than 426

prochloraz. In contrast the sterol profiles of malachite green-treated Saprolegnia 427

were unaltered indicating that the mode of action of malachite green was different 428

to azole antifungals and did not inhibit CYP51. 429

Searching the Broad Institute S. parasitica genome database for sterol 430

biosynthesis enzymes identified twelve enzymes (Table 4). For lanosterol 431

synthase and Δ5 sterol desaturase two potential candidates were identified for 432


21

each gene, in agreement with Jiang et al (51). For lanosterol synthase 433

SPRG_117832.2 (805 amino acids) and SPRG_17895.2 (472 amino acids) 434

shared 99% identity. SPRG_17895.2 was a truncated version of 435

SPRG_117832.2 starting at residue 334 and contained five point mutations. For 436

Δ5 sterol desaturase SPRG_11773.2 (270 amino acids) and SPRG_18544.2 (272 437

amino acids) also shared 99% identity with just three point mutations in the first 438

263 amino acid residues, however the C-termini (7 to 9 residues) of the two 439

proteins differed. Functional analysis of the lanosterol synthase and Δ5 sterol 440

desaturase candidates (expression, purification and reconstitution assays) will be 441

required to demonstrate the catalytic function of each protein. The S. parasitica 442

sterol biosynthesis enzymes identified here (Table 4) agree with those identified 443

by Jiang et al (51). However, Jiang et al's (51) proposed sterol pathway did not 444

relate to the sterols identified in this study. Our pathway (Fig. 11) shows the four 445

main sterols identified in Saprolegnia (desmosterol, 24-methylene cholesterol, 446

fucosterol and cholesterol) although the exact catalytic sequence of the enzymes 447

is not known. We could not identify a Δ3 sterol keto reductase candidate in S. 448

parasitica although the presence of desmosterol and 24-methylene cholesterol in 449

the cell membranes indicates that this chemical reaction occurs in vivo. This may 450

be due to the S. parasitica Δ3 sterol keto reductase having low sequence identity 451

to mammalian and fungal Δ3 sterol keto reductases or that this chemical reaction 452

is performed by a phylogenetically unrelated enzyme in S. parasitica. Jiang et al 453

(51) were unable to identify a Δ3 sterol keto reductase candidate in S. parasitica 454

as were Madoui et al (35) in A. euteiches. 455

456


22

DISCUSSION 457

Our phylogenetic analysis of the SpCYP51 protein suggested that S. parasitica 458

was more closely related to plants and animals than 'true' fungi in agreement with 459

previous findings that 'true' fungi and oomycetes were phylogenically distinct (35, 460

57). However, the low sequence identity between SpCYP51 and the fungal 461

CYP51 proteins suggests that azole antifungal agents commonly used to fight 462

fungal infection in the clinic and in agriculture may not be so efficacious against 463

oomycetes containing CYP51 enzymes. 464

SpCYP51 sterol binding affinities were similar to those reported for the 465

trypanosomal CYP51 enzymes of T. cruzi and L. infantum (58, 59). However, 466

purified SpCYP51 displayed 3- to 8-fold higher affinity for sterol than the CYP51 467

enzymes of C. albicans, A. fumigatus, M. graminicola and H. sapiens (60-62). 468

The ability of SpCYP51 to 14α-demethylate lanosterol, eburicol and obtusifoliol 469

confirmed the putative CYP51 was indeed as predicted and that SpCYP51 had 470

been isolated in a fully functional form. 471

SpCYP51 binding affinities for pharmazoles were similar to those reported 472

for C. albicans CYP51 (Kd 10 to 56 nM) (60). SpCYP51 binding affinities for the 473

agriazoles epoxiconazole, prochloraz, propiconazole and triadimenol were 5-, 8-, 474

6- and 4-fold tighter (Kd 4 to 16 nM) than those observed with C. albicans CYP51 475

(Kd 22 to 68 nM) (60). Therefore SpCYP51 should be strongly inhibited by azole 476

antifungal agents as no inherent resistance (high Kd values) towards azole 477

antifungals was evident unlike A. fumigatus CYP51A and CYP51B which both 478

bound fluconazole weakly (Kd 11930 and 4030 nM) (61). This was further 479


23

supported by the IC50 results which showed strong inhibition of CYP51 activity by 480

azole antifungal agents (Fig. 7), especially epoxiconazole, propiconazole and 481

prochloraz (IC50 values 0.17, 0.20 and 0.15 μM). The SpCYP51 IC50 values with 482

the agriazoles were two- to three-fold lower than the equivalent IC50 values with 483

C. albicans CYP51 (60) suggesting agriazoles would prove effective at inhibiting 484

Saprolegnia growth. However, the relatively poor performance of most azoles 485

against Saprolegnia, with the exception of clotrimazole, in terms of MIC100 values 486

was in agreement with the limited previous azole antifungal studies with 487

Saprolegnia species. 488

Heeres et al (63) established the MIC50 for terconazole was 10 μg ml-1 and 489

the MIC100 was 100 μg ml-1 whereas the MIC100 for clotrimazole was >100 μg ml-1 490

in Sabouraud broth (63) which was ~100-fold higher than that determined in this 491

study. Heeres et al (63) also determined the MIC100 for clotrimazole with C. 492

albicans to be >100 μg ml-1 in Sabouraud broth which was surprisingly high 493

considering clotrimazole is used for the treatment of Candida infections in 494

humans and calls the methodology into question. More recent MIC 495

determinations for clotrimazole against C. albicans range from 0.03 to 1 μg ml-1 496

(64-66), suggesting Heeres et al's (63) MIC determinations were over 100-fold 497

higher than present day determinations. In contrast, Marking et al (67) found the 498

MIC for clotrimazole to be 10 to 30 μg ml-1 after 15 min exposure which fell to 10 499

μg ml-1 after 60 min with Saprolegnia hypogyna. Our MIC determinations 500

exposed Saprolegnia to azoles for 72 h and included a number of different 501

species with similar results to Marking et al (67). Hu et al (56) determined the 502


24

Saprolegnia MIC for propiconazole to be 100 μg ml-1, compared to 64 μg ml-1 in 503

this study, and Saprolegnia MIC values for thiabendazole, difenoconazole and 504

diniconazole to be >100, 50 and 100 μg ml-1. High azole MIC100 values were 505

probably due to either poor uptake into the cells or the efficient efflux of the 506

azoles out of the cells. Clotrimazole is a relatively compact molecule, unlike many 507

of the ineffective azoles, suggesting size is important in efficient uptake of azole 508

antifungals in Saprolegnia. 509

For azole antifungal agents to be effective at combating Saprolegnia 510

infection they need to display high potency against the intended target CYP51 511

(SpCYP51) enzyme with minimal interaction with the host CYP51 (and other off-512

target host CYP enzymes). Morrison et al (68) determined the Kd values for 513

ketoconazole and propiconazole to be 260 and 640 nM with recombinant Danio 514

rerio (zebrafish) CYP51 by type II difference ligand binding spectroscopy and 76 515

and 3700 nM by in silico azole ligand docking experiments. Therefore the 516

apparent selectivities for SpCYP51 over the zebrafish homolog based on azole 517

Kd values were 20- and 112-fold for ketoconazole and propiconazole (azole 518

binding studies) or 6- and 649-fold (in silico studies). This is encouraging and it 519

would be of interest to determine the selectivity for the more potent Saprolegnia 520

inhibitor clotrimazole. 521

The change in sterol composition of Saprolegnia in response to exposure 522

to prochloraz and clotrimazole confirmed that CYP51 was being inhibited in vivo 523

by the accumulation of lanosterol in the cell membranes. Similar lanosterol 524

accumulation was observed in S. ferax when treated with triarimol, a CYP51 525


25

inhibiting pyrimidine fungicide (36) and accumulation of eburicol in fungi treated 526

with azole antifungals (69-71). Malachite green-treated Saprolegnia had 527

unaltered sterol profiles indicating that the mode of action of malachite green was 528

different to azole antifungals with no inhibition of CYP51 function. This is in 529

agreement with previous findings that malachite green acts independently of 530

known antifungal targets such as sterol biosynthesis and drug efflux pump 531

proteins in C. albicans (MIC50 0.1 μg ml-1) (72). Malachite green was found to 532

affect 207 genes in C. albicans with the up-regulation of 167 genes involved in 533

oxidative stress, virulence, carbohydrate metabolism, heat shock and amino acid 534

metabolism and the down-regulation of 37 genes involved in iron acquisition, 535

filamentous growth and mitochondrial respiration (72). Malachite green affects 536

multiple mechanisms to exert its antifungal effect and leads to a shift in 537

metabolism towards fermentation, increased generation of reactive oxygen 538

species, iron depletion and cell necrosis in C. albicans (72) and is likely to 539

behave similarly in Saprolegnia species. 540

There appear to be no published studies investigating the effectiveness of 541

clotrimazole at combating Saprolegnia infection of salmonid eggs and fry in vivo. 542

Bailey and Jeffrey (73) screened 215 candidate fungicides for suitability as 543

aquatic fungicides, 120 of which they deemed unsuitable, including clotrimazole 544

but did not elaborate why clotrimazole was deemed unsuitable. The OSPAR 545

commission (74) established that clotrimazole was not acutely or chronically toxic 546

to fish (Brachyodanio rerio and Oncorhynchus mykiss). The report, however, 547

concludes that at present there is no risk to the environment over the current 548


26

usage of clotrimazole. Azole antifungal agents are not presently used to treat 549

Saprolegnia infections in aquaculture. However, azole antifungal agents are 550

widely used in medicine to combat infections by human fungal pathogens such 551

as Candida and Aspergillus species and in agriculture to combat infections by 552

plant fungal pathogens such as Mycosphaerella graminicola and Fusarium 553

species. Clotrimazole proved to be as effective at inhibiting Saprolegnia growth in 554

vitro as malachite green (MIC ~1 µg ml-1). It is our contention that clotrimazole 555

would prove an effective control agent of Saprolegnia infection, especially if used 556

to pretreat salmonid eggs in commercial hatcheries. Fungal diseases have been 557

reported in many commercially important fish species besides salmonids, 558

including catfish, pike, bass, tilapia, roach, carp, mullet and sturgeon (3, 75-78), 559

such that treatment with clotrimazole analogs could potentially reduce financial 560

losses significantly by effective control of oomycete and fungal infections. 561

Therefore studies need to be performed on salmonid eggs in vivo to determine 562

optimum clotrimazole doses and pretreatment lengths for the prevention of 563

Saprolegnia infections as well as for other farmed species and for infected fish. 564

Hu et al (56) found that the two strobilurin antifungals kresoxim-methyl and 565

azoxystrobin were most effective against Saprolegnia with MIC values of 1 and 566

0.5 μg ml-1, however, both of these compounds are toxic to fish, highlighting the 567

problems of translating successful in vitro experiments into effective in vivo 568

treatments. 569

570

571


27

ACKNOWLEDGMENT 572

We are grateful to the Engineering and Physical Sciences Research 573

Council National Mass Spectrometry Service Centre at Swansea University for 574

assistance in GC/MS analyses. 575

This work was in part supported by the European Regional Development 576

Fund / Welsh Government funded BEACON research program (Swansea 577

University) and the National Science Foundation of the United States grant NSF-578

MCB-09020212 awarded to W. David Nes (Texas Tech University). 579

580

581


28

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3463. 766

56. Hu X-G, Liu L, Hu K, Yang X-L, Wang G-X. 2013. In vitro screening of 767

fungicidal chemicals for antifungal activity against Saprolegnia. J. World 768

Aquac. Soc. 44:528-535. 769

57. Baldauf SL, Roger AJ, Wenk-Siefert I, Doolittle WF. 2000. A kingdom-770

level phylogeny of eukaryotes based on combined protein data. Science 771

290:972-977. 772

58. Lepesheva GI, Zaitseva NG, Nes WD, Zhou W, Arase M, Liu J, Hill GC, 773

Waterman MR. 2006. CYP51 from Trypanosoma cruzi – a phyla-specific 774

residue in the B' helix defines substrate preferences of sterol 14α-775

demethylase. J. Biol. Chem. 281:3577-3585. 776

59. Hargrove TY, Wawrzak Z, Liu J, Nes WD, Waterman MR. 2011. 777

Substrate preferences and catalytic parameters determined by structural 778

characteristics of sterol 14α-demethylase (CYP51) from Leishmania 779

infantum. J. Biol. Chem. 286:26838-26848. 780


37

60. Warrilow AGS, Parker JE, Kelly DE, Kelly SL. 2013. Azole affinity of 781

sterol 14α-demethylase (CYP51) enzymes from Candida albicans and 782

Homo sapiens. Antimicrob. Agents Chemother. 57:1352-1360. 783

61. Warrilow AGS, Melo N, Martel CM, Parker JE, Nes WD, Kelly SL, Kelly 784

DE. 2010. Expression, purification, and characterization of Aspergillus 785

fumigatus sterol 14-α demethylase (CYP51) isoenzymes A and B. 786

Antimicrob. Agents Chemother. 54:4225-4234. 787

62. Parker JE, Warrilow AGS, Cools HJ, Martel CM, Nes WD, Fraaije BA, 788

Lucas JA, Kelly DE, Kelly SL. 2011. Mechanism of binding of 789

prothioconazole to Mycosphaerella graminicola CYP51 differs from that of 790

other azole antifungals. Appl. Environ. Microbiol. 77:1460-1465. 791

63. Heeres J, Hendrickx R, Van Cutsem J. 1983. Antimycotic azoles. 6. 792

Synthesis and antifungal properties of terconazole, a novel triazole ketal. 793

J. Med. Chem. 26:611-613. 794

64. Pelletier R, Peter J, Antin C, Gonzalez C, Wood L, Walsh TJ. 2000. 795

Emergence of resistance of Candida albicans to clotrimazole in human 796

immunodeficiency virus-infected children: in vitro and clinical correlations. 797

J. Clin. Microbiol. 38:1563-1568. 798

65. Bulik CC, Sobel JD, Nailor MD. 2011. Susceptibility profile of vaginal 799

isolates of Candida albicans prior to and following fluconazole induction - 800

impact of two decades. Mycoses 54:34-38. 801

66. Martel CM, Parker JE, Bader O, Weig M, Gross U, Warrilow AGS, 802

Rolley N, Kelly DE, Kelly SL. 2010. Identification and characterization of 803


38

four azole- resistant erg3 mutations of Candida albicans. Antimicrob. 804

Agents Chemother. 54:4527– 4533. 805

67. Marking LL, Rach JJ, Schreier TM. 1994. Search for antifungal agents in 806

fish culture. In Mueller GJ (ed), Salmon Saprolegniasis. US department of 807

energy, Bonneville Power administration, Portland Oregon. pp 131-148. 808

68. Morrison AMS, Goldstone JV, Lamb DC, Kubota A, Lemaire B, 809

Stegeman JJ. 2014. Identification, modeling and ligand affinity of early 810

deuterostome CYP51s, and functional characterization of recombinant 811

zebrafish sterol 14α-demethylase. Biochim. Biophys. Acta. 1840:1825-812

1836. 813

69. Kelly SL, Lamb DC, Kelly DE, Loeffler J, Einsele H. 1996. Resistance to 814

fluconazole and amphotericin in Candida albicans from AIDS patients. 815

Lancet 348:1523–1524. 816

70. Martel CM, Parker JE, Bader O, Weig M, Gross U, Warrilow AGS, 817

Kelly DE, Kelly SL. 2010. A clinical isolate of Candida albicans with 818

mutations in ERG11 (encoding sterol 14α-demethylase) and ERG5 819

(encoding C22 desaturase) is cross resistant to azoles and amphotericin 820

B. Antimicrob. Agents Chemother. 54:3578– 3583. 821

71. Watson PF, Rose ME, Ellis SW, England H, Kelly SL. 1989. Defective 822

sterol C5-6 desaturation and azole resistance: a new hypothesis for the 823

mode of action of azole antifungals. Biochem. Biophys. Res. Comm. 824

164:1170 –1175. 825


39

72. Dhamgaye S, Devaux F, Manoharlal R, Vandeputte P, Shah AH, Singh 826

A, Blugeon C, Sanglard D, Prasad R. 2012. In vitro effect of malachite 827

green on Candida albicans involves multiple pathways and transcriptional 828

regulators UPC2 and STP2. Antimicrob. Agents Chemother. 56:495-506. 829

73. Bailey TA, Jeffrey SM. 1989. Investigations in fish control: 99. Evaluation 830

of 215 candidate fungicides for use in fish culture. United States 831

Department of the Interior, Fish and Wildlife Service. ISSN 0565-0704. 832

74. Dallet M. 2013. Background document on clotrimazole (2013 update). A 833

report published by the OSPAR Commission, London. ISBN: 978-1-9-834

9159-28-0. 835

75. Bly JE, Lawson LA, Dale DJ, Szalai AJ, Durborow RM, Clem LW. 836

1992. Winter saprolegniosis in channel catfish. Dis. Aquat. Organ. 13:155-837

165. 838

76. Noga EJ. 1996. Fish disease diagnosis and treatment. Mosby-Year Book 839

Inc, St. Louis. 840

77. Willoughby LG. 1989. Continued defense of salmonid fish against 841

Saprolegnia fungus, after its establishment. J. Fish Dis. 12:63-67.. 842

78. Zaki MS, Fawzi OM, Jackey JE. 2008. Pathological and biochemical 843

studies in Tilapia nilotica infected with Saprolegnia parasitica and treated 844

with potassium permanganate. J. Agricul. Environ. Sci. 3:677-680. 845

846


40

847


41

FIG 1 Chemical structures of azole antifungal agents. The chemical structures of clotrimazole (molecular weight [MW], 848

345), econazole (MW, 445 as nitrate salt), fluconazole (MW, 306), itraconazole (MW, 706), ketoconazole (MW, 531), 849

miconazole (MW, 479 as nitrate salt), posaconazole (MW, 701), voriconazole (MW, 349), epoxiconazole (MW, 330), 850

prochloraz (MW, 377), propiconazole (MW, 342), prothioconazole (MW, 344), prothioconazole-desthio (MW, 312), 851

tebuconazole (MW, 308) and triadimenol (MW, 296) are shown. Also shown are the triazole and imidazole ring numbering 852

systems, using the voriconazole and econazole structures as examples. 853


42

854

855


43

FIG 2 Phylogenetic tree of eukaryotic CYP51 enzymes. A phylogenetic tree of 856

selected eukaryotic CYP51 proteins, including members from the fungi, plant and 857

animal kingdoms in addition to trypanosomal and oomycete CYP51 proteins, was 858

constructed. The individual CYP51 sequences used to construct the phylogenetic 859

tree are detailed in the supplementary table. Species names have been left non-860

italicized for clarity with (A) and (B) indicating CYP51A and CYP51B, 861

respectively. 862

863

864


44

865

FIG 3 Spectral properties of SpCYP51. The absolute oxidized absorption 866

spectrum (A) between 700 and 300 nm for 2 μM SpCYP51 is shown. Reduced 867

carbon monoxide difference spectra (B) between 500 and 400 nm using 2 μM 868

SpCYP51 is shown at sequential 45 second intervals with arrows indicating the 869

progressive fall in A447 and rise in A421 with time. Matched quartz semi-micro 870

cuvettes of light path 10 mm were used. 871


45

872

FIG 4 Sterol binding properties of SpCYP51. Type I difference spectra (A) were 873

obtained by progressive titration of lanosterol, eburicol and obtusifoliol against 4 874

μM SpCYP51 using 4.5 mm light path quartz semi-micro cuvettes. Sterol 875


46

saturation curves (B) were constructed for lanosterol (filled circles), eburicol 876

(hollow circles) and obtusifoliol (bullets). All spectral determinations were 877

performed in triplicate although only one replicate is shown. 878

879

880


47

881

FIG 5 Pharmazole binding to SpCYP51. Type II difference spectra were obtained 882

by progressive titration of 2 μM SpCYP51 with individual pharmazoles. Matched 883

quartz semi-micro cuvettes of light path 4.5 mm were used and all spectral 884

determinations were performed in triplicate although only one replicate is shown. 885

886

887


48

888

FIG 6 Agriazole binding to SpCYP51. Type II difference spectra were obtained by 889

progressive titration of 2 μM SpCYP51 with individual agriazoles. No reproducible 890

difference spectra were obtained between prothioconazole and 2 μM SpCYP51 891

(data not shown). Matched quartz semi-micro cuvettes of light path 4.5 mm were 892

used and all spectral determinations were performed in triplicate although only 893

one replicate is shown. 894

895

896


49

897

FIG 7 Azole saturation curves for SpCYP51. Saturation curves were constructed 898

for the imidazole-based pharmazoles (A), triazole-based pharmazoles (B) and 899

agriazoles (C) from the change in absorbance (ΔA428-412) against azole 900

concentration using a rearrangement of the Morrison equation (47) for the tight 901

ligand binding observed with SpCYP51 (Fig. 5 and 6). The imidazole-based 902

pharmazoles (A) used were clotrimazole (filled circles), econazole (hollow 903

circles), ketoconazole (bullets) and miconazole (crosses). The triazole-based 904

pharmazoles (B) used were fluconazole (filled circles), itraconazole (hollow 905


50

circles), posaconazole (bullets) and voriconazole (crosses). The agriazoles (C) 906

used were epoxiconazole (filled circles), prochloraz (hollow circles), 907

propiconazole (bullets), prothioconazole-desthio (crosses), tebuconazole 908

(asterisks) and triadimenol (filled triangles). All determinations were performed in 909

triplicate although only one replicate is shown. 910

911

912


51

913

FIG 8 Azole IC50 determinations for SpCYP51. IC50 values were determined for 914

the pharmazoles (A) clotrimazole (filled circles) fluconazole (hollow circles), 915

itraconazole (bullets), ketoconazole (crosses) and the agriazoles (B) 916

epoxiconazole (hollow circles), propiconazole (bullets), prochloraz (crosses) and 917

tebuconazole (asterisks) with clotrimazole (filled circles) as a reference in panel 918


52

B. The CYP51 reconstitution assays contained 0.5 μM SpCYP51 and 1 μM 919

AfCPR1 using 50 μM eburicol as substrate. Relative velocities of 1.00 920

corresponded to actual velocities of 0.87 ±0.21 min-1 for SpCYP51. The mean 921

values of two replicates are shown along with the associated standard error bars. 922

923

924


53

925

FIG 9 MIC determinations for Saprolegnia species. MIC100 values were 926

determined in duplicate after 72 h at 20°C using serial dilutions of 256, 128, 64, 927

32, 16, 8, 4, 2, 1, 0.5 and 0.25 µg ml-1 compound along with a dimethylsulfoxide 928

control. The MIC plates for epoxiconazole, prochloraz, clotrimazole and 929

malachite green are shown for S. parasitica, although identical results were 930

obtained with S. diclina and S. ferax. 931

932

933


54

934


55

FIG 10 Gas chromatograms of sterol content of Saprolegnia species. The 935

nonsaponifiable lipid fraction was isolated (50) from untreated and prochloraz-936

treated (16 µg ml-1) S. diclina, S. ferax and S. parasitica after 72 h growth at 937

20°C. Samples were derivatized with TMS prior to analysis by GC/MS (45). The 938

gas chromatograms are shown for untreated (solid lines) and prochloraz-treated 939

(dashed lines) samples. Gas chromatogram peaks were confirmed to be 940

cholesterol (1), desmosterol (2), 24-methylene cholesterol (3), fucosterol (4) and 941

lanosterol (5) by the mass fragmentation patterns obtained (see supplementary 942

figure S1). 943

944

945


56

946

FIG 11 Post-lanosterol sterol biosynthetic pathway in Saprolegnia 947

parasitica. The post-lanosterol biosynthetic pathway was elucidated based on 948

the sterol metabolites observed (Fig. 10) and searching the S. parasitica genome 949

database using homologs from A. euteiches, A. fumigatus and H. sapiens. 950

Dashed lines indicate reactions that involve several enzymes. 951


57

TABLE 1 Affinity of SpCYP51 for azole antifungal agents. 952 953 __________________________________________________________________________

Azole Kd (nM) IC50 (μM) Residual CYP51 activity a (%)

__________________________________________________________________________ Imidazole

Pharmazoles

Clotrimazole 24.6 ±5.2 0.95 27 Econazole ≤7.8 ±1.5 nd -

Ketoconazole ≤12.7 ±5.2 2.27 38 Miconazole ≤8.8 ±3.1 nd -

Triazole

Pharmazoles

Fluconazole 39.5 ±5.0 1.35 30 Itraconazole ≤8.9 ±3.7 0.84 19

Posaconazole ≤3.2 ±1.4 nd - Voriconazole ≤14.6 ±6.3 nd -

Imidazole Agriazole

Prochloraz ≤5.8 ±1.2 0.15 0

Triazole Agriazoles Epoxiconazole ≤3.5 ±1.1 0.17 0 Propiconazole ≤5.7 ±2.1 0.20 0

Prothioconazole-desthio

50.8 ±16.8 nd -

Tebuconazole ≤14.6 ±5.7 0.47 27 Triadimenol ≤16.4 ±11.8 nd -

__________________________________________________________________________ 954 a Residual CYP51 activity observed in the presence of 4 μM azole antifungal agent expressed as 955

a percentage of the CYP51 activity observed in the absence of azole. Prothioconazole did not 956

bind to SpCYP51. Mean Kd values of three replicates are shown along with the associated 957

standard deviations. 958

nd - not determined. 959

960

961


58

TABLE 2 MIC100 for azole antifungals against Saprolgenia species. 962 963 _____________________________________________________________________________

MIC100 (t = 72 h) a Inhibitor __________________________________________________

µg ml-1 µM _____________________________________________________________________________

Clotrimazole 1 to 2 3 to 6 Econazole 8 18

Epoxiconazole >256 >776 Fluconazole >256 >836 Itraconazole >256 >363

Ketoconazole 64 >120 Miconazole 8 17

Posaconazole >256 >365 Prochloraz 32 85

Propiconazole 64 187 Prothioconazole 16 46

Prothioconazole-desthio 32 to 64 103 to 205 Tebuconazole 64 208 Triadimenol 64 216

Malachite green 1 3

_____________________________________________________________________________ 964 a MIC100 experiments were performed in duplicate using S. parasitica, S. diclina and S. ferax. All 965

three Saprolegnia species displayed identical azole susceptibilities to the azoles tested and to the 966

malachite green positive control at 20°C. We have previously shown that prothioconazole is 967

progressively converted to prothioconazole-desthio in the media over time (45, 62) explaining 968

why a compound that can not bind to SpCYP51 can inhibit SpCYP51 activity and sterol 969

biosynthesis in Saprolegnia. 970

971


59

972 973 TABLE 3 Sterol compositions of untreated and azole-treated Saprolegnia species. 974 975 ____________________________________________________________________________________________________________________

Sterol composition (%) ___________________________________________________________________________________________

Sterol Untreated Prochloraz-treated (16 µg ml-1) Clotrimazole-treated (0.5 µg ml-1) ______________________________ ______________________________ __________________________ S. ferax S. parasitica S. diclina S. ferax S. parasitica S. diclina S. parasitica

____________________________________________________________________________________________________________________ Cholesterol 2.6 2.0 4.2 0.6 - - 0.8 Desmosterol 17.3 54.7 19.8 0.6 2.5 9.3 1.9

24-methylene cholesterol 46.3 39.4 49.1 5.9 0.6 - 4.9 Fucosterol 33.8 3.9 26.9 34.5 - - - Lanosterol trace trace trace 58.4 96.9 90.7 92.4

____________________________________________________________________________________________________________________ 976

The sterol compositions of untreated and prochloraz-treated S. ferax, S. parasitica and S. diclina, in addition to clotrimazole-treated S. parasitica, 977

were determined by extraction of the nonsaponifiable lipid fraction (50) followed by derivatization with BSTFA/TMS/pyridine and analysis by 978

GC/MS (45). All analyses were performed in duplicate. 979

980


60

TABLE 4 Sterol biosynthetic genes in Saprolegnia parasitica. 981 982 _________________________________________________________________________________

Enzyme Gene Locus ID Query Accession Number _________________________________________________________________________________

Squalene synthase SPRG_06233.2 B2ZWF5 Squalene epoxidase SPRG_11641.2 B2ZWF6 Lanosterol synthase SPRG_11783.2 & B2ZWF7

SPRG_17895.2

Δ14 sterol demethylase (CYP51) SPRG_09493.2 B2ZWE1 Δ14 sterol reductase SPRG_00418.2 B2ZWF8

Δ4 sterol methyl oxidase SPRG_01623.2 B2ZWF9 Δ3 sterol dehydrogenase SPRG_01499.2 Q4X017 Δ3 sterol keto reductase Not Identified -

Δ24 sterol methyltransferase SPRG_05001.2 B2ZWE2

Δ8 sterol isomerase SPRG_13330.2 Q15125 Δ5 sterol desaturase SPRG_11773.2 & Q4WDL3

SPRG_18544.2 Δ7 sterol reductase SPRG_01085.2 B2ZWG0 Δ24 sterol reductase SPRG_04988.2 B2ZWG1

_________________________________________________________________________________

983

The gene locus IDs of the most likely candidate(s) are shown along with the UniProtKB accession 984

number of the sequence used as the query in NCBI BLASTP searches. Sterol biosynthetic gene 985

homologs from A. euteiches (35), A. fumigatus (Q4WDL3 and Q4X017) and H. sapiens (Q15125) 986

were used for the BLASTP searches. 987

988

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