Multidrug transporters and alterations in sterol biosynthesis contribute to azole 1 antifungal ...

27 Pages·2015·0.81 MB·English

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
2 antifungal resistance in Candida parapsilosis
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4 Elizabeth L. Berkowa, Kayihura Manigabaa, Josie E. Parkerb, Katherine S. Barkera, Stephen L.
5 Kellyb, P. David Rogersa# D
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7 aDepartment of Clinical Pharmacy, College of Pharmacy, University of Tennessee Health a
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8 Science Center, Memphis, Tennessee, USA Department of Pediatrics, College of Medicine,
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9 University of Tennessee Health Science Center, Memphis, Tennessee, USA Children's m

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10 Foundation Research Center at Le Bonheur Children's Medical Center, Memphis, Tennessee, tt
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11 USA. a
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12 bInstitute of Life Science, Swansea University Medical School, Swansea, Wales, United .a
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13 Kingdom .
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14 /

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15 #Address correspondence to P. David Rogers, [email protected]
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27 Abstract
28 While much is known concerning azole resistance in Candida albicans, considerably
29 less is understood in Candida parapsilosis, an emerging species of Candida with clinical
30 relevance. We conducted a comprehensive analysis of azole resistance in a collection of
31 resistant C. parapsilosis clinical isolates in order to determine what genes might play a role in D
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32 this process within this species. We examined the relative expression of the putative drug n
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33 transporter genes CDR1 and MDR1, as well as that of ERG11. In isolates overexpressing a
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34 these genes, we sequenced the genes encoding their presumed transcriptional regulators,
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35 TAC1, MRR1, and UPC2, respectively. We also sequenced the sterol biosynthesis genes ERG3 m

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36 and ERG11 in these isolates in order to find mutations that might contribute to this phenotype in tt
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37 this Candida species. Our findings demonstrate that the putative drug transporters Cdr1 and a
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38 Mdr1 contribute directly to azole resistance, and suggest their overexpression is due to .a
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39 activating mutations in the genes encoding their transcriptional regulators. We also observe the .
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40 Y132F substitution in ERG11 as the only substitution occurring exclusively among azole /

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41 resistant isolates and correlate this with specific changes in sterol biosynthesis. Finally, sterol
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42 analysis of these isolates suggests other changes in sterol biosynthesis may contribute to azole v
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43 resistance in C. parapsilosis. b
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53 Introduction
54 Although Candida albicans classically has been, and currently remains, the most
55 commonly isolated of the species, approximately 50% of all Candida infections are attributable
56 to one of the non-albicans species of Candida. Reports of rising rates of these species are
57 increasingly common in the literature, and of these, Candida parapsilosis is of particular D
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58 concern.(1, 2) Population-based surveillance has determined that the incidence of C. n
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59 parapsilosis candidemia increased two-fold between 2008 and 2011 and is responsible for 10- a
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60 20% of all candidemia in North America.(3, 4) It can lead to a wide spectrum of manifestations,
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61 including endocarditis, meningitis, ocular infections, vulvovaginitis, and urinary tract infections. m

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62 This organism has a propensity for forming biofilms on catheters and other implanted medical tt
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63 devices and within high glucose-containing solutions, such as total parenteral nutrition. a
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64 Nosocomial acquisition is fostered by this organism’s unique ability to grow on inanimate objects .a
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65 and surfaces as well as by the documented spread by healthcare workers via hand carriage. .
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66 Also, as it is the predominant fungal pathogen recovered in neonatal intensive care units and is /

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67 associated with neonatal mortality, C. parapsilosis is of specific concern for the pediatric
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68 population.(5, 6) v
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69 The azole antifungals, and particularly fluconazole, are often first line therapy for b
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70 candidemia, and as such, there has been much investigation of azole resistance in Candida 1
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71 albicans, the most frequently isolated human fungal pathogen.(7) Indeed several mechanisms 2
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72 of azole resistance have been described. The overexpression of ERG11, which encodes the 8

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73 azole target enzyme sterol demethylase, leads to its increased production which overwhelms g
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74 the activity of the drug. This often occurs due to activating mutations in the gene encoding the s
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75 transcriptional regulator Upc2.(8, 9) Also, mutations in ERG11 that lead to amino acid
76 substitutions alter target protein structure, reduce drug binding affinity, and increase azole
77 resistance.(10) A less commonly observed mechanism is the inactivation of sterol desaturase
78 due to mutation of ERG3. This permits the fungal cell to bypass production of toxic methylated
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79 sterols in the presence of the azole antifungal and results in resistance to azoles and
80 amphotericin B.(11) In addition to these mechanisms, two types of transporters have been
81 shown to contribute to azole resistance in C. albicans. Overexpression of the ATP binding
82 cassette (ABC) transporters Cdr1 and Cdr2 result in efflux of all azole antifungals, whereas
83 overexpression of the major facilitator superfamily (MFS) transporter Mdr1 results in efflux of D
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84 fluconazole and voriconazole.(12, 13) Overexpression of CDR1 and CDR2 is due to activating n
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85 mutations in the transcription factor gene TAC1 whereas overexpression of MDR1 is due to a
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86 activating mutations in the transcription factor gene MRR1.(14, 15)
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87 Previous studies have provided limited information concerning azole resistance in C. m

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88 parapsilosis. One study has correlated the overexpression of MDR1 and mutations within tt
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89 MRR1 with fluconazole resistance in laboratory-derived resistant isolates of C. parapsilosis.(2) a
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90 More recently a surveillance study of a collection of clinical C. parapsilosis isolates again .a
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91 implicated MDR1 and MRR1 in fluconazole resistance in this species, yet a direct role for these .
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92 genes in azole resistance was not established. This study also identified a mutation in ERG11 /

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93 leading to the Y132F amino acid substitution in sterol demethylase in many of the resistant
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94 isolates.(16) This substitution has been shown to contribute directly to fluconazole resistance in v
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95 C. albicans.(17) b
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96 In the present study, we conducted a comprehensive analysis of azole resistance in a 1
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97 collection of resistant C. parapsilosis complex clinical isolates in order to determine what genes 2
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98 might play a role in this process within this species. We examined the relative expression of the 8

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99 putative drug transporter genes CDR1 and MDR1, as well as that of ERG11. In isolates g
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100 overexpressing these genes we sequenced the genes encoding their presumed transcriptional s
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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
103 phenotype in this Candida species. Our findings demonstrate that the putative drug
104 transporters Cdr1 and Mdr1 contribute directly to azole resistance, and suggest their
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105 overexpression is due to activating mutations in the genes encoding their transcriptional
106 regulators. We also observe the Y132F substitution as the only substitution occurring
107 exclusively among azole resistant isolates and correlate this with specific changes in sterol
108 biosynthesis. Finally, sterol analysis of these isolates suggests other changes in sterol
109 biosynthesis may contribute to azole resistance in C. parapsilosis. D
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110 n
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112 Materials and Methods
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113 Strains and media. All C. parapsilosis isolates used in this study are listed in Table 1. m

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114 Isolates were kept as frozen stock in 40% glycerol at −80°C and subcultured on YPD (1% yeast tt
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115 extract, 2% peptone, and 1% dextrose) agar plates at 30°C. YPD liquid medium was used for a
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116 routine growth of strains, while YPM (1% yeast extract, 2% peptone, 1% maltose) liquid medium .a
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117 was used for induction of the MAL2 promoter in constructed strains. Nourseothricin (200 μg/ml) .
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118 was added to YPD agar plates when needed for selection of isolates containing the SAT1- /

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119 flipper cassette.(18) One Shot Escherichia coli TOP10 chemically competent cells (Invitrogen)
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120 were used for plasmid construction. These strains were grown in Luria-Bertani (LB) broth or on v
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121 LB agar plates supplemented with 100 μg/ml ampicillin (Sigma) or 50 μg/ml kanamycin (Fisher b
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122 Bioreagents), when needed. 1
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123 2
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124 Drug susceptibility testing. Stock solutions of the fluconazole were prepared by dissolving 8

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125 drug in water to a concentration of 5mg/mL. Stock solutions of both voriconazole and g
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126 itraconazole were prepared by dissolving drug in dimethyl sulfoxide (DMSO) at a concentration s
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127 of 640μg/ml. MICs were obtained by performing broth microdilution as described in CLSI
128 document M27-A3.(19) Aliquots of 100 μl from the working drug stocks were used to inoculate
129 a series of RPMI medium dilutions, the highest being 256μg/ml for fluconazole and 8μg/ml for
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130 both itraconazole and voriconazole. Cultures were incubated at 35°C and MICs were recorded
131 at 48 hours.
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133 Construction of plasmids. All primers used are listed in Table 2. A MDR1 deletion construct
134 was generated by amplifying an ApaI-XhoI-containing fragment consisting of flanking regions - D
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135 822 to +116 relative to the start codon of MDR1 using primers MDR1-A and MDR1-B, as well as n
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136 a NotI-SacII-containing fragment of flanking regions +1601 to +2463 using primers MDR1-C and a
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137 MDR1-D. These upstream and downstream fragments of MDR1 were cloned on both sides of
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138 the SAT1 flipper cassette in plasmid pSFS2(18) to result in plasmid p317MDR1. A CDR1 m

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139 deletion construct was generated in a similar fashion. An ApaI-XhoI-containing fragment tt
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140 consisting of flanking regions -670 to +131 relative to the start codon of CDR1 using primers a
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141 CDR1-A and CDR1-B, as well as a SacII-SacI-containing fragment of flanking regions +4689 to .a
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142 +5219 using primers CDR1-C and CDR1-D. These upstream and downstream fragments of .
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143 CDR1 were cloned on both sides of the SAT1 flipper cassette in plasmid pBSS2 to result in /

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144 plasmid p317CDR1. The coding region of each gene was amplified with primers MDR1-A and
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145 MDR1-E or CDR1-A and CDR1-E. This ApaI-XhoI-containing fragment replaced the upstream v
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146 sequence in cassette p317MDR1 or p317CDR1 to reintroduce the native gene, creating b
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147 plasmids pMDR1comp and pCDR1comp. 1
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148 2
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149 Candida parapsilosis transformation. C. parapsilosis strains were transformed by 8

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150 electroporation as described previously but with some modifications.(18) Cells were grown for 6 g
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151 hours in 2mL YPD liquid medium and then 4μL of this cell suspension was passed to 50mL of s
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152 fresh YPD liquid medium and grown overnight at 30°C in a shaking incubator. When the optical
153 density of the culture reached an OD of 2.0, cells were centrifuged at 4000rpm for 5 minutes.
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154 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
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156 cell suspension and reincubated for a further 30 minutes. Cells were then diluted with 40mL of
157 ice cold water, pelleted at 4000rpm for 5 minutes, and washed twice, first with 25mL ice cold
158 sterile water and next with 5mL ice cold 1M sorbitol. Finally the cells were resuspended in
159 100μL of fresh ice cold 1mM sorbitol. Gel purified ApaI-SacI fragment from the correct plasmid
160 was mixed with 40μL of competent cells and transferred into a chilled 2-mm electroporation D
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161 cuvette. The reaction was carried out at 1.5kV, using a Cellject Pro Electroporator (Thermo). n
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162 Immediately following, 1mL of YPD containing 1M sorbitol was added and the mixture was a
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163 transferred to a 1.5mL centrifuge tube. Cells were allowed to recover at 30°C for 6 hours.
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164 Finally, 100μL was removed and plated to YPD agar plates containing 200μg/mL nourseothricin m

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165 and 1M sorbitol. Transformants were selected after at least 48 hours growth at 30°C. tt
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166 a
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167 RNA isolation. RNA was isolated using the hot phenol method of RNA isolation described .a
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168 previously.(20) Briefly, overnight cultures were diluted to an OD600 of 0.2 and then incubated at .o
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169 30°C with shaking for an additional 3 hours to mid-log phase. Cells were pelleted by /

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170 centrifugation, liquid medium was poured off, and cells were frozen at -80°C for a minimum of 1
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171 hour. Next, cell pellets were resuspended in 900μL sodium acetate-EDTA buffer, and then v
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172 transferred to a 2-ml microcentrifuge tube containing 950μL acid phenol (pH 4.3) and 80μL 20% b
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173 SDS. Cells were incubated at 65°C for 10 min with occasional inversion mixing, placed on ice 1
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174 for a minimum of 5 minutes, and centrifuged. The supernatant was then transferred into a new 2
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175 tube containing 900μl of chloroform and mixed. The sample was subjected to centrifugation 8

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176 again, and the supernatent was transferred to a fresh tube containing 1mL of isopropanol and g
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177 100μL of 2M sodium acetate. This RNA pellet was washed with 500μl of 70% ice cold ethanol s
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178 and collected by centrifugation. The RNA pellet was resuspended in DNase/RNase-free H O.
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179 RNA concentrations were determined using a Nanodrop spectrophotometer; RNA integrity was
180 verified using a Bioanalyzer 2100 (Agilent Technologies).
181
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182 qRT-PCR. Complementary DNA was synthesized from total RNA using the SuperScript First
183 Strand Synthesis System in accordance to manufacturer instructions. Synthesized cDNA was
184 used for both the amplification of ACT1 and the gene of interest by PCR, using SYBR Green
185 PCR Master Mix according to manufacturer instructions. Gene-specific primers were designed
186 using Primer Express software synthesized by Integrated DNA Technologies and are listed in D
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187 Table 2. The PCR conditions consisted of AmpliTaq Gold activation at 95°C for 10 min, n
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188 followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C. a
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189 Software for the 7000 detection system (Applied Biosystems) was used to determine the
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190 dissociation curve and threshold cycle (C ). The 2 –ΔΔCT method was used to calculate changes m
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191 in gene expression among isolates. All experiments include biological and technical replicates tt
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192 in triplicate. The standard error was calculated from ΔC values as previously described.(21) a
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193 .a
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194 Isolation of gDNA and Southern hybridization. Genomic DNA (gDNA) was isolated as .
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195 described previously(22) and digested with an appropriate restriction endonuclease, separated /

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196 on a 1% agarose gel, and, after staining with ethidium bromide, was transferred by vacuum
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197 blotting to a nylon membrane and fixed by UV crosslinking. Southern hybridization with v
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198 enhanced-chemiluminescence-labeled probes was performed with the Amersham ECL Direct b
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199 nucleic acid labeling and detection system according to the manufacturer’s instructions. 1
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200 2
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201 Sequence analysis of individual genes. Coding sequences from genes of interest in C. 8

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202 parapsilosis were amplified by PCR from C. parapsilosis genomic DNA using the primers listed g
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203 in Table 2. Products were cloned into pCR-BLUNTII-TOPO using a Zero Blunt TOPO PCR s
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204 cloning kit and transferred into Escherichia coli TOP10 cells with selection on LB agar plates
205 containing 50 μg/ml kanamycin. Plasmid DNA was purified (Qiagen) and sequenced on an ABI
206 model 3130XL genetic analyzer using sequencing primers resulting in a full-length sequence
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207 from both strands of the C. parapsilosis gene of interest. The sequencing was performed using
208 six sets of clones derived from three independent PCRs for each strain/isolate sequenced.
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210 Sterol Profiling. Non-saponifiable lipids were extracted using alcoholic KOH. Samples were
211 dried in a vacuum centrifuge (Heto) and were derivatized by the addition of 100µl 90 % BSTFA / D
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212 10% TMS (Sigma), 200µl anhydrous pyridine (Sigma) and heating for 2 hours at 80 °C. TMS- n
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213 derivatized sterols were analysed and identified using GC/MS (Thermo 1300 GC coupled to a a
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214 Thermo ISQ mass spectrometer, Thermo Scientific) with reference to retention times and
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215 fragmentation spectra for known standards. GC/MS data files were analysed using Xcalibur m

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216 software (Thermo Scientific) to determine sterol profiles for all isolates and for integrated peak tt
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217 areas.(23) a
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218 .a
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219 .
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220 Results /

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221 Collection of clinical isolates and susceptibility testing. Thirty five unrelated fluconazole
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222 resistant isolates and four susceptible isolates of Candida parapsilosis were selected from a v
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223 collection from the University of Iowa. These isolates vary in terms of source of infection, year of b
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224 collection, country of origin and patient demographic. In accordance with the most recent CLSI 1
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225 guidelines for C. parapsilosis, resistance is defined as a minimum inhibitory concentration (MIC) 2
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226 of fluconazole of ≥8μg/mL and voriconazole of ≥1μg/mL. There are currently no interpretive 8

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227 guidelines for in vitro susceptibility testing of C. parapsilosis against itraconazole. MICs were g
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228 confirmed in our laboratory and are listed in Table 3. The 35 resistant isolates exhibited MICs of s
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229 fluconazole ranging from 8-256μg/mL. As expected, the four remaining isolates were
230 susceptible to fluconazole with MICs of 0.5μg/mL and none were susceptible dose-dependent.
231 Of the 39 total isolates, fourteen isolates were susceptible to voriconazole, 13 were susceptible
232 dose-dependent, and the remaining 12 isolates were resistant to voriconazole with MICs
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233 ranging from 1-8μg/mL. Susceptibilities to all isolates to itraconazole ranged from 0.016-
234 0.5μg/mL.
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236 Expression of ERG11, CDR1, and MDR1. Orthologs of CaMDR1, CaCDR1 and CaERG11
237 were identified in C. parapsilosis by performing a BLAST search of these genes at the Candida D
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238 Genome Database website and the sequence with the highest homology to that of C. albicans n
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239 was selected for primer creation. CPAR2_301760, CPAR2_405290, and CPAR2_303740 a
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240 were identified as MDR1, CDR1, and ERG11, respectively. Quantitative real-time RT-PCR was
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241 used to determine whether transcript levels were increased in any resistant isolate as compared m

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242 to an average of the 4 susceptible isolates. (Figure 1) For the purposes of our investigation, we tt
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243 defined overexpression as a 2-fold increase in expression. For the four susceptible control a
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244 isolates, relative expression levels for CDR1, MDR1, and ERG11, as compared to the average .a
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245 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 .
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246 isolates, we observed varied expression of both drug transporter genes among the isolates, with /

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247 expression levels increased as much as 50-fold. More isolates showed CDR1-overexpression
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248 than MDR1-overexpression. Sixteen isolates showed a minimum of 2-fold increased expression v
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249 of CDR1. These isolates exhibited a fluconazole MIC of at least 16μg/mL. Isolates 29, 30, and b
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250 36 showed the highest expression levels of MDR1 with a minimum of 25-fold increase; each 1
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251 exhibited a MIC of fluconazole of at least 64μg/mL. Eight isolates exhibited a minimum of 2-fold 2
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252 increase in CpERG11 expression. These isolates ranged in susceptibility of fluconazole from 8

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253 16-128 μg/mL. Notably, a single isolate (isolate 27) exhibited the highest expression with an 11- g
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254 fold increase. This particular isolate exhibited a fluconazole MIC of 32 μg/mL. s
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256 Sequencing of transcriptional regulators. In C. albicans, activating mutations within
257 transcriptional regulator genes UPC2, MRR1, and TAC1 lead to upregulation of target genes
258 ERG11, MDR1, and CDR1, respectively, and in turn decrease fluconazole susceptibility.(8, 9,
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