Transcription factor ADS-4 regulates adaptive responses and resistance to antifungal azole stress

39 Pages·2015·7.72 MB·English

AAC Accepted Manuscript Posted Online 22 June 2015
Antimicrob. Agents Chemother. doi:10.1128/AAC.00542-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.

1 Title: Transcription factor ADS-4 regulates adaptive responses and resistance to
2 antifungal azole stress
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4 Kangji Wanga,b, Zhenying Zhanga, Xi Chena,b, Xianyun Suna, Cheng Jina, Hongwei Liua*,
5 Shaojie Lia* D
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8 a State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, fr
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9 Beijing 100101, China h
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10 b University of Chinese Academy of Sciences, Beijing 100049, China :/
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13 ∗Corresponding author address: /
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14 State Key Laboratory of Mycology, A
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15 Institute of Microbiology, Chinese Academy of Sciences, 4
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16 No.1 Beichen West Road, Chaoyang District, 1
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17 Beijing 100101, P.R.China. y

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18 Tel: [phone], Fax: [phone] e
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19 E-mail address: ([email protected] (S. Li) or
20 [email protected] (H. Liu)
21
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23 Abstract
24 Azoles are commonly used as antifungal drugs or pesticides to control fungal infections
25 in medicine and agriculture. Fungi adapt to azole stress by rapidly activating the transcription
26 of a number of genes and transcriptional increases in some azole responsive genes can elevate
27 azole resistance. The regulatory mechanisms that control transcriptional responses to azole D
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28 stress are not well understood in filamentous fungi. This study identified a bZIP transcription n
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29 factor, ADS-4 (antifungal drug sensitive-4), as a new regulator of adaptive responses and d
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30 resistance to antifungal azoles. Transcription of ads-4 in Neurospora crassa increased when it fr
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31 was subjected to ketoconazole treatment, whereas the deletion of ads-4 resulted in h
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32 hypersensitivity to ketoconazole and fluconazole. In contrast, the overexpression of ads-4 //
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33 increased resistance to fluconazole and ketoconazole in N. crassa. RNA-seq, followed by .
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34 qRT-PCR confirmation, showed that ADS-4 positively regulated the transcriptional responses .
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35 of at least six genes to ketoconazole stress in N. crassa. The gene products of four /
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36 ADS-4-regulated genes are known contributors to azole resistance, including the major efflux A
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37 pump CDR4 (Pdr5p ortholog), an ABC multidrug transporter (NcAbcB), sterol C-22 4
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38 desaturase ERG5 and a lipid transporter NcRTA2, which is involved in calcineurin-mediated 1
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39 azole resistance. Deletion of the ads-4 homologous gene Afads-4 in Aspergillus fumigatus y

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40 caused hypersensitivity to itraconazole and ketoconazole, which suggested that ADS-4 is a e
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41 functionally conserved regulator of adaptive responses to azoles. This study provides
42 important information on a new azole resistance factor that could be targeted by a new range
43 of anti-fungal pesticides and drugs.
44 Key words: drug resistance, transcription factor, Neurospora crassa，Aspergillus fumigatus，
45 stress adaptation
46 Introduction
47 Filamentous fungi cause over 70% of plant diseases and some can cause deadly infections
48 in humans(1-5). Azoles are the most commonly used antifungal drugs in medicine (e.g.
49 itraconazole, fluconazole and ketoconazole) and some azoles, such as triadimenol and D
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50 propiconazole, are also used to control fungal diseases in plants (6). Antifungal azoles inhibit n
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51 14α-methyl sterol demethylase (encoded by ERG11), a key enzyme involved in fungal d
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52 ergosterol biosynthesis. This leads to changes in membrane consistency(7, 8). In addition to fr
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53 blocking ergosterol production, the inhibition of ERG11 by azoles also results in the h
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54 accumulation of toxic 14α-methylated sterol intermediates (9, 10). //
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55 Fungi can adapt to azole stress by rapidly increasing the expression of a number of genes. .
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56 Increased expression of some azole-responsive genes, such as genes encoding azole efflux .
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57 pumps and genes involved in ergosterol biosynthesis, can increase resistance to azoles (11, /
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58 12). Previous studies in Saccharomyces cerevisiae and Candida albicans identified a number A
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59 of regulatory genes that mediate azole responses. Transcription factors Pdr1p and Pdr3p in S. 4
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60 cerevisiae, and their homologs in C. albicans, regulate azole responses by controlling 1
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61 multidrug efflux pump genes (13-15). However, filamentous fungi do not have their homologs. y

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62 Transcription factor Upc2p in C. albicans, and its ortholog Ecm22p in S. cerevisiae, regulate e
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63 azole responses by up-regulating ergosterol synthesis genes and multidrug efflux pump genes
64 (16-19). Although Upc2p homologs are present in filamentous fungi, the deletion mutant
65 (FGSC#11076) of Neurospora crassa Upc2p homolog gene (NCU03686) was not
66 hypersensitive to ketoconazole (20). To date, only one transcription factor, AP-1, is known to
67 play a role in the azole responses in both yeasts and filamentous fungi (21-23). It is possible
68 that filamentous fungi may have azole response regulation mechanisms that are different from
69 those found in yeasts.
70 Neurospora crassa, which has knockout mutants for over 6000 genes, was recently used
71 as a model to study how filamentous fungi respond to azole stress. This has led to the D
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72 discovery of a number of new azole responsive genes that play an important role in azole n
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73 resistance (20, 24, 25). One of the newly identified genes encodes a transcription factor d
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74 CCG-8 in N. crassa that regulates transcriptional responses to ketoconazole by 78 genes, fr
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75 including azole target coding gene erg11 and a Pdr5p-like ABC transporter coding gene cdr4. h
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76 Its homolog in another filamentous fungus, Fusarium verticillioides, also has a similar role //
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77 (20). .
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78 Using N. crassa as a model, this study identified another new transcription factor, ADS-4, .
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79 which is essential for normal azole resistance in both N. crassa and Aspergillus fumigatus. /
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80 ADS-4 regulated the transcriptional responses of at least six genes to ketoconazole. A
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81 Material and methods 4
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82 Strain cultivation 1
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83 The Neurospora crassa wild-type (WT) strain and knockout mutants used in this study y

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84 were purchased from the Fungal Genetics Stock Center (FGSC Kansas City, Missouri USA) e
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85 and are listed in Supplementary Table 1. Vogel’s medium (26), supplemented with 2% (w/v)
86 sucrose for slants or 2% glucose for liquid and plate media, were used to culture N. crassa.
87 All the N. crassa strains were cultured at 28oC.
88 Aspergillus fumigatus wild-type strain YJ407 and the CEA17 strain (△pyrG89) were
89 grown in complete medium (CM). All Aspergillus fumigatus cultures were grown at 37oC.
90 Drug sensitivity test
91 Ketoconazole, itraconazole and fluconazole were dissolved in dimethyl sulfoxide
92 (DMSO) and then aseptically added to the autoclaved medium before it was poured into agar
93 plates. The final DMSO concentration was below 0.25% (v/v). Two microliters of conidial D
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94 suspension were inoculated onto the plates (Φ = 9 cm), with or without antifungal drugs, and n
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95 incubated in the dark. d
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96 Complementation of the ads-4 deletion mutant fr
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97 To complement the ads-4 knockout mutant, the ads-4 knockout mutant (FGSC #11386) h
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98 was crossed with mutant FGSC#6103 (his-3; A), which can not synthesize histidine, to //
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99 generate the ads-4 knockout strain NCW#1, which has a his-3 background. To create the .
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100 complementary plasmid, the whole length of the ads-4 coding sequence (1260 bp), with a .
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101 1948 bp upstream region and a 1976 bp downstream region, was amplified using primers: /
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102 ads4F and ads4R (Table 1) to create a 5184 bp complementation fragment. The PCR product A
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103 was inserted into the pBM61 vector (27) at the SmaI site to form the complementary plasmid 4
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104 pBM61-ads4. The PBM61-ads4 construct was transformed into the ads-4 deletion mutant 1
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105 with a his-3 background (NCW#1) using a previously reported method (28). Transformants y

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106 were screened on Vogel’s medium without histidine and verified by PCR using primers: e
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107 ads4v-F and ad4v-R (Table 1).
108 Overexpression of ads-4
109 The cfp promoter was used to overexpress ads-4 (29). The cfp promoter (888 bp) was
110 amplified from the wild-type N. crassa genome by PCR using cfp-F and cfp-R primers (Table
111 1). The ads-4 coding region, tagged with 5×cMyc-6×His, was amplified from the
112 Qa5myc6his-ads4 vector (constructed by inserting the ads-4 coding sequence into the
113 Qa5myc6his plasmid) by PCR using the hismycads4-F and hismycads4-R primers (Table 1).
114 The trpc terminator (997 bp) was amplified from the pCSN43 vector using primers trpc-F and
115 trpc-R (Table 1). The three fragments were purified and fused together by fusion PCR. The D
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116 fused fragment (3423 bp) was ligated to the pCSN43 vector, which produced the ads-4 n
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117 overexpression vector pCSN43-ads4OE. The pCSN43-ads4OE vector was transformed into the d
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118 N. crassa wild-type strain (FGSC#4200) by protoplast transformation as previously reported fr
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119 (20). The transformants were screened on Vogel’s medium with hygromycin and verified by h
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120 PCR using primers cfp-F and trpc-R (Table 1). //
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121 RNA extraction and transcriptional analysis by qRT-PCR .
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122 RNA extraction and cDNA synthesis were performed according to previously described .
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123 methods (24). The qRT-PCR was carried out using the iQ5 Multicolor Real-Time PCR /
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124 Detection System (Bio-Rad, Hercules, CA) with SYBR-Green detection (SYBR PrimeScript A
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125 RT-PCR Kit, TaKaRa Biotechnology Co., Ltd.) according to the manufacturer’s instructions. 4
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126 Each cDNA sample was analyzed in triplicate and the average threshold cycle was calculated. 1
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127 Relative expression levels were calculated using the 2-ΔΔCt method (30). The results were y

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128 normalized to the expression level of β-tubulin. Gene-specific primers were shown in Table 1. e
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129 Transcript profile analysis
130 Briefly, conidia from the wild-type strain and the ads-4 deletion mutant were added to 20
131 mL Vogel’s liquid medium in a plate (Φ = 9 cm) and incubated at 28oC in the dark for 24 h
132 until a mycelial mat formed on the surface of the liquid medium. The mycelial mat was then
133 cut into small pieces (Φ = 10 mm) and transferred to the Vogel’s liquid medium (two
134 pieces/100 mL) in 150 mL flasks. The cultures were incubated at 28°C for 12 h with shaking.
135 KTC, at a final concentration of 2.5 µg/mL, was then added to the medium. After 24 h
136 incubation, total RNA was extracted and subjected to RNA-seq analysis. Genes with the
137 transcriptional ratios of more than 2.0 or less than 0.5 in two samples were considered to be D
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138 differentially expressed. n
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139 Knockout of the ads-4 homologous gene in Aspergillus fumigatus d
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140 A deletion cassette, containing the pyrG gene as the selectable marker, was constructed fr
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141 to knock out Afu1g16460 in Aspergillus fumigatus. PCR primers Afu1g16460U-XhoI-F and h
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142 Afu1g16460U-ClaI-SmaI-R (Table 1) were designed to amplify the upstream sequence (1462 //
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143 bp) of Afu1g16460 before the ATG start codon, and Afu1g16460D-ClaI-F and .
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144 Afu1g16460D-BamHI-R (Table 1) were used to amplify downstream flanking sequence .
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145 (821bp) of Afu1g16460 after the stop codon. The upstream and downstream non-coding /
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146 fragments were digested with XhoI/ ClaI and ClaI/ BamHI, respectively, and then cloned into A
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147 the pBlueScript (pSK) vector to form the pSK-UD vector. The pyrG gene selective marker, 4
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148 which was released from pCDA14 (31) by HpaI digestion, was inserted into pSK-UD at the 1
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149 SmaI site between the upstream and downstream Afu1g16460 sequences. The deletion vectors y

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150 were linearized by digesting them with NotI. They were then transformed into A. fumigatus e
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151 CEA17 protoplasts, and plated under uridine and uracil autotrophy selection. The deletion
152 mutants were confirmed using PCR analysis with primers Afu1g16460V-F and
153 Afu1g16460V-R (Table 1) to amplify the coding sequence for Afu1g16460 (32).
154 Results
155 ADS-4 transcriptionally responds to ketoconazole in Neurospora crassa
156 NCU08744 is a transcription factor composed of 430 amino acids with a bZIP
157 DNA-binding domain. NCU08744 was named ADS-4 (antifungal drug sensitive-4) according
158 to the azole hypersensitive phenotype of its deletion mutant. RNA-seq analysis showed that
159 KTC (2.5 μg/mL) treatment (24 h) resulted in a 6.7-fold increase in the ads-4 transcript levels D
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160 (Supplemental data S1). The transcriptional increase in ads-4 after treatment with KTC was n
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161 confirmed by a followed time course experiment, in which the ads-4 transcript levels were d
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162 measured after 2 h, 6 h, 12 h, 18 h, 24 h and 30 h of KTC treatment. Figure 1A shows that the fr
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163 ads-4 transcript levels increased by 0.68-fold after 2 h of KTC treatment and continued to h
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164 increase with time. Thus, ADS-4 is a transcriptional factor that responds to KTC stress. //
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165 Deletion of ads-4 causes hypersensitivity to azoles in N. crassa .
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166 To test whether ADS-4 makes a potential contribution to azole resistance, the .
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167 sensitivities of an ads-4 deletion mutant (FGSC #11386) and the WT (FGSC #4200) to two /
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168 azole drugs were comparatively analyzed. When grown on the medium without drugs, the A
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169 ads-4 mutant had a growth rate similar to that of the WT. However, when grown on media 4
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170 containing KTC or fluconazole (FLC), the ads-4 mutant grew significantly slower than the 1
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171 WT (Figure 2). The inhibition rates for the WT when treated with KTC and FLC were 72.29% y

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172 and 61.25%, respectively, while the inhibition rates for the mutant were 90.33% and 69.96%, e
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173 respectively. Statistical analysis indicated that ads-4 deletion significantly increased
174 sensitivity to KTC and FLC (Table 2).
175 To confirm the role of ads-4 in azole resistance, the NcW#2 (△ads-4;ads-4) strain, which
176 complements the ads-4 deletion, was created. As shown in Figure 2 and Table 2, the
177 complemented strain restored the wild-type sensitivities to KTC and FLC, which indicated
178 that ADS-4 is required if normal resistance to azoles is to be maintained.
179 Overexpression of ads-4 increases azole resistance in N. crassa
180 To identify the role of ads-4 during azole resistance, an ads-4 overexpression strain,
181 ads-4OE, was generated, in which ADS-4 was tagged with 5×his-6×myc at its N terminal and D
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182 was driven by a cfp promoter (29). In ads-4OE, the ads-4 transcriptional levels were 5.25-fold n
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183 higher than in the WT in liquid medium without azoles (Figure 1B). When treated with KTC d
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184 (2.5 μg/mL) for 24 h, the ads-4 transcriptional levels in ads-4OE were 17.26-fold higher than fr
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185 in the WT (Figure 1B). On plates without azoles, ads-4OE grew at a similar rate to the WT. On h
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186 plates with KTC or FLC, ads-4OE grew faster than the WT (Figure 2). The inhibition rates for ://
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187 the WT when treated with KTC and FLC were 72.29% and 61.25%, respectively, while the .
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188 inhibition rates for ads-4oe were 67.93%, and 58.51%, respectively. The Waller-Duncan t test .
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189 showed that the ads-4oe inhibition rates for these azoles were significantly lower than the WT /
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190 rates (Table 2). These results indicate that the transcriptional increase in ads-4 during azole A
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191 stress improves azole resistance. 4
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192 Deletion of ADS-4 affects genome-wide transcriptional responses to ketoconazole in N. 1
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193 crassa y

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194 The ads-4 deletion strain and the WT genome-wide transcriptional responses to KTC e
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195 were comparatively analyzed by RNA-seq in order to ascertain whether ADS-4 mediates
196 transcriptional responses to azoles. The RNA-seq data showed that, under KTC treatment, 488
197 genes were up-regulated in the WT, while only 398 genes were up-regulated in the ads-4
198 deletion mutant. KTC treatment also caused down-regulation of 427 genes in the WT,
199 whereas only 342 genes were down-regulated by KTC treatment in the ads-4 deletion mutant
200 (Supplemental data S1).
201 ADS-4 regulates the transcriptional response by sterol C-22 desaturase ERG5 to
202 ketoconazole
203 ERG5 is a sterol C-22 desaturase and plays a role in ergosterol biosynthesis. Deletion of D
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204 erg5 in N. crassa and Fusarium verticillioides caused hypersensitivity to azoles (25). The n
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205 RNA-seq data showed that erg5 transcript level in the KTC-treated WT was 2.1-fold higher d
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206 than in the none-KTC treated WT. In the ads-4 knockout mutant, KTC treatment did not fr
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207 significantly changed the erg5 transcript level (Supplemental data S1). Consistent with the h
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208 RNA-seq data, qRT-PCR analysis showed that the expression of erg5 in the KTC-treated WT //
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209 was 3.76 folds of that in non-treated WT. In contrast, no significant transcriptional increase in .
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210 erg5 was detected in the ads-4 knockout mutant after KTC treatment (Figure 3A). .
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211 In the ads-4 overexpression strain ads-4OE, the erg5 transcript level was 2.06-fold higher /
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212 than the WT in the liquid medium without KTC. When treated with KTC, the erg5 transcript A
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213 level in the ads-4OE strain was also significantly higher than in the WT (Figure 3A). il 4
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214 These results show that ADS-4 is a transcription factor that regulates the expression of 1
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215 erg5 and is essential for the erg5 transcriptional response to KTC stress. The transcriptional y

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216 increase in ads-4 during KTC stress should promote the expression of erg5. Promotion of e
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217 erg5 expression is probably a mechanism by which ADS-4 regulates the adaptation to azole
218 stress.
219 ADS-4 activates transcriptional responses by the azole efflux pump CDR4 to
220 ketoconazole