AAC Accepted Manuscript Posted Online 13 July 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.01358-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

1

Multidrug transporters and alterations in sterol biosynthesis contribute to azole

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antifungal resistance in Candida parapsilosis

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Elizabeth L. Berkowa, Kayihura Manigabaa, Josie E. Parkerb, Katherine S. Barkera, Stephen L.

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Kellyb, P. David Rogersa#

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a

8

Science Center, Memphis, Tennessee, USA Department of Pediatrics, College of Medicine,

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University of Tennessee Health Science Center, Memphis, Tennessee, USA Children's

Department of Clinical Pharmacy, College of Pharmacy, University of Tennessee Health

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Foundation Research Center at Le Bonheur Children's Medical Center, Memphis, Tennessee,

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USA.

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b

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Kingdom

Institute of Life Science, Swansea University Medical School, Swansea, Wales, United

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#Address correspondence to P. David Rogers, [email protected]

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Abstract While much is known concerning azole resistance in Candida albicans, considerably

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less is understood in Candida parapsilosis, an emerging species of Candida with clinical

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relevance. We conducted a comprehensive analysis of azole resistance in a collection of

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resistant C. parapsilosis clinical isolates in order to determine what genes might play a role in

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this process within this species. We examined the relative expression of the putative drug

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transporter genes CDR1 and MDR1, as well as that of ERG11. In isolates overexpressing

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these genes, we sequenced the genes encoding their presumed transcriptional regulators,

35

TAC1, MRR1, and UPC2, respectively. We also sequenced the sterol biosynthesis genes ERG3

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and ERG11 in these isolates in order to find mutations that might contribute to this phenotype in

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this Candida species. Our findings demonstrate that the putative drug transporters Cdr1 and

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Mdr1 contribute directly to azole resistance, and suggest their overexpression is due to

39

activating mutations in the genes encoding their transcriptional regulators. We also observe the

40

Y132F substitution in ERG11 as the only substitution occurring exclusively among azole

41

resistant isolates and correlate this with specific changes in sterol biosynthesis. Finally, sterol

42

analysis of these isolates suggests other changes in sterol biosynthesis may contribute to azole

43

resistance in C. parapsilosis.

44 45 46 47 48 49 50 51 52 2

53

Introduction

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Although Candida albicans classically has been, and currently remains, the most

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commonly isolated of the species, approximately 50% of all Candida infections are attributable

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to one of the non-albicans species of Candida. Reports of rising rates of these species are

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increasingly common in the literature, and of these, Candida parapsilosis is of particular

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concern.(1, 2) Population-based surveillance has determined that the incidence of C.

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parapsilosis candidemia increased two-fold between 2008 and 2011 and is responsible for 10-

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20% of all candidemia in North America.(3, 4) It can lead to a wide spectrum of manifestations,

61

including endocarditis, meningitis, ocular infections, vulvovaginitis, and urinary tract infections.

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This organism has a propensity for forming biofilms on catheters and other implanted medical

63

devices and within high glucose-containing solutions, such as total parenteral nutrition.

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Nosocomial acquisition is fostered by this organism’s unique ability to grow on inanimate objects

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and surfaces as well as by the documented spread by healthcare workers via hand carriage.

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Also, as it is the predominant fungal pathogen recovered in neonatal intensive care units and is

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associated with neonatal mortality, C. parapsilosis is of specific concern for the pediatric

68

population.(5, 6)

69

The azole antifungals, and particularly fluconazole, are often first line therapy for

70

candidemia, and as such, there has been much investigation of azole resistance in Candida

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albicans, the most frequently isolated human fungal pathogen.(7) Indeed several mechanisms

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of azole resistance have been described. The overexpression of ERG11, which encodes the

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azole target enzyme sterol demethylase, leads to its increased production which overwhelms

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the activity of the drug. This often occurs due to activating mutations in the gene encoding the

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transcriptional regulator Upc2.(8, 9) Also, mutations in ERG11 that lead to amino acid

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substitutions alter target protein structure, reduce drug binding affinity, and increase azole

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resistance.(10) A less commonly observed mechanism is the inactivation of sterol desaturase

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due to mutation of ERG3. This permits the fungal cell to bypass production of toxic methylated 3

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sterols in the presence of the azole antifungal and results in resistance to azoles and

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amphotericin B.(11) In addition to these mechanisms, two types of transporters have been

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shown to contribute to azole resistance in C. albicans. Overexpression of the ATP binding

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cassette (ABC) transporters Cdr1 and Cdr2 result in efflux of all azole antifungals, whereas

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overexpression of the major facilitator superfamily (MFS) transporter Mdr1 results in efflux of

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fluconazole and voriconazole.(12, 13) Overexpression of CDR1 and CDR2 is due to activating

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mutations in the transcription factor gene TAC1 whereas overexpression of MDR1 is due to

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activating mutations in the transcription factor gene MRR1.(14, 15)

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Previous studies have provided limited information concerning azole resistance in C. One study has correlated the overexpression of MDR1 and mutations within

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parapsilosis.

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MRR1 with fluconazole resistance in laboratory-derived resistant isolates of C. parapsilosis.(2)

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More recently a surveillance study of a collection of clinical C. parapsilosis isolates again

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implicated MDR1 and MRR1 in fluconazole resistance in this species, yet a direct role for these

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genes in azole resistance was not established. This study also identified a mutation in ERG11

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leading to the Y132F amino acid substitution in sterol demethylase in many of the resistant

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isolates.(16) This substitution has been shown to contribute directly to fluconazole resistance in

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C. albicans.(17)

96

In the present study, we conducted a comprehensive analysis of azole resistance in a

97

collection of resistant C. parapsilosis complex clinical isolates in order to determine what genes

98

might play a role in this process within this species. We examined the relative expression of the

99

putative drug transporter genes CDR1 and MDR1, as well as that of ERG11.

In isolates

100

overexpressing these genes we sequenced the genes encoding their presumed transcriptional

101

regulators, TAC1, MRR1, and UPC2, respectively. We also sequenced the sterol biosynthesis

102

genes ERG3 and ERG11 in these isolates in order to find mutations that might contribute to this

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phenotype in this Candida species.

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transporters Cdr1 and Mdr1 contribute directly to azole resistance, and suggest their

Our findings demonstrate that the putative drug

4

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overexpression is due to activating mutations in the genes encoding their transcriptional

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regulators.

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exclusively among azole resistant isolates and correlate this with specific changes in sterol

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biosynthesis.

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biosynthesis may contribute to azole resistance in C. parapsilosis.

We also observe the Y132F substitution as the only substitution occurring

Finally, sterol analysis of these isolates suggests other changes in sterol

110 111 112

Materials and Methods

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Strains and media. All C. parapsilosis isolates used in this study are listed in Table 1.

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Isolates were kept as frozen stock in 40% glycerol at −80°C and subcultured on YPD (1% yeast

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extract, 2% peptone, and 1% dextrose) agar plates at 30°C. YPD liquid medium was used for

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routine growth of strains, while YPM (1% yeast extract, 2% peptone, 1% maltose) liquid medium

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was used for induction of the MAL2 promoter in constructed strains. Nourseothricin (200 μg/ml)

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was added to YPD agar plates when needed for selection of isolates containing the SAT1-

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flipper cassette.(18) One Shot Escherichia coli TOP10 chemically competent cells (Invitrogen)

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were used for plasmid construction. These strains were grown in Luria-Bertani (LB) broth or on

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LB agar plates supplemented with 100 μg/ml ampicillin (Sigma) or 50 μg/ml kanamycin (Fisher

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Bioreagents), when needed.

123 124

Drug susceptibility testing. Stock solutions of the fluconazole were prepared by dissolving

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drug in water to a concentration of 5mg/mL. Stock solutions of both voriconazole and

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itraconazole were prepared by dissolving drug in dimethyl sulfoxide (DMSO) at a concentration

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of 640μg/ml. MICs were obtained by performing broth microdilution as described in CLSI

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document M27-A3.(19) Aliquots of 100 μl from the working drug stocks were used to inoculate

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a series of RPMI medium dilutions, the highest being 256μg/ml for fluconazole and 8μg/ml for

5

130

both itraconazole and voriconazole. Cultures were incubated at 35°C and MICs were recorded

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at 48 hours.

132 133

Construction of plasmids. All primers used are listed in Table 2. A MDR1 deletion construct

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was generated by amplifying an ApaI-XhoI-containing fragment consisting of flanking regions -

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822 to +116 relative to the start codon of MDR1 using primers MDR1-A and MDR1-B, as well as

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a NotI-SacII-containing fragment of flanking regions +1601 to +2463 using primers MDR1-C and

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MDR1-D. These upstream and downstream fragments of MDR1 were cloned on both sides of

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the SAT1 flipper cassette in plasmid pSFS2(18) to result in plasmid p317MDR1. A CDR1

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deletion construct was generated in a similar fashion. An ApaI-XhoI-containing fragment

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consisting of flanking regions -670 to +131 relative to the start codon of CDR1 using primers

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CDR1-A and CDR1-B, as well as a SacII-SacI-containing fragment of flanking regions +4689 to

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+5219 using primers CDR1-C and CDR1-D. These upstream and downstream fragments of

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CDR1 were cloned on both sides of the SAT1 flipper cassette in plasmid pBSS2 to result in

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plasmid p317CDR1. The coding region of each gene was amplified with primers MDR1-A and

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MDR1-E or CDR1-A and CDR1-E. This ApaI-XhoI-containing fragment replaced the upstream

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sequence in cassette p317MDR1 or p317CDR1 to reintroduce the native gene, creating

147

plasmids pMDR1comp and pCDR1comp.

148 149

Candida parapsilosis transformation. C. parapsilosis strains were transformed by

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electroporation as described previously but with some modifications.(18) Cells were grown for 6

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hours in 2mL YPD liquid medium and then 4μL of this cell suspension was passed to 50mL of

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fresh YPD liquid medium and grown overnight at 30°C in a shaking incubator. When the optical

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density of the culture reached an OD600 of 2.0, cells were centrifuged at 4000rpm for 5 minutes.

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The pellet was resuspended in 1mL 10x TE buffer, 1mL lithium acetate, and 8mL of deionized

155

water and then reincubated at 30°C for 1 hour. Freshly prepared 1M dithiothreitol was added to 6

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cell suspension and reincubated for a further 30 minutes. Cells were then diluted with 40mL of

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ice cold water, pelleted at 4000rpm for 5 minutes, and washed twice, first with 25mL ice cold

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sterile water and next with 5mL ice cold 1M sorbitol. Finally the cells were resuspended in

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100μL of fresh ice cold 1mM sorbitol. Gel purified ApaI-SacI fragment from the correct plasmid

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was mixed with 40μL of competent cells and transferred into a chilled 2-mm electroporation

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cuvette. The reaction was carried out at 1.5kV, using a Cellject Pro Electroporator (Thermo).

162

Immediately following, 1mL of YPD containing 1M sorbitol was added and the mixture was

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transferred to a 1.5mL centrifuge tube. Cells were allowed to recover at 30°C for 6 hours.

164

Finally, 100μL was removed and plated to YPD agar plates containing 200μg/mL nourseothricin

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and 1M sorbitol. Transformants were selected after at least 48 hours growth at 30°C.

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RNA isolation. RNA was isolated using the hot phenol method of RNA isolation described

168

previously.(20) Briefly, overnight cultures were diluted to an OD600 of 0.2 and then incubated at

169

30°C with shaking for an additional 3 hours to mid-log phase. Cells were pelleted by

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centrifugation, liquid medium was poured off, and cells were frozen at -80°C for a minimum of 1

171

hour. Next, cell pellets were resuspended in 900μL sodium acetate-EDTA buffer, and then

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transferred to a 2-ml microcentrifuge tube containing 950μL acid phenol (pH 4.3) and 80μL 20%

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SDS. Cells were incubated at 65°C for 10 min with occasional inversion mixing, placed on ice

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for a minimum of 5 minutes, and centrifuged. The supernatant was then transferred into a new

175

tube containing 900μl of chloroform and mixed. The sample was subjected to centrifugation

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again, and the supernatent was transferred to a fresh tube containing 1mL of isopropanol and

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100μL of 2M sodium acetate. This RNA pellet was washed with 500μl of 70% ice cold ethanol

178

and collected by centrifugation. The RNA pellet was resuspended in DNase/RNase-free H2O.

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RNA concentrations were determined using a Nanodrop spectrophotometer; RNA integrity was

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verified using a Bioanalyzer 2100 (Agilent Technologies).

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qRT-PCR. Complementary DNA was synthesized from total RNA using the SuperScript First

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Strand Synthesis System in accordance to manufacturer instructions. Synthesized cDNA was

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used for both the amplification of ACT1 and the gene of interest by PCR, using SYBR Green

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PCR Master Mix according to manufacturer instructions. Gene-specific primers were designed

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using Primer Express software synthesized by Integrated DNA Technologies and are listed in

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Table 2. The PCR conditions consisted of AmpliTaq Gold activation at 95°C for 10 min,

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followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C.

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Software for the 7000 detection system (Applied Biosystems) was used to determine the

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dissociation curve and threshold cycle (CT). The 2 –ΔΔCT method was used to calculate changes

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in gene expression among isolates. All experiments include biological and technical replicates

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in triplicate. The standard error was calculated from ΔCT values as previously described.(21)

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Isolation of gDNA and Southern hybridization. Genomic DNA (gDNA) was isolated as

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described previously(22) and digested with an appropriate restriction endonuclease, separated

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on a 1% agarose gel, and, after staining with ethidium bromide, was transferred by vacuum

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blotting to a nylon membrane and fixed by UV crosslinking. Southern hybridization with

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enhanced-chemiluminescence-labeled probes was performed with the Amersham ECL Direct

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nucleic acid labeling and detection system according to the manufacturer’s instructions.

200 201

Sequence analysis of individual genes. Coding sequences from genes of interest in C.

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parapsilosis were amplified by PCR from C. parapsilosis genomic DNA using the primers listed

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in Table 2. Products were cloned into pCR-BLUNTII-TOPO using a Zero Blunt TOPO PCR

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cloning kit and transferred into Escherichia coli TOP10 cells with selection on LB agar plates

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containing 50 μg/ml kanamycin. Plasmid DNA was purified (Qiagen) and sequenced on an ABI

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model 3130XL genetic analyzer using sequencing primers resulting in a full-length sequence

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from both strands of the C. parapsilosis gene of interest. The sequencing was performed using

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six sets of clones derived from three independent PCRs for each strain/isolate sequenced.

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Sterol Profiling. Non-saponifiable lipids were extracted using alcoholic KOH. Samples were

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dried in a vacuum centrifuge (Heto) and were derivatized by the addition of 100µl 90 % BSTFA /

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10% TMS (Sigma), 200µl anhydrous pyridine (Sigma) and heating for 2 hours at 80 °C. TMS-

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derivatized sterols were analysed and identified using GC/MS (Thermo 1300 GC coupled to a

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Thermo ISQ mass spectrometer, Thermo Scientific) with reference to retention times and

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fragmentation spectra for known standards. GC/MS data files were analysed using Xcalibur

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software (Thermo Scientific) to determine sterol profiles for all isolates and for integrated peak

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areas.(23)

218 219 220

Results

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Collection of clinical isolates and susceptibility testing. Thirty five unrelated fluconazole

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resistant isolates and four susceptible isolates of Candida parapsilosis were selected from a

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collection from the University of Iowa. These isolates vary in terms of source of infection, year of

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collection, country of origin and patient demographic. In accordance with the most recent CLSI

225

guidelines for C. parapsilosis, resistance is defined as a minimum inhibitory concentration (MIC)

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of fluconazole of ≥8μg/mL and voriconazole of ≥1μg/mL. There are currently no interpretive

227

guidelines for in vitro susceptibility testing of C. parapsilosis against itraconazole. MICs were

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confirmed in our laboratory and are listed in Table 3. The 35 resistant isolates exhibited MICs of

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fluconazole ranging from 8-256μg/mL. As expected, the four remaining isolates were

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susceptible to fluconazole with MICs of 0.5μg/mL and none were susceptible dose-dependent.

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Of the 39 total isolates, fourteen isolates were susceptible to voriconazole, 13 were susceptible

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dose-dependent, and the remaining 12 isolates were resistant to voriconazole with MICs 9

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ranging from 1-8μg/mL. Susceptibilities to all isolates to itraconazole ranged from 0.016-

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0.5μg/mL.

235 236

Expression of ERG11, CDR1, and MDR1. Orthologs of CaMDR1, CaCDR1 and CaERG11

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were identified in C. parapsilosis by performing a BLAST search of these genes at the Candida

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Genome Database website and the sequence with the highest homology to that of C. albicans

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was selected for primer creation. CPAR2_301760, CPAR2_405290, and CPAR2_303740

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were identified as MDR1, CDR1, and ERG11, respectively. Quantitative real-time RT-PCR was

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used to determine whether transcript levels were increased in any resistant isolate as compared

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to an average of the 4 susceptible isolates. (Figure 1) For the purposes of our investigation, we

243

defined overexpression as a 2-fold increase in expression. For the four susceptible control

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isolates, relative expression levels for CDR1, MDR1, and ERG11, as compared to the average

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of these four isolates, ranged from 0.8-1.3 fold, 0.4-2.1 fold, and 0.3-1.8 fold. For the resistant

246

isolates, we observed varied expression of both drug transporter genes among the isolates, with

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expression levels increased as much as 50-fold. More isolates showed CDR1-overexpression

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than MDR1-overexpression. Sixteen isolates showed a minimum of 2-fold increased expression

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of CDR1. These isolates exhibited a fluconazole MIC of at least 16μg/mL. Isolates 29, 30, and

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36 showed the highest expression levels of MDR1 with a minimum of 25-fold increase; each

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exhibited a MIC of fluconazole of at least 64μg/mL. Eight isolates exhibited a minimum of 2-fold

252

increase in CpERG11 expression. These isolates ranged in susceptibility of fluconazole from

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16-128 μg/mL. Notably, a single isolate (isolate 27) exhibited the highest expression with an 11-

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fold increase. This particular isolate exhibited a fluconazole MIC of 32 μg/mL.

255 256

Sequencing of transcriptional regulators. In C. albicans, activating mutations within

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transcriptional regulator genes UPC2, MRR1, and TAC1 lead to upregulation of target genes

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ERG11, MDR1, and CDR1, respectively, and in turn decrease fluconazole susceptibility.(8, 9, 10

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12-15) As such, we questioned whether any of our ERG11, MDR1, or CDR1-overexpressing

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isolates contain SNPs within the gene encoding its putative regulator. We identified the

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orthologs of CaTAC1, CaMRR1, and CaUPC2 in the manner described above and determined

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their sequence in the isolates which overexpressed CDR1, MDR1, and ERG11, respectively, as

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compared to that of the reference strain CDC317. CPAR2_807270, CPAR2_303510, and

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CPAR2_207280 were identified as MRR1, TAC1, and UPC2, respectively. Although ERG11

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expression was observed to increase in eight of these isolates, among them we found a single

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heterozygous UPC2 mutation in only a single isolate (isolate 36). While this mutation may

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influence Upc2 activity, these data suggest that activating mutations are not the mechanism by

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which ERG11 is overexpressed in most of these isolates and, unlike in C. albicans, does not

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represent a common genetic mechanism of azole resistance in C. parapsilosis.

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Among the 16 CDR1-overexpressing isolates, mutations leading to amino acid

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substitutions were detected in TAC1 among isolates 35, 38, and 40. Both isolates 35 and 38

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contained the amino acid substitution G650E whereas isolate 40 contained a heterozygous

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L978W substitution. None of these SNPs correspond to a documented activating mutation in

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CaTAC1. TAC1 mutations were not observed in the remaining 13 isolates that overexpress

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CDR1.

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We know from C. albicans that overexpression of MDR1 must be quite high in order to

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impact on azole susceptibility.(24) As such, we focused on the 3 isolates (isolates 29, 30, and

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36) which overexpress MDR1 to the greatest degree for further evaluation. Mutations leading to

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amino acid substitutions were detected in MRR1 among MDR1-overexpressing isolates 29, 30,

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and 36. Isolate 29 contained an A854V substitution, isolate 30 contained an R479K

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substitution, and isolate 36 contained an I283R substitution. As was observed with TAC1, none

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of these SNPs correspond to a documented activating mutation in CaMRR1 and as such their

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direct role in constitutively activating the transcription of MRR1 in C. parapsilosis remains to be

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determined. 11

285 286

Targeted disruption of drug transporter genes. To determine if the putative drug

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transporters Mdr1 and Cdr1 directly contribute to azole resistance, we selected those isolates

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which overexpressed each drug transporter gene to the highest degree and deleted both alleles

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of the corresponding gene from those strains. (Figure 2) Two independent replicate mutant

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strains were created for each and MICs were determined according to CLSI guidelines.

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CDR1 was deleted in isolates 35, 38, and 40 and led to a 1-dilution decrease in

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fluconazole susceptibility in all 3 isolates. The fluconazole MIC for both isolates 35 and 40

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dropped from 128μg/mL to 64μg/mL and for isolate 38 dropped from 256μg/mL to 128μg/mL.

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All phenotypes reverted upon complementation of the deleted alleles.

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MDR1 was deleted from isolates 29, 30, and 36 and this conferred a reproducible 1-

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dilution decrease in fluconazole susceptibility in both isolates 30 and 36. The fluconazole MIC

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for isolate 30 dropped from 128μg/mL to 64μg/mL and for isolate 36 dropped from 64μg/mL to

298

32μg/mL. Isolate 29 showed no change in susceptibility upon MDR1 deletion; it maintained its

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fluconazole MIC of 64μg/mL.

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These data suggest that increased expression of MDR1 or CDR1 contributes only in part

301

to the observed azole resistant phenotype of these isolates, but cannot completely explain the

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high levels of resistance observed. Therefore, other as yet determined mechanisms are also

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impacting azole resistance in these isolates.

304 305

Alterations in the ergosterol biosynthesis pathway. In C. albicans, alterations among genes

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of the ergosterol biosynthesis pathway contribute to changes in azole susceptibility.(25) In

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order to determine whether similar resistance mechanisms are operative among the thirty-five

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C. parapsilosis isolates within this collection, we sequenced both ERG11 and ERG3 in all

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isolates and compared resulting sequences to reference strain CDC317. We also acquired

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comprehensive sterol profiles by GC/MS for each isolate in order to determine whether 12

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alterations in sterol biosynthesis may be associated with azole resistance in these clinical

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isolates.

313

We found no mutations present in ERG3 among any isolate within the collection as

314

compared to the reference strain. This conclusion is supported by the sterol profile data; no

315

isolate exhibited increases in any sterol which would point to a change in the functionality of

316

Erg3, namely ergosta-7,22-dienol, episterol, and ergosta 7-enol. We did however observe three

317

distinct SNPs in ERG11 within 16 of the isolates: S216L, Y132F, and R398I. As S216L was

318

found in only one susceptible isolate and R398I was identified in both susceptible and resistant

319

isolates, we presume that alone these mutations do not contribute to azole resistance. The

320

Y132F substitution, however, is present alone in only one of these resistant isolates whereas it

321

occurred together with the R398I substitution in 10 resistant isolates. Interestingly, nine of the

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11 total isolates containing this amino acid substitution in ERG11 showed measureable levels of

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14-methyl fecosterol, a sterol undetected in most isolates within this collection. (Table 4)

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Additionally, these isolates exhibit increases in a sterol denoted to be either lanosterol or

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obtusfoliol; these are indistinguishable by our methodology of detection.

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We also observed additional variations within the sterol profiles of isolates within this

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collection. For example, isolates 4, 15, and 19 each exhibited increases in fecosterol, isolates

328

15 and 19 displayed higher than average levels of 4-methyl fecosterol, and isolate 4 exhibited

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an increase in fecosterol in addition to increase in ergosterol. The significance of these

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alterations in sterol biosynthesis in the context of azole resistance remains to be determined.

331

(Table 4)

332 333 334 335 336

Discussion We reasoned that mechanisms of azole resistance operative in clinical isolates of C. parapsilosis would be similar to those observed in C. albicans. As such, we focused our efforts 13

337

on the genes encoding the drug transporters Cdr1 and Mdr1, those encoding the sterol

338

biosynthesis enzymes Erg11 and Erg3, and those encoding their transcriptional regulators

339

Tac1, Mrr1, and Upc2. While similarities between these species were indeed observed,

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important and unexpected differences are apparent.

341

We found that both drug transporters Mdr1 and Cdr1 appear to play a modest role in

342

azole resistance in C. parapsilosis. For Mdr1, this observation is consistent with what has been

343

observed previously in C. albicans where deletion of CaMDR1 in resistant isolates

344

overexpressing this gene led to only a 1-dilution decrease in fluconazole susceptibility.(14) The

345

same was found to be true in the MDR1-overexpressing isolates of C. parapsilosis examined in

346

the present study. We only observed marked overexpression of MDR1 in three of the isolates in

347

this collection. This is also consistent with our observations in a collection of azole-resistant

348

isolates of C. albicans.(9) We were, however, surprised that the deletion of CDR1 in isolates

349

overexpressing this gene did not increase susceptibility to fluconazole to a greater degree, as

350

has been observed in C. albicans.(15) As amino acid substitutions in both regulators of these

351

transporters, TAC1 and MRR1, are correlated with overexpression of their target genes, it is

352

tempting to speculate that other targets of these regulators may contribute to azole resistance in

353

these isolates.

354

As has been observed in C. albicans, ERG11 was found to be overexpressed in many of

355

the azole resistant clinical C. parapsilosis isolates in this collection. However, this does not

356

appear to be mediated by activating mutations in UPC2. Only one isolate, isolate 36, contained

357

a heterozygous mutation in UPC2. Unlike previously described CaUPC2 activating mutations,

358

the substitution is not located in the C-terminal domain and therefore may not contribute to

359

increased ERG11 expression.(17) The mechanism by which this increased expression occurs

360

is currently under investigation.

361 362

We sequenced both ERG3 and ERG11 in all isolates of this collection and found no amino acid substitutions in ERG3 in any isolate as compared to the reference strain CDC317. 14

363

Sterol profiles corroborate this finding. This azole resistance mechanism is considered

364

uncommon in clinical isolates of C. albicans and according to this analysis, the same might be

365

concluded in C. parapsilosis. We did, however, identify 3 individual SNPs in the ERG11 gene

366

leading to the S216L, R398I, and Y132F amino acid substitutions. The Y132F substitution was

367

found only in resistant isolates but not susceptible isolates. Amino acid position 132 is located

368

in the predicated Erg11 catalytic site and most likely interacts with fluconazole. Furthermore, in

369

C. albicans its introduction into a susceptible strain reduces susceptibility to the azoles.(17)

370

Interestingly, sterol profiles of the isolates in this collection indicate that isolates which

371

contain the Y132F substitution in ERG11 have higher than average levels of 14-methyl

372

fecosterol. Increases in this sterol have previously been identified in Erg3-defective strains of C.

373

albicans following Erg11 inhibition by azole treatment.(26) It should be reiterated that isolates of

374

this study contain no mutation within the gene encoding Erg3 and the sterol profiles as a whole

375

confirm this fact. These Y132F-containing isolates also showed particularly high abundance of

376

lanosterol/obtusfoliol, as compared to other isolates among the collection. This pattern

377

suggests a potential bottleneck in sterol biosynthesis at the point of Erg11 in these isolates.

378

(Figure 3) It is possible that Erg11 is altered in such a way by the Y132F mutation that the

379

function of this enzyme has been compromised, forcing higher sterol turnover at the point of

380

Erg25/Erg26/Erg27.

381

The low variability observed in ERG11 sequence in this collection is both surprising and

382

intriguing. We previously found 26 distinct amino acid substitutions in a collection of 63

383

fluconazole-resistant C. albicans clinical isolates.(17) It is therefore somewhat unexpected that

384

we only observed one Erg11 substitution associated with azole resistance in this collection of

385

azole resistant C. parapsilosis isolates. It is important to note that many azole resistant isolates

386

from which ERG11 mutations have been identified in C. albicans have come from oral isolates,

387

whereas the C. parapsilosis isolates in this collection were collected from bloodstream isolates

388

where higher doses of azoles are used and higher concentrations at the site of infection are 15

389

achieved. Indeed, in C. albicans, the Y132F substitution was among the strongest individual

390

mutations with regard to impact on fluconazole susceptibility increasing the MIC of fluconazole

391

from 0.5μg/mL to 2μg/mL.(17) It is possible that weaker mutations fail to arise in the presence

392

of azole concentrations achieved during the treatment of bloodstream infections. It is

393

worthwhile to note that the ERG11 sequence for the reference strain CDC317 contains the

394

Y132F substitution in Erg11 and the MIC of fluconazole for this strain is 4 μg/mL.

395

The remaining sterol data point to changes which may give insight into the ability of

396

these isolates to cope with the effects of azole treatment. Isolate 4 has a relatively normal

397

ergosterol abundance but a high fecosterol abundance. This could indicate a higher turnover

398

through the enzymes upstream of fecosterol and less rapid consumption/conversion through the

399

enzymes encoded by ERG2/3/4/5.(Figure 3) Isolates 22 and 37 have higher than average

400

amounts of 4-methyl fecosterol with slight decreases in ergosterol abundance. This could

401

indicate a bottleneck in the pathway around ERG25/26/27, leading to reduced ergosterol

402

production. Whereas ergosta-8-enol is detected in quite low quantities in most isolates, it is

403

relatively high level in Isolate 39. Excess amounts of this sterol would be expected with

404

impaired function of Erg2.(27) Whether this alteration in sterol composition is a consequence of

405

a mutation in ERG2, and in turn impacting azole resistance, is not known.

406

A recent investigation of C. parapsilosis azole resistance mechanisms by Grossman et

407

al also examined the sequences of ERG11 for presence of amino acid substitutions.(16) This

408

group utilized the sequence of a different reference strain as the comparator, ATCC 22019.

409

They identified the Y132F substitution as well and in fact observed it in 56.7% of their

410

fluconazole resistant isolates. They concluded that this mutation is perhaps largely responsible

411

for a majority of fluconazole resistance observed within this species. Our findings differ slightly

412

with respect to frequency and in that we observe this polymorphism most frequently in

413

combination with another substitution, R398I. The R398I substitution occurs alone in 1

414

susceptible isolate (isolate 3), and therefore does not appear to directly influence azole 16

415

susceptibility. As this substitution is often observed in combination with the Y132F substitution,

416

it is tempting to speculate that it may mitigate a cost in fitness that may occur in the presence of

417

the Y132F substitution alone.

418

These data strongly indicate that known molecular mechanisms of resistance do not

419

completely account for the azole resistance observed in this collection. Although it is likely that

420

an individual isolate contains more than one mechanism, there is still insufficient cause for the

421

high level resistance observed for numerous isolates. This finding highlights the importance of

422

continued investigation into the resistance mechanisms of C. parapsilosis to the azole drug

423

class.

424 425 426

Acknowledgements

427

This work was supported by NIH NIAID grant R01 AI058145 awarded to PDR.

428

We would like to thank Dr. Daniel J. Diekema for generously providing the clinical

429

isolates used in this study and Qing Zhang for her assistance in the laboratory. We also thank

430

Dr. Tom Cunningham in the Molecular Resource Center of Excellence at the University of

431

Tennessee Health Science Center.

432 433 434

References

435 436 437 438 439 440 441 442 443

1. 2.

3.

Diekema D, Arbefeville S, Boyken L, Kroeger J, Pfaller M. 2012. The changing epidemiology of healthcare-associated candidemia over three decades. Diagn Microbiol Infect Dis 73:45-48. Silva AP, Miranda IM, Guida A, Synnott J, Rocha R, Silva R, Amorim A, Pina-Vaz C, Butler G, Rodrigues AG. 2011. Transcriptional profiling of azole-resistant Candida parapsilosis strains. Antimicrob Agents Chemother 55:3546-3556. Lockhart SR, Iqbal N, Cleveland AA, Farley MM, Harrison LH, Bolden CB, Baughman W, Stein B, Hollick R, Park BJ, Chiller T. 2012. Species identification and antifungal susceptibility testing of Candida bloodstream isolates from population-based surveillance studies in two U.S. cities from 2008 to 2011. J Clin Microbiol 50:3435-3442.

17

444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490

4. 5.

6. 7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18. 19.

Pfaller MA, Diekema DJ. 2010. Epidemiology of invasive mycoses in North America. Crit Rev Microbiol 36:1-53. van Asbeck EC, Clemons KV, Stevens DA. 2009. Candida parapsilosis: a review of its epidemiology, pathogenesis, clinical aspects, typing and antimicrobial susceptibility. Crit Rev Microbiol 35:283-309. Trofa D, Gacser A, Nosanchuk JD. 2008. Candida parapsilosis, an emerging fungal pathogen. Clin Microbiol Rev 21:606-625. Pappas PG, Kauffman CA, Andes D, Benjamin DK, Jr., Calandra TF, Edwards JE, Jr., Filler SG, Fisher JF, Kullberg BJ, Ostrosky-Zeichner L, Reboli AC, Rex JH, Walsh TJ, Sobel JD, Infectious Diseases Society of A. 2009. Clinical practice guidelines for the management of candidiasis: 2009 update by the Infectious Diseases Society of America. Clin Infect Dis 48:503-535. MacPherson S, Akache B, Weber S, De Deken X, Raymond M, Turcotte B. 2005. Candida albicans zinc cluster protein Upc2p confers resistance to antifungal drugs and is an activator of ergosterol biosynthetic genes. Antimicrob Agents Chemother 49:1745-1752. Flowers SA, Barker KS, Berkow EL, Toner G, Chadwick SG, Gygax SE, Morschhauser J, Rogers PD. 2012. Gain-of-function mutations in UPC2 are a frequent cause of ERG11 upregulation in azole-resistant clinical isolates of Candida albicans. Eukaryot Cell 11:1289-1299. Warrilow AG, Martel CM, Parker JE, Melo N, Lamb DC, Nes WD, Kelly DE, Kelly SL. 2010. Azole binding properties of Candida albicans sterol 14-alpha demethylase (CaCYP51). Antimicrob Agents Chemother 54:4235-4245. Kelly SL, Lamb DC, Kelly DE, Manning NJ, Loeffler J, Hebart H, Schumacher U, Einsele H. 1997. Resistance to fluconazole and cross-resistance to amphotericin B in Candida albicans from AIDS patients caused by defective sterol delta5,6-desaturation. FEBS Lett 400:80-82. Sanglard D, Kuchler K, Ischer F, Pagani JL, Monod M, Bille J. 1995. Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrob Agents Chemother 39:2378-2386. Lopez-Ribot JL, McAtee RK, Lee LN, Kirkpatrick WR, White TC, Sanglard D, Patterson TF. 1998. Distinct patterns of gene expression associated with development of fluconazole resistance in serial candida albicans isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis. Antimicrob Agents Chemother 42:2932-2937. Morschhauser J, Barker KS, Liu TT, Bla BWJ, Homayouni R, Rogers PD. 2007. The transcription factor Mrr1p controls expression of the MDR1 efflux pump and mediates multidrug resistance in Candida albicans. PLoS Pathog 3:e164. Coste A, Turner V, Ischer F, Morschhauser J, Forche A, Selmecki A, Berman J, Bille J, Sanglard D. 2006. A mutation in Tac1p, a transcription factor regulating CDR1 and CDR2, is coupled with loss of heterozygosity at chromosome 5 to mediate antifungal resistance in Candida albicans. Genetics 172:2139-2156. Grossman NT, Pham CD, Cleveland AA, Lockhart SR. 2015. Molecular Mechanisms of Fluconazole Resistance in Candida parapsilosis Isolates from a U.S. Surveillance System. Antimicrob Agents Chemother 59:1030-1037. Flowers SA, Colon B, Whaley SG, Schuler MA, Rogers PD. 2015. Contribution of clinically derived mutations in ERG11 to azole resistance in Candida albicans. Antimicrob Agents Chemother 59:450-460. Reuss O, Vik A, Kolter R, Morschhauser J. 2004. The SAT1 flipper, an optimized tool for gene disruption in Candida albicans. Gene 341:119-127. (CLSI) CaLSI. 2008. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard - Third Edition.

18

491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512

20. 21.

22. 23.

24.

25. 26.

27.

Schmitt ME, Brown TA, Trumpower BL. 1990. A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res 18:3091-3092. Dunkel N, Liu TT, Barker KS, Homayouni R, Morschhauser J, Rogers PD. 2008. A gain-offunction mutation in the transcription factor Upc2p causes upregulation of ergosterol biosynthesis genes and increased fluconazole resistance in a clinical Candida albicans isolate. Eukaryot Cell 7:1180-1190. Amberg DC, Burke DJ, Strathern JN. 2006. Isolation of yeast genomic DNA for southern blot analysis. CSH Protoc 2006. Kelly SL, Lamb DC, Corran AJ, Baldwin BC, Kelly DE. 1995. Mode of action and resistance to azole antifungals associated with the formation of 14 alpha-methylergosta-8,24(28)-dien-3 beta,6 alpha-diol. Biochem Biophys Res Commun 207:910-915. Hiller D, Sanglard D, Morschhauser J. 2006. Overexpression of the MDR1 gene is sufficient to confer increased resistance to toxic compounds in Candida albicans. Antimicrob Agents Chemother 50:1365-1371. White TC, Marr KA, Bowden RA. 1998. Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 11:382-402. Watson PF, Rose ME, Ellis SW, England H, Kelly SL. 1989. Defective sterol C5-6 desaturation and azole resistance: a new hypothesis for the mode of action of azole antifungals. Biochem Biophys Res Commun 164:1170-1175. Hull CM, Bader O, Parker JE, Weig M, Gross U, Warrilow AG, Kelly DE, Kelly SL. 2012. Two clinical isolates of Candida glabrata exhibiting reduced sensitivity to amphotericin B both harbor mutations in ERG2. Antimicrob Agents Chemother 56:6417-6421.

513 514 515 516 517 518 519 520 521 522 523 524 525 19

526 527 528 529

Figure Legends

530 531

Figure 1. Relative fold change in expression of MDR1, CDR1, or ERG11 obtained from qRT-

532

PCR for each resistant isolate of the collection. All experiments include biological and technical

533

replicates in triplicate. Fold expression of genes was compared to the average of the

534

expression levels of the 4 susceptible isolates within the collection. The dotted line indicates 2-

535

fold relative change in gene expression. Isolates are listed in order of increasing MIC to

536

fluconazole.

537 538

Figure 2. Fluconazole MICs for isolates in which either Mdr1 (A) or Cdr1(B) have been deleted.

539

Susceptibilities were performed according to CLSI guidelines and the 48hr MICs are reported

540

here in µg/mL. Bars are grouped by specific isolate, listing the wild type isolate, the deletion

541

mutant, and the complemented derivative isolate.

542 543

Figure 3. Schematic representation of the ergosterol biosynthesis pathway for Candida species.

544

Solid arrows indicate a single enzymatic step while notched arrows represent multiple

545

enzymatic steps.

546

20

Table 1. C. parapsilosis strains used in this study Strain

Strain Background

Relevant Characteristics

Source or Reference

CDC317

N/A

N/A

ATCC

N/A

Fluconazole susceptible

University of Iowa

N/A

Fluconazole resistant

University of Iowa

CP29

mdr1Δ::FRT/mdr1Δ::FRT

Clinical Isolates CP3, 5, 13, 23 CP1-2, 4, 6-12, 1422, 24-40 Constructed Laboratory Strains 29I23N1

This study

mdr1Δ::FRT/ mdr1Δ::MDR1 FRT

CP29

-

N1A2G3

29I23N1

30B6A1

CP30

mdr1Δ::FRT/mdr1Δ::FRT

This study

A1B2G1

30B6A1

mdr1Δ::FRT/ mdr1Δ::MDR1CP30FRT

This study

36O3EE12

CP36

mdr1Δ::FRT/mdr1Δ::FRT

This study

EE12B4D1

36O3EE12

mdr1Δ::FRT/ mdr1Δ::MDR1CP36FRT

This study

35A1B1

CP35

cdr1Δ::FRT/cdr1Δ::FRT

This study

B1H2M10

35A1B1

cdr1Δ::FRT/cdr1Δ::CDR1CP35FRT

This study

38A1B1

CP38

cdr1-1Δ::FRT/cdr1-2Δ::FRT

This study

CP38

B1T4V1

38A1B1

cdr1Δ::FRT/cdr1Δ::CDR1 FRT

40B1G1

CP40

cdr1-1Δ::FRT/cdr1-2Δ::FRT

G1A2C12

40B1G1

cdr1Δ::FRT/cdr1Δ::CDR1 FRT

-

This study

This study This study

CP40

-

This study

Table 2. Primers used in this study. Underlined segments indicate introduced restriction sites. Primer qRT-PCR ACT1 – F ACT1 – R ERG11 – F ERG11 – R CDR1 – F CDR1 – R MDR1 – F MDR1 – R

Sequence 5’-TGGTTGGTATGGGTCAAA-3’ 5’-TGACGAAGCCCAATCA -3’ 5’-ATCAGCATCCACCAATGACG-3’ 5’-TCGTATTTCTAATTTGGTGG -3’ 5’-GCGTTTGACCATCGGAGTT-3’ 5’-AGATTCGCAAACAGC-3’ 5’-ATTGCCTCGGTGTTTCCAA-3’ 5’-CCTGTGGCTTGGGGTTCTC-3’

Mutant Construction MDR1 – A MDR1 – B MDR1 – C MDR1 – D MDR1 – E CDR1 – A CDR1 – B CDR1 – C CDR1 – D CDR1 – E

5’-ATATTGCAACCAGCGGGCCCGGATATAAGT-3’ 5’-AGACTATGTCATTCTCGAGAAATATTTGAA-3’ 5’-GGGAATGGTCGCTATAGCGGCCGCATTTTATTTG-3’ 5-GAGAGAAAGTGCCGCGGCCAGATATCACTA-3’ 5’-GAGAGAAAGTGCTCGAGCCAGATATCACTA-3’ 5’-GAAGTGGGGCCCATATGCATTAATTTTGTC-3’ 5’- CTGAACTGGTGTCTTCTCGAGTGTATGTTCTT -3’ 5’-AGAAACCGCGGTTTAGTCATTTGTTTTATT-3’ 5’-AATATCGGATGAGCTCAACTAGACTTTATC-3’ 5’- AATATCGGATGGTATCCTCGAGACTTTATC -3’

Sequencing ERG11 – A ERG11 – B ERG11 – C ERG11 – D ERG11 – E ERG11 – F ERG3 – A ERG3 – B ERG3 – C ERG3 – D UPC2 – A UPC2 – B UPC2 – C UPC2 – D UPC2 – E UPC2 – F UPC2 – G UPC2 – H TAC1 – A TAC1 – B TAC1 – C TAC1 – D TAC1 – E TAC1 – F TAC1 – G TAC1 – H MRR1 – A MRR1 – B MRR1 – C MRR1 – D MRR1 – E MRR1 – F MRR1 – G MRR1 - H MRR1 – I MRR1 - J

5’-CATACGACTGAGTTTCCCATCG-3’ 5’-GAAACAGAAAAGTGGCGTTGTTG-3’ 5’-GAGGACACCACGTATTGGTG-3’ 5’-CAACGAACATTCTGCATTAAACC -3’ 5’- GTAGTGGCACTAGTATGCTGTC -3’ 5’- CACGACATTGTTCAAAAAACCC -3’ 5’-CCCACGTTTATTTCACTAGATCC-3’ 5’-GGTTGCCTTGACCAACCC-3’ 5’-GTGTCCCTATTGCCCATTCCC -3’ 5’-GGGTTGGTCAAGGCAACC -3’ 5’-CCATCCTCAGAGTGAGAGACA-3’ 5’-GGACAGTTCGGTACCACCTG-3’ 5’-CTATGGCACAAGCAATGAATTCG-3’ 5’-CGAATATTTGCATTTCCGGCATTG-3’ 5’- GCCATTTGAAGTTGACCCACTAG-3’ 5’-CTAGTGGGTCAACTTCAAATGGC-3’ 5’- CCTTGGGAGTCCAAGTTGATG -3’ 5’- CGTTGAAGAGTTCAACCCATCC -3’ 5’-TGAACCATATCTGGGAGTTTAACAG-3’ 5’-GGATATGCACTGTATATCGGTACC-3’ 5’-GATGATGTCACAACCTGTACAGAG -3’ 5’-CGATTTTGCCAAACCCGATAAG-3’ 5’- CTAAACACCCCACTTGAGATGC -3’ 5’- CTTATCGGGTTTGGCAAAATCG -3’ 5’- CTCTGTACAGGTTGTGACATCATC - 3’ 5’- GGTACCGATATACAGTGCATATCC-3’ 5’-CTCGCTCTTACTTAAAGCGGAAATAC-3’ 5’-CCGGCTAATAAGCATCTCCAATTAG -3’ 5’-GAAGAAGAGTTTATCGAGTGGACGG-3’ 5’-GAGTGCTTGCAGGCAAATACATAC-3’ 5’-CATCAATTGGTGAATTCACCAAGGAG-3’ 5’- GAAAACAAGAAACACTGGGGTGG -3’ 5’-CTCCTTGGTGAATTCACCAATTGATG -3’ 5’-GTATGTATTTGCCTGCAAGCACTC -3’ 5’-CCGTCCACTCGATAAACTCTTCTTC -3’ 5’- CTAATTGGAGATGCTTATTAGCCGG -3’

Table 3. Azole antifungal susceptibilities for C. parapsilosis isolates in this collection.

Isolate 3 5 13 23 21 1 2 6 8 9 10 11 12 15 16 17 18 19 20 25 24 27 31 4 14 28 29 36 39 7 26 30 34 35 40 22 32 37 38

Fluconazole (µg/mL) 0.5 0.5 0.5 0.5 8 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 32 32 32 64 64 64 64 64 64 128 128 128 128 128 128 256 256 256 256

Voriconazole (µg/mL) 0.016 0.016 0.016 0.016 4 0.25 0.25 0.5 0.125 0.0313 0.25 0.0625 0.0625 0.25 0.0625 0.125 0.125 0.0625 0.25 1 0.5 0.25 0.5 0.25 0.125 0.25 1 0.5 1 0.5 1 2 2 4 8 1 0.0313 4 4

Itraconazole (µg/mL) 0.0313 0.0625 0.0313 0.0625 0.5 0.5 0.25 0.5 0.5 0.125 0.25 0.25 0.25 0.5 0.125 0.25 0.25 0.125 0.25 0.5 0.5 0.5 0.5 0.25 0.5 0.25 0.5 0.25 0.25 0.25 0.5 1 0.5 0.5 0.5 0.5 0.125 0.5 0.5

Table 4. Sterol compositions of C. parapsilosis isolates. Those isolates which contain ERG11 Y132F mutations are indicated with gray shading.

% Sterol Composition per Isolate MIC 0.5 0.5 0.5 0.5 8 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 32 32 32 64 64 64 64 64 64 128 128 128 128 Isolate Number 3 5 13 23 21 1 2 6 8 9 10 11 12 15 16 17 18 19 20 25 24 27 31 4 14 28 29 36 39 7 26 30 34 Ergosta-5,7,24(28)-tetraenol 0.6 0.3 0.5 0.4 0.4 0.8 Ergosta-5,8,22-trienol 0.8 0.8 0.3 0.2 0.5 0.5 0.8 0.9 1.1 0.3 0.8 0.7 1.2 Zymosterol 1.5 0.9 2.7 1.5 1.9 1.0 1.0 0.9 0.7 0.4 0.8 1.0 2.0 3.4 1.9 0.9 1.1 1.3 1.6 2.2 1.6 2.1 2.6 2.9 2.1 1.9 2.0 2.1 1.2 1.2 1.9 2.9 3.2 Ergosterol 70.5 71.1 77.2 79.2 75.8 79.2 83.6 79.0 78.2 89.6 83.3 81.4 83.1 72.4 81.8 76.4 71.2 84.8 85.7 72.8 74.8 85.6 84.2 85.1 84.8 82.3 88.0 92.0 68.3 82.3 83.2 89.1 77.9 Ergosta-7,22-dienol 3.7 3.8 2.4 1.9 1.5 2.3 1.8 2.9 2.5 1.4 1.2 2.2 1.2 0.8 2.1 3.0 0.8 0.7 2.5 2.4 0.9 1.1 0.7 0.5 1.8 1.5 0.4 0.3 1.1 0.6 0.1 1.9 Fecosterol (E8,24(28)) 0.6 0.7 0.6 1.1 1.1 1.3 0.1 0.8 0.7 0.4 3.2 0.6 0.5 0.5 3.8 0.7 1.3 0.7 1.5 3.2 1.5 2.4 0.4 1.3 0.1 2.4 1.1 1.7 2.5 Ergosta-8-enol 1.0 0.4 0.3 0.7 0.4 0.4 0.3 0.4 0.5 4.2 0.5 0.3 3.4 0.3 14-methyl fecosterol 1.2 2.8 2.1 0.8 0.8 1.6 1.5 1.2 Ergosta 5,7 dienol 11.1 15.2 4.2 10.0 7.0 7.9 6.8 10.7 11.0 3.6 6.2 5.0 2.4 7.7 4.4 11.6 15.7 8.9 4.2 5.9 5.3 1.9 2.7 3.5 3.3 1.7 7.7 2.9 3.5 1.8 2.5 Episterol (E7,24(28)) 0.5 1.0 0.4 0.6 0.6 0.5 0.8 0.9 1.9 0.6 1.0 4.3 0.7 0.4 2.9 0.5 0.4 0.7 0.9 0.4 0.2 0.4 0.6 0.4 Ergosta-7-enol 7.0 5.8 2.1 2.1 2.3 4.1 3.6 3.7 5.0 2.3 2.4 2.5 2.0 1.7 1.8 2.5 0.9 0.7 1.7 1.3 1.1 1.2 1.0 2.0 1.8 1.3 0.3 1.1 0.9 1.1 1.5 unknown m/z 484 0.6 0.5 0.5 0.9 0.4 0.2 1.2 0.7 Lanosterol 2.8 3.3 4.0 1.6 5.9 1.9 1.7 1.5 2.5 0.9 1.4 1.1 1.4 1.8 0.8 1.3 2.1 0.8 10.2 9.0 1.2 3.3 2.2 1.6 2.0 1.0 1.3 10.6 6.3 5.1 1.4 7.6 4-methyl fecosterol 0.4 0.9 0.5 1.2 1.3 1.5 1.3 1.1 0.7 1.6 1.4 0.3 0.8 0.9 0.3 4,4,-dimethyl cholesta-8,24-dienol 1.2 4.6 1.2 0.3 1.3 1.5 2.1 1.4 4.1 2.8 5.2 6.0 2.2 1.5 5.3 0.7 1.2 1.7 0.6 0.6 2.0 0.7 0.5 0.9 0.9 0.7 1.3 0.3 Eburicol 3.2 0.3 4,4-dimethyl-ergosta-8,24(28)-dienol 1.0 0.0 0.1 0.1 0.0 0.1 0.1 0.1 0.4 0.0 Total 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

128 128 256 256 256 256 35 40 22 32 37 38 0.9 0.6 0.6 0.4 0.8 1.9 1.2 1.3 1.8 0.7 2.0 74.4 57.4 69.7 60.6 62.4 80.4 2.2 2.3 3.3 3.8 3.0 1.3 1.1 0.8 0.7 0.9 0.4 2.0 1.8 0.3 0.6 0.4 0.4 3.9 6.3 2.2 1.9 3.2 7.2 1.9 10.2 8.1 1.9 1.1 6.5 0.3 3.2 0.3 2.4 3.3 4.7 1.7 0.9 1.4 1.5 0.5 0.5 0.9 11.4 20.0 10.8 8.7 14.4 6.5 0.4 1.4 0.3 1.7 0.5 0.5 0.7 0.7 0.8 1.2 0.8 0.0 0.0 100 100 100 100 100 100