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MODEL

Research in Microbiology xx (2015) 1e9 www.elsevier.com/locate/resmic

Original article

Mechanisms of azole resistance in Candida albicans clinical isolates from Shanghai, China Jin-Yan Liu a,1, Ce Shi b,1, Ying Wang b, Wen-Jing Li b, Yue Zhao b, Ming-Jie Xiang a,b,* a

Department of Laboratory Medicine, Ruijin Hospital Luwan Branch, Shanghai Jiao Tong University School of Medicine, No. 149 South Chongqing Road, Shanghai 200020, China b Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Received 14 December 2014; accepted 24 February 2015 Available online ▪ ▪ ▪

Abstract This study was undertaken to characterize the mechanism(s) of azole resistance in clinical isolates of Candida albicans collected in Shanghai, China, focusing on the role of efflux pumps, target enzymes of fluconazole (Erg11), respiratory status and the ergosterol biosynthetic pathway. Clinical isolates of C. albicans (n ¼ 30) were collected from 30 different non-HIV-infected patients in four hospitals in Shanghai. All 30 C. albicans isolates were susceptible to amphotericin B and 5-fluorocytosine. Twelve C. albicans isolates showed resistance to at least one type of triazole antifungal. Flow cytometry analysis of rhodamine 6G efflux showed that azole-resistant isolates had greater efflux pump activity, which was consistent with elevated levels of CDR1 and CDR2 genes that code for ABC efflux pumps. However, we did not observe increased expression of ERG11 and MDR1 or respiratory deficiency. Several mutations of ERG11 and TAC1 genes were detected. The F964Y mutation in the TAC1 gene was identified for the first time. Two main sterols, ergosterol and lanosterol, were identified by GCeMS chromatogram, and no missense mutations were found in ERG3. Furthermore, seven amino acid substitutions in ERG11, A114S, Y132H, Y132F, K143Q, K143R, Y257H and G448E were found, by Type II spectral quantitative analysis, to contribute to low affinity binding between Erg11 and fluconazole. © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Triazole resistance; ERG11; Efflux pumps; Transcription factor; Ergosterol; Biosynthetic pathway

1. Introduction Candida sp. are common opportunistic pathogens. In immunocompetent patients, Candida sp. infects the skin, nails and oral-pharyngeal, gastrointestinal and vaginal mucosae. In immunocompromised or immunodeficient patients, Candida sp. can cause invasive life-threatening infections such as disseminated candidiasis [1,2]. Although the number of infections with other Candida sp. has been increasing lately,

* Corresponding author. Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, No. 197 Ruijin Second Road, Shanghai 200025, China. Tel.: þ86 21 64370045, þ86 21 63867643; fax: þ86 21 64311744, þ86 21 63867643. E-mail address: [email protected] (M.-J. Xiang). 1 Jin-Yan Liu and Ce Shi contributed equally to this work.

Candida albicans remains the most prevalent. According to the SENTRY Antimicrobial Surveillance Program of 2008e2009 in the United States, among 2085 cases of Candida-associated bloodstream infections in five different regions, 48.4% were attributable to C. albicans [3]. Recently, epidemiological studies on nosocomial Candida sp. bloodstream infections in China have been reported [4e7]. These studies showed that C. albicans was the Candida species that led to invasive candidiasis in 37.2e57.4% of cases. Due to its broad spectrum, high efficiency, good bioavailability and safety profile, fluconazole is recommended as the first choice for treating Candida infections by the Infectious Disease Society of America (2004) Candida treatment guidelines [8]. However, long-term or repeated treatment with fluconazole can lead to resistance, possibly leading to azole cross-resistance, which could seriously hamper the clinical

http://dx.doi.org/10.1016/j.resmic.2015.02.009 0923-2508/© 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Liu J-Y, et al., Mechanisms of azole resistance in Candida albicans clinical isolates from Shanghai, China, Research in Microbiology (2015), http://dx.doi.org/10.1016/j.resmic.2015.02.009

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J.-Y. Liu et al. / Research in Microbiology xx (2015) 1e9

treatment of candidiasis. In one multicenter China-SCAN study, the rate of fluconazole resistance in C. albicans was almost 14.1% [6]. Thus, understanding the molecular mechanism(s) of resistance to azoles in C. albicans has become an active area of research. Currently, several mechanism(s) are thought to lead to azole resistance in C. albicans [9e12]. The primary hypotheses include: (1) upregulation of transporters, either in the ATP binding cassette (ABC) family, such as Cdr1p or Cdr2p, or in the major facilitator superfamily (MFS), such as Mdr1p; (2) alterations in expression of the structure of the azole target enzyme, lanosterol 14a-demethylase (Erg11p); (3) mitochondrial defects leading to decreased ATP and ROS; and (4) a defect in sterol △ 5,6 desaturase (encoded by ERG3), leading to alterations in the ergosterol biosynthetic pathway. Interestingly, multiple mechanisms of resistance usually contribute to azole resistance in a single isolate [13e15]. Here we evaluated the resistance mechanisms of azoleresistant strains of C. albicans isolated from non-HIVinfected patients from four different medical hospitals in Shanghai. We focused on characterizing alterations in the azole target enzyme, the activity of the efflux pumps and their associated transcription factors, respiratory status and mutations to ERG3. 2. Materials and methods 2.1. Strains and medium C. albicans isolates (n ¼ 30) were collected from non-HIV infected patients (n ¼ 30) with vulvovaginal candidiasis, mucocutaneous candidiasis and candida infections of the skin, respiratory tract and digestive tract at four hospitals in Shanghai over a two-year period. The isolates were identified as C. albicans using germ tube formation in serum-containing medium, morphology analysis on CHROMagar Candida medium (CHROMagar, Paris, France) and carbohydrate assimilation tests (API 20C AUX BioMerieux, Marcy I'Etoile, France). All isolates were stored in YPD (yeast extract peptone dextrose) liquid medium (1 g/L yeast extract, 2 g/L peptone, 2 g/L glucose) with 25% glycerol at 20  C. The Escherichia coli strain DH5a and LB (lysogeny broth) medium were used for transformation and plasmid DNA preparation. Saccharomyces cerevisiae strain INVsc-1 (MATa his3△1/ his3△1 leu2/leu2trp1-289/trp-289ura3-52/ura3-52, His-, Leu, Trp-, Ura-) and plasmid pYES2 were purchased from Invitrogen (Carlsbad CA, USA). SD-URA3-Minus-2% glucose medium (Genmed, Minneapolis, MN, USA) was used to propagate the transfectants and SD-URA3-Minus-Gla-Raf medium (Genmed, Minneapolis, MN, USA) was used to induce heterogeneous expression of the Erg11 protein. 2.2. Antifungal susceptibility tests Fluconazole and voriconazole were purchased from Pfizer (Shanghai, China) and itraconazole, amphotericin B and

5-fluorocytosine were purchased from SigmaeAldrich (St. Louis, MO, USA). The minimum inhibitory concentrations (MICs) of these antifungal drugs were determined using the broth microdilution method established by the CLSI M27-A2 standard guideline (2002) [16]. C. albicans (ATCC 90028), Candida parapsilosis (ATCC 22019) and Candida krusei (ATCC 6258) were used as internal controls. C. albicans was scored as “susceptible,” “susceptible-dose-dependent/ intermediate,” or “resistant” to fluconazole, itraconazole, or voriconazole using the following MIC threshold values: fluconazole-susceptible ¼ 8 mg/mL, susceptible-dosedependent (SDD) ¼ 16e32 mg/mL and resistant ¼ 64 mg/ mL; itraconazole-susceptible ¼ 0.125 mg/mL, SDD ¼ 0.25e0.5 mg/mL, resistant ¼ 1 mg/mL; and voriconazole-susceptible ¼ 1 mg/mL, intermediate ¼ 2 mg/mL and resistant ¼ 4 mg/mL. 2.3. Real-time PCR RNA was extracted from cultures in the mid-log exponential growth phase, grown in YPD medium at 35  C and shaking at 200 rpm using the yeast RNAiso reagent kit (TaKaRa, Tokyo, Japan) according to the manufacturer's recommendations. The RNA concentration was determined using a Nanodrop 8000 (Thermo-Scientific, Waltham, MA, USA). The isolated RNA was quantitatively reverse-transcribed to cDNA using the PrimeScript RT Reagent kit (TaKaRa, Tokyo, Japan). Expression levels of azole resistance genes CDR1, CDR2, MDR1, ERG11 and of housekeeping genes PMA1 and ACT1 were determined by quantitative RT-PCR. The analysis was performed on an Mx3000P instrument (Stratagene, USA) with the SYBR Premix Ex Taq kit (TaKaRa) under the following conditions: denaturation at 94  C for 3 min, followed by 40 cycles consisting of 10 s at 94  C and 20 s at 55  C. The primers used for RT-PCR analysis are shown in Table 1. Each sample was processed in triplicate. Relative gene expression was measured quantitatively after normalization to an 18S RNA control that was amplified at the same time as each target gene. The RNA transcript levels for each isolate were compared with the average expression level of a collection of 18 azole-susceptible isolates. RNA transcripts were considered significantly overexpressed when the △Ct value [Ct(gene of interest)Ct(18S RNA)] exceeded 3.0 standard deviations. 2.4. Rhodamine 6G efflux The activity of efflux pumps was evaluated using flow cytometry as previously described [17] with the following modifications. C. albicans cells were grown overnight at 30  C to 5  107 cells/mL in YPD. Cells were centrifuged at 3000  g for 5 min and washed three times with PBS. The cells were then incubated in PBS for 4 h at 30  C under continuous shaking (200 rpm/min) to deplete the energy reserves of the cells. Rhodamine 6G (SigmaeAldrich, St Louis, MO, USA) at a final concentration of 10 mM was added and the cells were incubated for 2 h at 30  C. The fluorescence uptake was measured immediately using a Beckman Coulter

Please cite this article in press as: Liu J-Y, et al., Mechanisms of azole resistance in Candida albicans clinical isolates from Shanghai, China, Research in Microbiology (2015), http://dx.doi.org/10.1016/j.resmic.2015.02.009

J.-Y. Liu et al. / Research in Microbiology xx (2015) 1e9 Table 1 Primers used in the study. Gene

Sequence (50 e30 )

Reference

ERG11-1-F ERG11-1-R ERG11-2-F ERG11-2-R ERG11-F for RT-PCR ERG11-R for RT-PCR MDR1-F MDR1-R CDR2-F CDR2-R CDR1-F CDR1-R PMA1-F PMA1-R ACT1-F ACT1-R 18SeF 18S-R TAC1-1-F TAC1-1-R TAC1-2-F TAC1-2-R ERG3-F ERG3-R

atg gct att gtt gaa act gtc att gga tca ata tca cca cgt tct c att gga gac gtg atg ctg ctc aa cca aat gat ttc tgc tgg ttc agt aac tac ttt tgt tta taa ttt aag atg gac tat tga

[20] [20] [20] [20] [23]

aat gat ttc tgc tgg ttc agt agg t

[23]

tta cct gaa act ttt ggc aaa aca act tgt gat tct gtc gtt acc g ggt att ggc tgg tcc taa tgt ga gct tga atc aaa taa gtg aat gga ttac ttt agc cag aac ttt cac tca tga tt tat tta ttt ctt cat gtt cat atg gat tga ttg aag atg acc acc caa tcc gaa acc tct gga agc aaa ttg g ttg gtg atg aag ccc aat cc cat atc gtc cca gtt gga aac a gag aaa cgg cta cca cat att cca att aca aga ccc atg gac act tca ctg tca ctg gga act ggt att gat tac tgg agg ttt acc gga t atc tag aag agg caa cga aaa gac gt tta aat ccc caa att att gtc aaa ga ggt caa cct tcc cat cac att a cgt aaa tca aag cca acc aga t

[23] [23] [23] [23] [23] [23] [23] [23] [23] [23] This This This This This This This This

3

Alto, USA) to assess changes in the sterol biosynthesis pathways of C. albicans isolates. Cholesterol was added as internal standard. Each experiment was processed in triplicate. 2.7. PCR amplification and sequencing of ERG11, TAC1 and ERG3 C. albicans genomic DNA was extracted as described by Versalovic et al. [19] and used as a template to amplify fulllength ERG11, TAC1 and ERG3 genes. The primers used were designed with Primer-BLAST (http://www.ncbi.nlm.nih. gov/tools/primer-blast/) and are shown in Table 1. The PCR products were sequenced by BGI Inc. (Shanghai, China) and compared with the sequences published in GenBank for ERG11 (AY856352), TAC1 (DQ393587) and ERG3 (AF069752). 2.8. Testing affinity for fluconazole

study study study study study study study study

Epics XL FACScan flow cytometer at 535 nm. Samples of cells (2.5  106) from the original culture were washed with PBS and resuspended in 1 mL PBS or 1 mL PBS containing 2 mM glucose for another 30 min. Then the change in fluorescence indicative of active efflux was evaluated by FACScan flow cytometry. The fluorescence of cells incubated without rhodamine 6G was used as an unstained control. Each experiment was performed in triplicate. 2.5. Evaluation of respiratory status Rhodamine 123 (SigmaeAldrich), a fluorochrome that can be enriched in mitochondria in a membrane-potentialdependent manner, was used to measure the respiratory capacity of C. albicans isolates with or without sodium azide (NaN3), using the method described by Skowronek et al. [18]. Flow cytometry was performed as described above and each sample was processed in triplicate. 2.6. Analysis of sterol composition Sterols were extracted from lyophilized C. albicans cells grown to the stationary phase in RPMI 1640 medium. The cells were centrifuged and dried under a vacuum. Methanol (0.5 mL) and 20% KOH (7 mL) were added and the cells were sonicated at 95  C for 2.5 h. Then, 7 mL of diethyl ether was added and the sonicated cells were vibrated for 15 min. Then the supernatant was dried in a vacuum and dissolved in 50 mL of hexane. The solution was processed using a gas chromatograph mass spectrometer (Agilent technologies, Palo

C. albicans Erg11 was expressed from a yeast system using the S. cerevisiae GAL1 promoter in vector pYES2. For our previous study, we had constructed S. cerevisiae INVSc1 transfectants transformed with pYES2-ERG11-wild type (pYES2-ERG11-WT) and pYES2-ERG11 mutants (pYES2ERG11M) using a frozen EZ yeast transformation kit (ZMYO Research, Orange, CA, USA) [20]. To construct expression plasmids, ERG11 ORF of C. albicans ATCC 90028 was amplified and ligated into pMD18-T, creating plasmid-TERG11. Then the pMD18-T-ERG11 mutants were created by site-directed mutagenesis. The wild-type and pMD18-TERG11 mutant constructs were used as templates to amplify the ERG11 gene, flanked with KpnI and XhoI restriction sites. Finally, the amplified ERG11 fragments were subcloned into pYES2 pre-cut with KpnI and XhoI, to produce the final pYES2-ERG11-WT and pYES2-ERG11M expression plasmids. The S. cerevisiae transfectants were added to 25 mL SD-URA3-Minus 2% glucose medium and incubated at 30  C overnight. Cells (108 cells/mL) were centrifuged in 50 mL Corning tubes at 1500  g for 5 min and washed twice with sterile water. Then, 25 mL SD-URA3-Minus medium was added to the pellet and the cells were incubated at 30  C for 5 h with constant shaking to induce starvation. Expression of the Erg11 protein was induced by adding 50 mL of 1  SD-URA3-Minus-Gla-Raf medium for 24 h. The cells were collected and washed twice with PBS, then resuspended in 10 mL of PBS (containing 1 M DTT and 1 M PMSF). Then a cell disruptor (FastPrep-24, MP Biomedicals, USA) was used to break apart the resuspended cells. Cellular debris was removed by centrifugation and the supernatant containing the isolated proteins was collected. Erg11 was then purified using a nickel column that bound the polyhistidine region coded by the plasmid. The quality of the protein was assessed by SDSPAGE and western blot. Protein was quantified using the Bradford protein assay kit (Generay, Shanghai, China). The affinity between Erg11 and fluconazole was measured by adding different concentrations of fluconazole (0.2, 0.5, 2.5, or

Please cite this article in press as: Liu J-Y, et al., Mechanisms of azole resistance in Candida albicans clinical isolates from Shanghai, China, Research in Microbiology (2015), http://dx.doi.org/10.1016/j.resmic.2015.02.009

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No. Site FLC of strainsa of isolation

ITRA

VRC

Amino acid substitution(s) in Tac1b

Missense mutation in TAC1 gene

Amino acid substitution(s) in Erg11b

Missense mutation in ERG11 gene

Gene overexpression

G596A, C2207T, T2804C T310G, C2207T, T2804C G2326A, T2804C

A114S, Y205E, Y257H, V437I

CDR2

G596A, G2326A, T2804C e

Y132H, Y205E, V437I, G472R

G340T, T613G or C615A, T769C, G1309A G340T, T613G or C615A, T769C, G1309A T394C, T613G or C615A, G1309A, G1343A T394C, T613G or C615A, G1309A, G1414A T394C, T613G or C615A, A1303G, G1343A, C1506A T394C, T613G or C615A, T769C, A779T, G1309A, G1343A T613G or C615A, G1309A T613G or C615A, G1309A T348A, A383C, T613G or C615A, G1309A T613G or C615A, G1309A T348A T613G or C615A, G1309A T348A, A383C, A428G, T613G or C615A, G1309A T394C, T613G or C 615A, G1309A, G1343A T348A, A395T, A427C, T613G or C615A, T769C T348A, A395T, A427C, T613G or C615A, G1309A T348A, A395T, A427C, T613G or C615A, T769C T348A T613G or C615A, G1309A T348A T613G or C615A, A789C, G1309A T613G or C615A, G1309A T348A, G1309A T613G or C615A, C764T, G1309A T348A, T613G or C615A T348A, G1309A T613G or C615A, G1309A T348A T613G or C615A, G1309A T613G or C615A, G1309A

Y141

Vagina

16(SDD) >16(R)

0.5(S)

S199N, A736V, L935S

Y201

Vagina

16(SDD) 16(R)

1(S)

F104V, A736V, L935S

Y205

Vagina

16(SDD) 0.5(SDD)

2(I)

D776N, L935S

Y206

Vagina

16(SDD) 16(R)

2(I)

S199N, D776N, L935S

Y208

Vagina

32(SDD) 16(R)

1(S)

e

Y210

Vagina

16(SDD) 0.25(SDD)

4(R)

Y203 J026 J509

Vagina Sputum Vagina

0.5(S) 0.0313(S) 0.5(S) 0.0313(S) 32(SDD) 0.25(SDD)

16(R) 8(R) e e D116E, Y132F, K143Q, Y205E, Y257H >16(R) 16(R) S199N, N772K, N896S G596A, T2316A, D116E, Y132F, K143Q, Y205E, A2687G V437I 16(R) 4(R) e e D116E, Y132F, K143Q, Y205E, Y257H