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FLO8 deletion leads to azole resistance by upregulating CDR1 and CDR2 in Candida albicans
Journal Pre-proof FLO8 deletion leads to azole resistance by upregulating CDR1 and CDR2 in Candida albicans L.I. Wen-Jing, L.I.U. Jin-Yan, Ce SHI, Yue ZHAO, Ling-ning MENG, Fang Wu, X.I.A.N.G. Ming-Jie PII:
S0923-2508(19)30089-0
DOI:
https://doi.org/10.1016/j.resmic.2019.08.005
Reference:
RESMIC 3735
To appear in:
Research in Microbiology
Received Date: 23 June 2018 Revised Date:
17 March 2019
Accepted Date: 20 August 2019
Please cite this article as: L. Wen-Jing, L. Jin-Yan, C. SHI, Y. ZHAO, L.-n. MENG, F. Wu, X. MingJie, FLO8 deletion leads to azole resistance by upregulating CDR1 and CDR2 in Candida albicans, Research in Microbiologoy, https://doi.org/10.1016/j.resmic.2019.08.005. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Masson SAS on behalf of Institut Pasteur.
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FLO8 deletion leads to azole resistance by upregulating CDR1 and CDR2 in
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Candida albicans
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Wen-Jing LI a,b,1, Jin-Yan LIU b,1, Ce SHI a, Yue ZHAO a,b, Ling-ning MENG a,b, Fang Wuc,
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Ming-Jie XIANG a,b,*
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a Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School
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of Medicine, 197 Ruijin Second Road, Shanghai 200025, China
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b Department of Laboratory Medicine, Ruijin Hospital Luwan Branch, Shanghai Jiao Tong
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University School of Medicine, 149 Chongqing South Road, Shanghai 200020, China
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c Department of Geriatric, Ruijin Hospital, Shanghai Jiao Tong University School of
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Medicine , Shanghai, China
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* Corresponding author:Dr. Ming-Jie XIANG, Department of Laboratory Medicine, Ruijin
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Hospital, Shanghai Jiao Tong University School of Medicine, 197 Ruijin Second Road,
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Shanghai 200025, China. Tel.: +86 21 6437 0045; fax: +86 21 6431 1744. E-mail address:
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[email protected](M.-J.Xiang)
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These authors contributed equally to this work.
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Abstract Candida albicans has the ability to switch reversibly between budding yeast, filamentous,
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pseudohypha, and hyphal forms, a process in which the transcription factor Flo8 plays an
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important role. This ability is important for the virulence and pathogenicity of Candida
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albicans. To determine whether Flo8 plays a role in the regulation of drug sensitivity, we
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constructed a FLO8 null mutant flo8/flo8 from the parental strain SN152 and a
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Flo8-overexpressing strain, flo8/flo8::FLO8. The susceptibility of the isolates to antifungal
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agents was then evaluated using the agar dilution and broth microdilution methods.
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Expression of drug resistance-related genes by the isolates was investigated by real-time PCR.
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The flo8/flo8 mutation isolates exhibited increased resistance to fluconazole, voriconazole,
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and itraconazole compared with the wild-type and drug sensitivity was restored by FLO8
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overexpression (flo8/flo8::FLO8). Of seven drug resistance-related genes, the FLO8 null
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mutation resulted in increased CDR1 and CDR2 expression (1.60-fold and 5.27-fold,
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respectively) compared with SN152, while FLO8 overexpression resulted in decreased CDR1
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expression (0.63-fold). These results suggest that Flo8 is involved in the susceptibility of
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Candida albicans to antifungal azoles, with FLO8 deletion leading to constitutive
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overexpression of CDR1 and CDR2 and resistance to antifungal azoles.
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Keywords: FLO8 deletion; Resistance; CDR1 and CDR2; Candida albicans
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1. Introduction Candida albicans (C. albicans) is one of the most important and common opportunistic
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pathogens found in humans and causes oropharyngeal, esophageal and vaginal candidiasis.
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This frequently progresses to invasive and systemic candidiasis in individuals with
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compromised or immature immune systems due to infections such as HIV, diabetes,
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radiotherapy or chemotherapy, and aging [1-3]. Flo8 was originally identified in
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Saccharomyces cerevisiae before subsequent classification as Saccharomyces diastaticus, and
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was originally reported to be a dominant flocculation gene[4]. The FLO8 gene is highly
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conserved in yeast strains with a variety of flocculation genotypes and phenotypes and can
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confer a haploid cell-specific flocculation ability via transcriptional activation of the FLO1
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gene[5]. Flo8 induces invasive growth in haploid yeast strains via transcriptional activation of
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Flo11, which has been linked to adhesion to polystyrene surfaces and biofilm formation in C.
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albicans[6-8]. Flo8 also regulates the expression of extracellular glucoamylase production by
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transcriptional activation of Sta1 [6, 7]. Furthermore, in C. albicans, Flo8 is also required for
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hyphal development, binding to epithelial surfaces and for virulence in animal models [9, 10].
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Flo8 functions as a downstream component of the cAMP/PKA pathway to regulate the
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process of switching between multiple morphological states (yeast, filamentous,
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pseudohyphal and hyphal forms) in different environments, which is important for the
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pathogenicity and virulence of this species [11, 10]. However, the relationship between Flo8
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and susceptibility to antifungal agents remains to be elucidated. Our previous study of the
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relationship between Flo8 and the virulence of C. albicans strains yielded the incidental
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observation that the flo8/flo8 mutant was more resistant to azole antifungal agents than the
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parental SC5314 strain. Therefore, we constructed the FLO8 null mutant flo8/flo8 and the
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Flo8-overexpressing strain flo8/flo8::FLO8 to investigate the function of Flo8 in the
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regulation of drug susceptibility in C. albicans.
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2. Materials and methods
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2.1. Strains, media and culture conditions
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The C. albicans strains used in this study are listed in Table 1. We also screened 200 C.
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albicans clinical isolates from seven hospitals in Shanghai, China. The FLO8/flo8 and
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flo8/flo8 strains were constructed from the parental strain FLO8/FLO8 (SN152) by
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PCR-based homologous recombination according to the method described by Noble and
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Johnson[12]. The Flo8-overexpressing strain flo8/flo8::FLO8 was constructed by integrating
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gene fragment ADH1-FLO8-LoxP-ARG4-LoxP-ADE2, in which the FLO8 gene is under the
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control of the ADH1 promoter, into the ADE2 locus in flo8/flo8. The genotype of the empty
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vector EV was the same as that of flo8/flo8::FLO8 except that EV had no FLO8 gene. All
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yeast strains were routinely maintained at 30°C on yeast-peptone-dextrose (YPD) medium (1%
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yeast extract, 2% peptone and 2% glucose) or on solid YPD medium, with the addition of 1.5%
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agar as previously described[13].
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2.2. Antifungal susceptibility testing
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The antifungal susceptibility of C. albicans strains was evaluated using the agar dilution
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methods as previously described[14]. Briefly, C. albicans cells cultured overnight on YPD
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plates were diluted to 0.5×108 cells/mL. Stock solutions of the antifungal drugs fluconazole,
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voriconazole and itraconazole were prepared in dimethyl sulfoxide (DMSO) and added to
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YPD plates at final concentrations of 10 mg/L, 5 mg/L and 2.5 mg/L, respectively. Five
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ten-fold dilutions (0.5×108 cells/mL–0.5×104 cells/mL) of each samples were prepared and
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spotted (2 μL per spot) onto YPD plates with fluconazole, voriconazole or itraconazole. The
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plates were incubated for 2 days at 30°C.
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The broth microdilution method was then used to evaluate the susceptibility of the
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isolates to antifungal agents according to the guidelines published by the Clinical and
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Laboratory Standards Institute (CLSI) [15]. C. albicans ATCC 90028 was used as a control.
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Two-fold serial dilutions of the drugs were prepared in the following ranges: fluconazole,
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128–0.25 mg/L and voriconazole and itraconazole, 16–0.03125 mg/L. Triplicate samples of
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the isolates(1.5×105 cells/mL) were incubated in tubes or 96-well plates in the presence of
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the antifungal agents for 2 days at 30°C, and the growth of each isolate was measured by a
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spectrophotometer (OD=600; Mapada Instruments, Shanghai, China). The relative growth of
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cells was normalized according to growth of the control cells in the absence of drug (defined
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arbitrarily as 100%). The drug concentration inhibiting 80% of cell growth in the control was
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defined as the minimum inhibitory concentration (MIC).
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2.3. RNA isolation and quantitative analysis of mRNA levels by real-time PCR
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C. albicans cells were harvested after being grown in 3 mL YPD liquid medium at 30°C
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for 16 h. Total RNA was isolated using the Yeast RNAiso Kit (TaKaRa, Tokyo, Japan) and
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then treated with gDNA Eraser (TaKaRa) to digest the contaminating DNA. First-strand
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cDNA was synthesized from 500ng of total RNA in a 10 μL reaction mixture using the
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PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa). The mRNA levels of the drug
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resistance-related genes were determined by real-time PCR with a 7300 Real-Time PCR
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System (Applied Biosystems, Shanghai, China) as described previously [16]. The primers
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used in the PCR cycling are listed in Table 2. Triplicate technical replicates of each gene were
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analyzed in reaction mixtures prepared with SYBR Premix ExTaq (Tli RNaseH Plus)
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(TaKaRa). Real-time PCR was performed using previously described conditions [3]. The
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change of the SYBR Green dye fluorescence was monitored as the PCR cycle numbers
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increased. The dissociation curves representing the amplification of each product were
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generated at the end of each PCR cycle, and the cycle threshold (CT) above the background
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was also calculated. 18S rRNA was used as a reference gene and relative mRNA levels were
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calculated using the 2−∆∆CT method.
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2.4. Rhodamine 6G efflux
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The R6G assay measures the efflux activity of individual yeast cells. The C. albicans
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ATP-binding cassette (ABC) transporters, Cdr1 and Cdr2, expel azoles and the fluorescent
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substrate R6G from the interior of the cells. Therefore, the flow cytometric R6G efflux assay
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was adopted to measure the function of the transporters according to the method described by
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Peralta[17]. A suspension of 108 yeast cells/mL in YPD medium was incubated overnight at
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30°C with shaking (200 rpm). Cells were washed, resuspended in PBS(108 yeast cells/mL),
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and incubated for 4 h to exhaust energy, before incubation with R6G (10 mol/L) for 2 h. Cells
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were then washed twice and resuspended in ice pre-cooled PBS. A sample (100 μL) of the
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suspension was then added to 900 μL ice pre-cooled PBS and analyzed for R6G uptake using
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the beckman boulter Epics XL flow cytometer(Beckman Coulter, USA). A further sample (1
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mL) of the suspension was incubated with glucose (4 mmol/L) at 30°C for 1 h, washed twice,
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and then diluted 10-fold in ice pre-cooled PBS for analysis of R6G efflux. Experiments were
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replicated on three independent occasions.
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2.5. Statistical analysis
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Data were analyzed by IBM SPSS statistics version 22. The comparison of FLO8 expression levels between the fluconazole-susceptible and fluconazole-resistant clinical
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strains were analyzed by t-test for two independent samples. The correlations between FLO8
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and CDR1 or CDR2 gene expression were analyzed by Spearman non-parametric correlation
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test.
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3. Results
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3.1. FLO8 null mutation decreased susceptibility of C. albicans to antifungal drugs
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Decreased susceptibility of C. albicans carrying the FLO8 null mutation to antifungal
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drugs was confirmed using the agar dilution method. The FLO8/FLO8, FLO8/flo8, flo8/flo8,
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flo8/flo8::FLO8 and EV strains showed similar patterns of strong growth on YPD plates in the
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absence of antifungal drugs (Fig. 1, left panel), suggesting that the growth of C. albicans is
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not affected by the flo8/flo8 mutation.
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Interestingly, growth of FLO8/flo8 mutant cells was superior to that of the wild-type
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FLO8/FLO8 (SN152), and the growth of the flo8/flo8 mutant was superior to that of the
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FLO8/flo8 mutant on YPD plates containing fluconazole, voriconazole or itraconazole (Fig.
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1). The drug sensitivity of the flo8/flo8 mutant cells was restored by FLO8 overexpression
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(flo8/flo8::FLO8), and the flo8/flo8::FLO8 mutant cells exhibited less growth compared with
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the other strains. This suggested that the antifungal drug resistance of the flo8/flo8 mutant
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cells results from the null mutation in FLO8. The flo8/flo8 and EV cells showed similar
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patterns of growth on YPD plates with or without antifungal drugs, suggesting that the
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inserted fragment ADH1-LoxP-ARG4-LoxP-ADE2 did not affect the growth rate, growth
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ability and drug susceptibility of C. albicans. Thus, FLO8 overexpression increased the
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susceptibility of C. albicans to fluconazole, voriconazole and itraconazole, while the null
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mutation in Flo8 decreased susceptibility to these antifungal drugs.
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We also evaluated the FLO8/FLO8, flo8/flo8 and flo8/flo8::FLO8 strains using the broth
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microdilution method to confirm the effect of Flo8 on antifungal drug resistance. The flo8/flo8
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mutants were also found to be more resistant to fluconazole (Fig. 2A), voriconazole (Fig. 2B)
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and itraconazole (Fig. 2C) than the FLO8/FLO8 and flo8/flo8::FLO8. Similarly,
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flo8/flo8::FLO8 exhibited less growth compared with the other strains and showed a little
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more sensitivity to the three antifungal drugs compared with FLO8/FLO8. After 2 days in
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culture at 30°C, flo8/flo8 cells incubated in the presence of 128 µg/mL fluconazole, 16 µg/mL
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voriconazole or 16 µg/mL itraconazole exhibited 60% growth relative to that of flo8/flo8
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incubated in the absence of drug, 20% growth relative to that of FLO8/FLO8 and 5%–10%
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growth relative to that of flo8/flo8::FLO8. Furthermore, the antifungal drug resistance of the
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C. albicans flo8/flo8 isolate was reflected by the MICs of >128 µg/mL for fluconazole, 16
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µg/mL for itraconazole and 16 µg/mL for voriconazole (Table 3). Hence, these findings were
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consistent with the results of the agar dilution assay.
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3.2. Expression levels of seven drug resistance-related genes in flo8/flo8 and flo8/flo8::FLO8
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compared with those in FLO8/FLO8
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To investigate whether the genes responsible for antifungal drugs resistance are regulated
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directly or indirectly by Flo8, real-time PCR was performed to compare the mRNA levels of
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seven antifungal drug resistance-related genes (CDR1, CDR2, MDR1, FLU1, ERG11, UPC2
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and FCR1) in FLO8/FLO8, flo8/flo8 and flo8/flo8::FLO8. The 2−∆∆CT values of the genes
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which have statistically significant differences (P<0.05) in FLO8/flo8 or flo8/flo8 , or
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flo8/flo8::FLO8 compared with FLO8/FLO8 are summarized in Figure 3. The
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others(MDR1,MRR1, FLU1, ERG11, UPC2 and FCR1) have no significant differences and
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are not shown. As shown in Figure 3, in FLO8/flo8, flo8/flo8 and flo8/flo8::FLO8, the
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expression levels of CDR1 were 1.28-fold, 1.75-fold and 0.64-fold compared with that in
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FLO8/FLO8. CDR2 were 2.28-fold, 5.44-fold and 0.97-fold, and TAC1 were 1.4-fold,
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1.85-fold and 0.94-fold respectively. In flo8/flo8, the disruption of FLO8 gene increased the
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expression of CDR1, CDR2, and TAC1 by 1.75-fold, 5.44-fold and 1.85-fold, respectively,
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and they all had statistically significant differences (P<0.05) compared with that in
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FLO8/FLO8. However, in flo8/flo8::FLO8, the expression of CDR1 was decreased (0.64-fold)
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compared with that in FLO8/FLO8 (P<0.05), while overexpression of FLO8 had no
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significant effects on the expression of CDR2(0.97-fold) and TAC1(0.94-fold).
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3.3. R6G efflux
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As is shown in Figure 4, there was no significant difference in the R6G uptake of
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FLO8/FLO8, FLO8/flo8, flo8/flo8 and flo8/flo8::FLO8 after glucose exhaustion. However,
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after incubation in the presence of glucose for 1 h, the R6G efflux of flo8/flo8 was higher than
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that of FLO8/FLO8, FLO8/flo8 and flo8/flo8::FLO8 (P < 0.05), while the efflux rate of
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flo8/flo8::FLO8 was lower than that of FLO8/FLO8 (P < 0.05). And, the differences were
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more significant after incubation with glucose for 2 h. The 2-h R6G excretion rate of
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FLO8/FLO8, FLO8/flo8, flo8/flo8 and flo8/flo8::FLO8 were 31%, 40%, 72% and 23%.
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3.4. FLO8 expression levels and fluconazole susceptibility in clinical isolates To identify the relationship between FLO8 expression levels and fluconazole
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susceptibility in C. albicans clinical isolates, a collection of 200 C. albicans isolates from
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seven hospitals in Shanghai, China, was obtained. FLO8 expression levels were identified by
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real-time PCR, and the expression level of FLO8 in SC5314 was defined as 1. In all the 200
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isolates, the FLO8 expression levels were from 0.34-fold to 5.9-fold. We defined the isolates
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with ＜0.8-fold FLO8 expression levels(only 16 isolates) to low-FLO8 group, and randomly
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selected 16 isolates with＞2-fold FLO8 expression levels(78 isolates) to high-FLO8 group. As
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is shown in Figure 5, C1-C16 belong to low-FLO8 group, and C17-C32 high-FLO8 group. Fluconazole susceptibility was evaluated by the broth microdilution method, and the
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MICs of C.albicans ATCC90028 and C.krusei ATCC6258 were within the scope of quality
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controls(not shown). As is shown in Figure 6, the MICs in low-FLO8 group were significantly
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higher than those in high-FLO8 group(P < 0.05). In low-FLO8 group, 8 isolates were
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fluconazole resistant, and 7 isolates were susceptible-dose dependent. And, all the 16 isolates
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in high-FLO8 group were fluconazole sensitive.
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3.5. CDR1, CDR2 and TAC1 expression levels in low-FLO8 group and high-FLO8 group
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To verify the association of FLO8 expression with CDR1, CDR2, and TAC1 in clinical
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strains, and that the expression relationships were consistent with those in the engineered
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isolates, we performed real-time quantitative RT-PCR analysis of a panel of 32 clinical
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isolates, comprising 16 (C1–C16) low-FLO8 strains and 16 (C17–C32) high-FLO8 strains.
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Expression levels of CDR1, CDR2, and TAC1 in the 32 clinical isolates relative to
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SC5314 are shown in Figure 7A(CDR1), B(CDR2), and C(TAC1). CDR1 expression levels
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ranged from 0.83-fold to 6.0-fold(in low-FLO8 group), and from 0.52-fold to 1.98-fold(in
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high-FLO8 group). CDR2 expression levels ranged from 1.3-fold to 8.18-fold(in low-FLO8
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group), and from 0.58-fold to 4.75-fold(in high-FLO8 group). TAC1 expression levels ranged
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from 0.87-fold to 1.93-fold(in low-FLO8 group), and from 0.67-fold to 1.23-fold(in
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high-FLO8 group).The expression levels of CDR1, CDR2, and TAC1 in low-FLO8 group
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were higher than those in high-FLO8 group(P<0.05).
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4. Discussion
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In the present study, we confirmed that Flo8 is associated with the antifungal azole
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susceptibility of C. albicans, with FLO8 deletion leading to constitutive overexpression of the
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multidrug efflux transporters CDR1 and CDR2 and resistance to antifungal azoles.
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To confirm the hypothesis that FLO8 expression is involved in regulation of the azole
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susceptibility of C. albicans via expression of drug resistance related genes, we constructed
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the FLO8/flo8, flo8/flo8 and flo8/flo8::FLO8 strains and evaluated their antimicrobial
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susceptibility using three different methods. Using the agar dilution method, flo8/flo8 showed
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superior resistance to fluconazole, voriconazole and itraconazole compared with that of
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FLO8/flo8, while flo8/flo8::FLO8 was the least resistant strain. A similar pattern of drug
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resistance was identified using the broth microdilution method. Furthermore, the MIC values
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of the three antifungal drugs for flo8/flo8 were >128 µg/mL for fluconazole, 16 µg/mL for
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itraconazole and 16 µg/mL for voriconazole, which reflected the drug-resistant phenotype of
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flo8/flo8. The consistency of the phenotypes of FLO8/flo8, flo8/flo8 and flo8/flo8::FLO8 in
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these tests indicates the involvement of FLO8 expression in the mechanism of susceptibility
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to antifungal azoles in C. albicans, with FLO8 deletion associated with a marked increase the
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resistance of C. albicans to antifungal azoles. This resistance was reversed by FLO8
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overexpression.
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To explore the mechanism underlying the resistance to antifungal azoles caused by FLO8
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deletion in C. albicans, we analyzed the expression levels of nine drug resistance-related
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genes (CDR1, CDR2, TAC1, MDR1, MRR1, FLU1, ERG11, UPC2 and FCR1) in FLO8/FLO8,
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FLO8/flo8, flo8/flo8 and flo8/flo8::FLO8. As shown in Figure 3, the expression of
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CDR1,CDR2 and TAC1 was increased in flo8/flo8, with 1.75-fold, 5.44-fold and 1.85-fold
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compared with that in FLO8/FLO8, while only the expression of CDR1 was decreased in
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flo8/flo8::FLO8, with 0.64-fold compared with that in FLO8/FLO8. The changes in the
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expression of CDR1,CDR2 and TAC1 in flo8/flo8 and flo8/flo8::FLO8 could explain the
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changes in the susceptibility of these strains to antifungal azoles. These results indicate that
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FLO8 deletion results in activation of the transcription of TAC1, CDR1 and especially CDR2,
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while FLO8 overexpression just decreases CDR1 expression. Because TAC1 controls the
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upregulation of CDR1 and CDR2 at the transcriptional level[18], we guess FLO8 deletion
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increases the expression of TAC1, and then TAC1 activates the transcription of CDR1 and
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CDR2. CDR1 is regulated not only by TAC1 but also by MRR2 and NDT80[19], other
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regulation mechanisms between FLO8 and CDR1 may exist.
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The function of the changes in expression of CDR1,CDR2 and TAC1 caused by FLO8
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deletion or overexpression were then confirmed by the R6G efflux assay. We set the time of
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efflux with glucose at 1h and 2h. As is shown in Figure 4, there were no significant
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differences in R6G uptake by FLO8/FLO8, FLO8 /flo8, flo8/flo8 and flo8/flo8::FLO8, and for
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each of them, the 2h-efflux rate was higher than the corresponding 1h-efflux rate. In flo8/flo8,
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the 1h-efflux rate was 40%, and 2h-efflux-rate 72%, significantly higher than the others(P <
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0.05). And, flo8/flo8::FLO8 exhibited the lowest 1h-efflux and 2h-efflux rates (P < 0.05).
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Similarly, the results of the R6G efflux assay were consistent with those of the antifungal drug
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sensitivity tests, and also corresponded with the drug resistance-related genes expression
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levels.
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Based on our findings, we hypothesized that the removal or low expression of FLO8
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results in upregulations of TAC1, CDR1 and especially CDR2, and finally leads to the
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decreased sensitivity to antifungal azoles. While FLO8 overexpression can reversed this
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phenotype.To verify that the relationship between FLO8 expression and fluconazole
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susceptibility, and the correlations of FLO8 expression with that of CDR1, CDR2 and TAC1
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in C. albicans clinical isolates were consistent with those of the engineered isolates, we
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collected and analyzed FLO8 expression levels in 200 C. albicans clinical isolates. As is
316
shown in Figure 5, C1-C16 belong to low-FLO8 group, and C17-C32 high-FLO8 group.And
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fluconazole susceptibility is shown in Figure 6. The MICs in low-FLO8 group(with 8
318
fluconazole resistant, and 7 susceptible-dose dependent) were significantly higher than those
319
in high-FLO8 group(all fluconazole sensitive)(P < 0.05). Based on this result, it can be
320
considered that low expression of FLO8 could reduce the susceptibility of C.albicans clinical
321
strains to fluconazole, which is consistent with that in the engineered isolates. The
322
correlations of FLO8 expression with that of CDR1, CDR2 and TAC1 in C. albicans clinical
323
isolates are shown in Figure 7. The expression levels of CDR1, CDR2, and TAC1 in
324
low-FLO8 group were higher than those in high-FLO8 group(P<0.05). Based on the results in
325
clinical isolates, it can be considered Flo8 is associated with the antifungal azole susceptibility
326
of C. albicans. The low expression of FLO8 increases the expression of TAC1, CDR1 and
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CDR2, and can finally decrease azoles susceptibility in C.albicans.
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There are numerous reports describing the function of FLO8 and Flo8 in C. albicans
329
[9-11,20]. Cao et al. found that FLO8 deletion in C. albicans blocked hypha-specific gene
330
expression and hyphal development, and the flo8/flo8 mutant was avirulent in a mouse
331
systemic infection model[10]. Liu et al. showed that the T751D and G723R Flo8 mutants
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enhanced the Flo8 C-terminus activation, thereby increasing virulence and promoting
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filamentous growth[13]. In C.albicans clinical isolates, the overexpression of Cdr1 and Cdr2
334
is responsible for azole resistance. Cdr1 and Cdr2 upregulation are primarily controlled by
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TAC1 at the transcriptional level, and are coordinately regulated[18,21]. TAC1 binds to the
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drug-responsive element(DRE) of CDR1 and CDR2 and is localized in the nucleus. In
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C.albicans clinical strains, the gain-of-function mutations in TAC1 can result in high
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expression of CDR1 and CDR2[22], and then azole resistance. As compared with CDR2,
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CDR1 is a prime contributor of azole resistance in clinical strains[23]. In C.albicans clinical
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strains resistant to azoles, the removal of TAC1 can abolish CDR1/CDR2 expression and
341
therefore drug resistance, and this phenotype can be reversed when the TAC1 allele
342
reintroduced[18]. A TAC1 allele from an azole-resistant clinical strain can confer the
343
phenotype to a susceptible strain[18].
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It can be considered that the removal or low expression of FLO8 can increase the
345
transcriptional regulation of TAC1, CDR1 and CDR2, and then decreases azoles susceptibility
346
in C.albicans. And this phenotype can be reversed when FLO8 allele reintroduced.
347
Approximately 50%–60% of candidiasis cases are caused by the opportunistic C. albicans
348
infections[24]. And treatment failure caused by the emergence of azole-resistant clinical
349
isolates has become a serious problem[25,26]. Several mechanisms that account for azole
350
resistance in C. albicans clinical isolates have been elucidated, including upregulation of
351
CDR1 and CDR2. Our discoveries provide new insights into the mechanisms of azole
352
resistance and the functions of Flo8 in C. albicans. Treatment failure caused by azole
353
resistance has become an emergence. And, our discoveries represent the basis of further
17
354
research and development of new antifungal agents for the treatment of C. albicans. The
355
researchers can develop drugs especially according to the regulation mechanisms between
356
Flo8 and CDR1 and CDR2 for the patients infected by azole resistance C.albicans, in which
357
the resistance comes from the overexpressions of CDR1 and CDR2.
358
Some limitations of our study should be noted. On the one hand, the overexpression of
359
FLO8 in flo8/flo8::FLO8 decreases the expression of CDR1(0.64-fold compared with that in
360
FLO8/FLO8) , while it has no significant effects on the expression of CDR2(0.97-fold) and
361
TAC1(0.94-fold). CDR1 is regulated not only by TAC1 but also by MRR2 and NDT80[19].
362
The regulation of CDR1 may be not all attributed to TAC1, and additional mechanisms
363
between FLO8 and CDR1 can be expected. On the other hand, it can be determined that the
364
removal or low expression of FLO8 can increase the transcriptional regulation of TAC1,
365
CDR1 and CDR2, and then decrease azoles susceptibility in C.albicans. Whether the
366
upregulation of CDR1 and CDR2 comes from the increased TAC1 or by other mechanisms,
367
needs further research.
368
To sum up, we confirmed that Flo8 is associated with the antifungal azole susceptibility
369
of C. albicans, with the removal or low expression of FLO8 leading to constitutive
370
overexpression of TAC1, CDR1 and CDR2, and azoles resistance or decreased azoles
371
susceptibility. And the reintroduced FLO8 allele can reverse this phenotype. Of course, further
372
studies are required to fully clarify the regulation mechanisms between FLO8 and TAC1,
373
CDR1 and CDR2 expression in C. albicans.
374 375
18
376 377
Conflict of interest The authors declare no conflicts of interest.
378 379
Acknowledgements
380
This work was financed by grants from the Program of Shanghai Natural Science
381
Foundation [#15ZR1426900],the Program of Shanghai Key Specialty [#ZK2012A21] and
382
Excellent Youth of HuangPu District of Shanghai [#RCPY1407].
383 384 385 386 387 388
Ethical approval Not required.
19
389
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390
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452 453 454
22
455
Legends to figures
456
Fig. 1. Susceptibilities of five C. albicans strains [FLO8/FLO8, FLO8/flo8, flo8/flo8, flo8/flo8::FLO8 and
457
EV] to fluconazole, voriconazole and itraconazole were determined using the agar dilution assay. Five
458
10-fold serial dilutions (0.5×108–0.5×104 cells/mL) of each sample grown on YPD plates overnight were
459
spotted (2 µL per spot) onto YPD plates with or without fluconazole (10 mg/L), voriconazole (5 mg/L) or
460
itraconazole (2.5 mg/L). (Left panel) YPD medium without antifungal drugs was used as a control. The
461
plates were incubated for 2 days at 30°C.
462 463
Fig. 2. Susceptibilities of three C. albicans strains (FLO8/FLO8, flo8/flo8 and flo8/flo8::FLO8) to
464
fluconazole (A), voriconazole (B) and itraconazole (C) were determined by the broth microdilution assay.
465
Cells were incubated for 2 days at 30°C, The relative growth of cells was normalized according to cell
466
growth in the absence of drug (defined arbitrarily as 100%).
467 468
Fig. 3. Fold changes in the expression levels of CDR1, CDR2 and TAC1 in FLO8/flo8, flo8/flo8 and
469
flo8/flo8::FLO8. In flo8/flo8, the disruption of FLO8 gene increased the expression of CDR1, CDR2, and
470
TAC1 by 1.75-fold, 5.44-fold and 1.85-fold, respectively, compared with that in FLO8/FLO8 (P<0.05) . In
471
flo8/flo8::FLO8, only the expression of CDR1 was decreased (0.64-fold) compared with
472
FLO8/FLO8(P<0.05).
473 474
Fig. 4. Flow cytometric analysis of R6G uptake and efflux. There was no significant difference in the R6G
475
uptake of FLO8/FLO8, FLO8/flo8, flo8/flo8 and flo8/flo8::FLO8 after glucose exhaustion. After incubation
476
in the presence of glucose for 1 h and 2h, the R6G efflux of flo8/flo8 was significantly higher than that of
23
477
FLO8/FLO8, FLO8/flo8 and flo8/flo8::FLO8 (P < 0.05).
478 479
Fig. 5. Fold changes in the expression levels of FLO8 in low-FLO8 group (C1–C16) and high-FLO8 group
480
(C17–C32) of C. albicans relative to the value of SC5314.
481 482
Fig. 6. The MICs of fluconazole against 32 C.albicans clinical isolates. The MICs in low-FLO8 group
483
(C1–C16) were significantly higher than those in high-FLO8(C17–C32) group(P < 0.05).
484 485
Fig. 7. Fold changes in the expression levels of CDR1(A), CDR2(B) and TAC1(C) in 32 C. albicans clinical
486
isolates relative to SC5314. The expression levels of CDR1, CDR2, and TAC1 in low-FLO8 group were
487
higher than those in high-FLO8 group(P<0.05).
488 489 490 491 492
Table 1. Candida albicans strains used in this study Strain
Genotype or description
Source or reference
SC5314
Wild-type
(Liu et al., 2015)
ATCC90028
Candida albicans ATCC strain
ATCC
ATCC6258
Candida krusei ATCC strain
ATCC
ura3::imm434::URA3/ura3::imm434 iro1::IRO1/iro1::imm434 his1::hisG/his1::hisG leu2/leu2
(Noble and Johnson,
FLO8/FLO8
arg4/arg4
2005)
FLO8/flo8
ura3::imm434::URA3/ura3::imm434 iro1::IRO1/iro1::imm434 his1::hisG/his1::hisG leu2/leu2
This study
arg4/arg4 flo8::CdHIS1 flo8/flo8
ura3::imm434::URA3/ura3::imm434 iro1::IRO1/iro1::imm434 his1::hisG/his1::hisG leu2/leu2
This study
arg4/arg4 flo8::CdHIS1/flo8::CmLEU2 flo8/flo8::FLO8
ura3::imm434::URA3/ura3::imm434 iro1::IRO1/iro1::imm434 his1::hisG/his1::hisG leu2/leu2
This study
arg4/arg4 flo8::CdHIS1/flo8::CmLEU2 ADE2/ade2::ADH1p-FLO8-PAP EV
ura3::imm434::URA3/ura3::imm434 iro1::IRO1/iro1::imm434 his1::hisG/his1::hisG leu2/leu2 arg4/arg4 flo8::CdHIS1/flo8::CmLEU2 ADE2/ade2::ADH1p-PAP
Table 2. Primers used in this study Primers
Sequence (5ʹ-3ʹ)
18SRNA-F
TCTTGTACGGCACATATCTCGT
18SRNA-R
GTTTCGAGAGGACCTTCAGTC
MDR1-QF
GTTAAACATTTCACCCTCG
MDR1-QR
AACAAGATACCACCGACA
MRR1-QF
AACGCTGGTTATGGGTGA
MRR1-QR
TTTGCTGTTGGGCTTCTT
ERG11-QF
TTGGTGGTGGTAGACATA
ERG11-QR
TCTGCTGGTTCAGTAGGT
FCR1-QF
CCACTGGTCCAATCCTTA
FCR1-QR
CTGTAGACTCCATTCGTT
UPC2-QF
AATGCCTCGTTTCCTTCG
UPC2-QR
CACGGTCAATTCCTTTCG
FLU1 -QF
AGAAGAGCCACCAGAAGT
FLU1 -QR
CGATAAGGCAGCAAGACC
CDR1-QF
GGTCAACTTGTAATGGGTC
CDR1-QR
AGGACGATAAAGGGCATA
CDR2-QF
GCCAATGCTGAACCGACA
This study
CDR2-QR
ACCAGCCAATACCCCACA
TAC1-QF
GTCAATTAGGCGAGACAC
TAC1-QR
CTGCCACCAATACAAGATA
Table 3. Minimum inhibitory concentrations of fluconazole, itraconazole and voriconazole for the C. albicans isolates Strain
fluconazole (µg/mL)
itraconazole (µg/mL)
voriconazole (µg/mL)
ATCC90028 FLO8/FLO8 FLO8/flo8 flo8/flo8
0.25 0.25 32 >128 0.25 >128
≤0.125 ≤0.125 0.5 >16 ≤0.125 >16
≤0.125 ≤0.125 2 >16 ≤0.125 >16
flo8/flo8::FLO8 EV
S0923-2508(19)30089-0
DOI:
https://doi.org/10.1016/j.resmic.2019.08.005
Reference:
RESMIC 3735
To appear in:
Research in Microbiology
Received Date: 23 June 2018 Revised Date:
17 March 2019
Accepted Date: 20 August 2019
Please cite this article as: L. Wen-Jing, L. Jin-Yan, C. SHI, Y. ZHAO, L.-n. MENG, F. Wu, X. MingJie, FLO8 deletion leads to azole resistance by upregulating CDR1 and CDR2 in Candida albicans, Research in Microbiologoy, https://doi.org/10.1016/j.resmic.2019.08.005. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Masson SAS on behalf of Institut Pasteur.
1
1
FLO8 deletion leads to azole resistance by upregulating CDR1 and CDR2 in
2
Candida albicans
3
Wen-Jing LI a,b,1, Jin-Yan LIU b,1, Ce SHI a, Yue ZHAO a,b, Ling-ning MENG a,b, Fang Wuc,
4
Ming-Jie XIANG a,b,*
5
a Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School
6
of Medicine, 197 Ruijin Second Road, Shanghai 200025, China
7
b Department of Laboratory Medicine, Ruijin Hospital Luwan Branch, Shanghai Jiao Tong
8
University School of Medicine, 149 Chongqing South Road, Shanghai 200020, China
9
c Department of Geriatric, Ruijin Hospital, Shanghai Jiao Tong University School of
10
Medicine , Shanghai, China
11 12
* Corresponding author:Dr. Ming-Jie XIANG, Department of Laboratory Medicine, Ruijin
13
Hospital, Shanghai Jiao Tong University School of Medicine, 197 Ruijin Second Road,
14
Shanghai 200025, China. Tel.: +86 21 6437 0045; fax: +86 21 6431 1744. E-mail address:
15
[email protected](M.-J.Xiang)
16
1
17 18 19 20 21 22
These authors contributed equally to this work.
2
23 24
Abstract Candida albicans has the ability to switch reversibly between budding yeast, filamentous,
25
pseudohypha, and hyphal forms, a process in which the transcription factor Flo8 plays an
26
important role. This ability is important for the virulence and pathogenicity of Candida
27
albicans. To determine whether Flo8 plays a role in the regulation of drug sensitivity, we
28
constructed a FLO8 null mutant flo8/flo8 from the parental strain SN152 and a
29
Flo8-overexpressing strain, flo8/flo8::FLO8. The susceptibility of the isolates to antifungal
30
agents was then evaluated using the agar dilution and broth microdilution methods.
31
Expression of drug resistance-related genes by the isolates was investigated by real-time PCR.
32
The flo8/flo8 mutation isolates exhibited increased resistance to fluconazole, voriconazole,
33
and itraconazole compared with the wild-type and drug sensitivity was restored by FLO8
34
overexpression (flo8/flo8::FLO8). Of seven drug resistance-related genes, the FLO8 null
35
mutation resulted in increased CDR1 and CDR2 expression (1.60-fold and 5.27-fold,
36
respectively) compared with SN152, while FLO8 overexpression resulted in decreased CDR1
37
expression (0.63-fold). These results suggest that Flo8 is involved in the susceptibility of
38
Candida albicans to antifungal azoles, with FLO8 deletion leading to constitutive
39
overexpression of CDR1 and CDR2 and resistance to antifungal azoles.
40 41 42 43 44 45
Keywords: FLO8 deletion; Resistance; CDR1 and CDR2; Candida albicans
3
46 47
1. Introduction Candida albicans (C. albicans) is one of the most important and common opportunistic
48
pathogens found in humans and causes oropharyngeal, esophageal and vaginal candidiasis.
49
This frequently progresses to invasive and systemic candidiasis in individuals with
50
compromised or immature immune systems due to infections such as HIV, diabetes,
51
radiotherapy or chemotherapy, and aging [1-3]. Flo8 was originally identified in
52
Saccharomyces cerevisiae before subsequent classification as Saccharomyces diastaticus, and
53
was originally reported to be a dominant flocculation gene[4]. The FLO8 gene is highly
54
conserved in yeast strains with a variety of flocculation genotypes and phenotypes and can
55
confer a haploid cell-specific flocculation ability via transcriptional activation of the FLO1
56
gene[5]. Flo8 induces invasive growth in haploid yeast strains via transcriptional activation of
57
Flo11, which has been linked to adhesion to polystyrene surfaces and biofilm formation in C.
58
albicans[6-8]. Flo8 also regulates the expression of extracellular glucoamylase production by
59
transcriptional activation of Sta1 [6, 7]. Furthermore, in C. albicans, Flo8 is also required for
60
hyphal development, binding to epithelial surfaces and for virulence in animal models [9, 10].
61
Flo8 functions as a downstream component of the cAMP/PKA pathway to regulate the
62
process of switching between multiple morphological states (yeast, filamentous,
63
pseudohyphal and hyphal forms) in different environments, which is important for the
64
pathogenicity and virulence of this species [11, 10]. However, the relationship between Flo8
65
and susceptibility to antifungal agents remains to be elucidated. Our previous study of the
66
relationship between Flo8 and the virulence of C. albicans strains yielded the incidental
67
observation that the flo8/flo8 mutant was more resistant to azole antifungal agents than the
4
68
parental SC5314 strain. Therefore, we constructed the FLO8 null mutant flo8/flo8 and the
69
Flo8-overexpressing strain flo8/flo8::FLO8 to investigate the function of Flo8 in the
70
regulation of drug susceptibility in C. albicans.
71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89
5
90
2. Materials and methods
91
2.1. Strains, media and culture conditions
92
The C. albicans strains used in this study are listed in Table 1. We also screened 200 C.
93
albicans clinical isolates from seven hospitals in Shanghai, China. The FLO8/flo8 and
94
flo8/flo8 strains were constructed from the parental strain FLO8/FLO8 (SN152) by
95
PCR-based homologous recombination according to the method described by Noble and
96
Johnson[12]. The Flo8-overexpressing strain flo8/flo8::FLO8 was constructed by integrating
97
gene fragment ADH1-FLO8-LoxP-ARG4-LoxP-ADE2, in which the FLO8 gene is under the
98
control of the ADH1 promoter, into the ADE2 locus in flo8/flo8. The genotype of the empty
99
vector EV was the same as that of flo8/flo8::FLO8 except that EV had no FLO8 gene. All
100
yeast strains were routinely maintained at 30°C on yeast-peptone-dextrose (YPD) medium (1%
101
yeast extract, 2% peptone and 2% glucose) or on solid YPD medium, with the addition of 1.5%
102
agar as previously described[13].
103
2.2. Antifungal susceptibility testing
104
The antifungal susceptibility of C. albicans strains was evaluated using the agar dilution
105
methods as previously described[14]. Briefly, C. albicans cells cultured overnight on YPD
106
plates were diluted to 0.5×108 cells/mL. Stock solutions of the antifungal drugs fluconazole,
107
voriconazole and itraconazole were prepared in dimethyl sulfoxide (DMSO) and added to
108
YPD plates at final concentrations of 10 mg/L, 5 mg/L and 2.5 mg/L, respectively. Five
109
ten-fold dilutions (0.5×108 cells/mL–0.5×104 cells/mL) of each samples were prepared and
110
spotted (2 μL per spot) onto YPD plates with fluconazole, voriconazole or itraconazole. The
111
plates were incubated for 2 days at 30°C.
6
112
The broth microdilution method was then used to evaluate the susceptibility of the
113
isolates to antifungal agents according to the guidelines published by the Clinical and
114
Laboratory Standards Institute (CLSI) [15]. C. albicans ATCC 90028 was used as a control.
115
Two-fold serial dilutions of the drugs were prepared in the following ranges: fluconazole,
116
128–0.25 mg/L and voriconazole and itraconazole, 16–0.03125 mg/L. Triplicate samples of
117
the isolates(1.5×105 cells/mL) were incubated in tubes or 96-well plates in the presence of
118
the antifungal agents for 2 days at 30°C, and the growth of each isolate was measured by a
119
spectrophotometer (OD=600; Mapada Instruments, Shanghai, China). The relative growth of
120
cells was normalized according to growth of the control cells in the absence of drug (defined
121
arbitrarily as 100%). The drug concentration inhibiting 80% of cell growth in the control was
122
defined as the minimum inhibitory concentration (MIC).
123
2.3. RNA isolation and quantitative analysis of mRNA levels by real-time PCR
124
C. albicans cells were harvested after being grown in 3 mL YPD liquid medium at 30°C
125
for 16 h. Total RNA was isolated using the Yeast RNAiso Kit (TaKaRa, Tokyo, Japan) and
126
then treated with gDNA Eraser (TaKaRa) to digest the contaminating DNA. First-strand
127
cDNA was synthesized from 500ng of total RNA in a 10 μL reaction mixture using the
128
PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa). The mRNA levels of the drug
129
resistance-related genes were determined by real-time PCR with a 7300 Real-Time PCR
130
System (Applied Biosystems, Shanghai, China) as described previously [16]. The primers
131
used in the PCR cycling are listed in Table 2. Triplicate technical replicates of each gene were
132
analyzed in reaction mixtures prepared with SYBR Premix ExTaq (Tli RNaseH Plus)
133
(TaKaRa). Real-time PCR was performed using previously described conditions [3]. The
7
134
change of the SYBR Green dye fluorescence was monitored as the PCR cycle numbers
135
increased. The dissociation curves representing the amplification of each product were
136
generated at the end of each PCR cycle, and the cycle threshold (CT) above the background
137
was also calculated. 18S rRNA was used as a reference gene and relative mRNA levels were
138
calculated using the 2−∆∆CT method.
139
2.4. Rhodamine 6G efflux
140
The R6G assay measures the efflux activity of individual yeast cells. The C. albicans
141
ATP-binding cassette (ABC) transporters, Cdr1 and Cdr2, expel azoles and the fluorescent
142
substrate R6G from the interior of the cells. Therefore, the flow cytometric R6G efflux assay
143
was adopted to measure the function of the transporters according to the method described by
144
Peralta[17]. A suspension of 108 yeast cells/mL in YPD medium was incubated overnight at
145
30°C with shaking (200 rpm). Cells were washed, resuspended in PBS(108 yeast cells/mL),
146
and incubated for 4 h to exhaust energy, before incubation with R6G (10 mol/L) for 2 h. Cells
147
were then washed twice and resuspended in ice pre-cooled PBS. A sample (100 μL) of the
148
suspension was then added to 900 μL ice pre-cooled PBS and analyzed for R6G uptake using
149
the beckman boulter Epics XL flow cytometer(Beckman Coulter, USA). A further sample (1
150
mL) of the suspension was incubated with glucose (4 mmol/L) at 30°C for 1 h, washed twice,
151
and then diluted 10-fold in ice pre-cooled PBS for analysis of R6G efflux. Experiments were
152
replicated on three independent occasions.
153
2.5. Statistical analysis
154 155
Data were analyzed by IBM SPSS statistics version 22. The comparison of FLO8 expression levels between the fluconazole-susceptible and fluconazole-resistant clinical
8
156
strains were analyzed by t-test for two independent samples. The correlations between FLO8
157
and CDR1 or CDR2 gene expression were analyzed by Spearman non-parametric correlation
158
test.
159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177
9
178
3. Results
179
3.1. FLO8 null mutation decreased susceptibility of C. albicans to antifungal drugs
180
Decreased susceptibility of C. albicans carrying the FLO8 null mutation to antifungal
181
drugs was confirmed using the agar dilution method. The FLO8/FLO8, FLO8/flo8, flo8/flo8,
182
flo8/flo8::FLO8 and EV strains showed similar patterns of strong growth on YPD plates in the
183
absence of antifungal drugs (Fig. 1, left panel), suggesting that the growth of C. albicans is
184
not affected by the flo8/flo8 mutation.
185
Interestingly, growth of FLO8/flo8 mutant cells was superior to that of the wild-type
186
FLO8/FLO8 (SN152), and the growth of the flo8/flo8 mutant was superior to that of the
187
FLO8/flo8 mutant on YPD plates containing fluconazole, voriconazole or itraconazole (Fig.
188
1). The drug sensitivity of the flo8/flo8 mutant cells was restored by FLO8 overexpression
189
(flo8/flo8::FLO8), and the flo8/flo8::FLO8 mutant cells exhibited less growth compared with
190
the other strains. This suggested that the antifungal drug resistance of the flo8/flo8 mutant
191
cells results from the null mutation in FLO8. The flo8/flo8 and EV cells showed similar
192
patterns of growth on YPD plates with or without antifungal drugs, suggesting that the
193
inserted fragment ADH1-LoxP-ARG4-LoxP-ADE2 did not affect the growth rate, growth
194
ability and drug susceptibility of C. albicans. Thus, FLO8 overexpression increased the
195
susceptibility of C. albicans to fluconazole, voriconazole and itraconazole, while the null
196
mutation in Flo8 decreased susceptibility to these antifungal drugs.
197
We also evaluated the FLO8/FLO8, flo8/flo8 and flo8/flo8::FLO8 strains using the broth
198
microdilution method to confirm the effect of Flo8 on antifungal drug resistance. The flo8/flo8
199
mutants were also found to be more resistant to fluconazole (Fig. 2A), voriconazole (Fig. 2B)
10
200
and itraconazole (Fig. 2C) than the FLO8/FLO8 and flo8/flo8::FLO8. Similarly,
201
flo8/flo8::FLO8 exhibited less growth compared with the other strains and showed a little
202
more sensitivity to the three antifungal drugs compared with FLO8/FLO8. After 2 days in
203
culture at 30°C, flo8/flo8 cells incubated in the presence of 128 µg/mL fluconazole, 16 µg/mL
204
voriconazole or 16 µg/mL itraconazole exhibited 60% growth relative to that of flo8/flo8
205
incubated in the absence of drug, 20% growth relative to that of FLO8/FLO8 and 5%–10%
206
growth relative to that of flo8/flo8::FLO8. Furthermore, the antifungal drug resistance of the
207
C. albicans flo8/flo8 isolate was reflected by the MICs of >128 µg/mL for fluconazole, 16
208
µg/mL for itraconazole and 16 µg/mL for voriconazole (Table 3). Hence, these findings were
209
consistent with the results of the agar dilution assay.
210
3.2. Expression levels of seven drug resistance-related genes in flo8/flo8 and flo8/flo8::FLO8
211
compared with those in FLO8/FLO8
212
To investigate whether the genes responsible for antifungal drugs resistance are regulated
213
directly or indirectly by Flo8, real-time PCR was performed to compare the mRNA levels of
214
seven antifungal drug resistance-related genes (CDR1, CDR2, MDR1, FLU1, ERG11, UPC2
215
and FCR1) in FLO8/FLO8, flo8/flo8 and flo8/flo8::FLO8. The 2−∆∆CT values of the genes
216
which have statistically significant differences (P<0.05) in FLO8/flo8 or flo8/flo8 , or
217
flo8/flo8::FLO8 compared with FLO8/FLO8 are summarized in Figure 3. The
218
others(MDR1,MRR1, FLU1, ERG11, UPC2 and FCR1) have no significant differences and
219
are not shown. As shown in Figure 3, in FLO8/flo8, flo8/flo8 and flo8/flo8::FLO8, the
220
expression levels of CDR1 were 1.28-fold, 1.75-fold and 0.64-fold compared with that in
221
FLO8/FLO8. CDR2 were 2.28-fold, 5.44-fold and 0.97-fold, and TAC1 were 1.4-fold,
11
222
1.85-fold and 0.94-fold respectively. In flo8/flo8, the disruption of FLO8 gene increased the
223
expression of CDR1, CDR2, and TAC1 by 1.75-fold, 5.44-fold and 1.85-fold, respectively,
224
and they all had statistically significant differences (P<0.05) compared with that in
225
FLO8/FLO8. However, in flo8/flo8::FLO8, the expression of CDR1 was decreased (0.64-fold)
226
compared with that in FLO8/FLO8 (P<0.05), while overexpression of FLO8 had no
227
significant effects on the expression of CDR2(0.97-fold) and TAC1(0.94-fold).
228
3.3. R6G efflux
229
As is shown in Figure 4, there was no significant difference in the R6G uptake of
230
FLO8/FLO8, FLO8/flo8, flo8/flo8 and flo8/flo8::FLO8 after glucose exhaustion. However,
231
after incubation in the presence of glucose for 1 h, the R6G efflux of flo8/flo8 was higher than
232
that of FLO8/FLO8, FLO8/flo8 and flo8/flo8::FLO8 (P < 0.05), while the efflux rate of
233
flo8/flo8::FLO8 was lower than that of FLO8/FLO8 (P < 0.05). And, the differences were
234
more significant after incubation with glucose for 2 h. The 2-h R6G excretion rate of
235
FLO8/FLO8, FLO8/flo8, flo8/flo8 and flo8/flo8::FLO8 were 31%, 40%, 72% and 23%.
236 237
3.4. FLO8 expression levels and fluconazole susceptibility in clinical isolates To identify the relationship between FLO8 expression levels and fluconazole
238
susceptibility in C. albicans clinical isolates, a collection of 200 C. albicans isolates from
239
seven hospitals in Shanghai, China, was obtained. FLO8 expression levels were identified by
240
real-time PCR, and the expression level of FLO8 in SC5314 was defined as 1. In all the 200
241
isolates, the FLO8 expression levels were from 0.34-fold to 5.9-fold. We defined the isolates
242
with ＜0.8-fold FLO8 expression levels(only 16 isolates) to low-FLO8 group, and randomly
243
selected 16 isolates with＞2-fold FLO8 expression levels(78 isolates) to high-FLO8 group. As
12
244 245
is shown in Figure 5, C1-C16 belong to low-FLO8 group, and C17-C32 high-FLO8 group. Fluconazole susceptibility was evaluated by the broth microdilution method, and the
246
MICs of C.albicans ATCC90028 and C.krusei ATCC6258 were within the scope of quality
247
controls(not shown). As is shown in Figure 6, the MICs in low-FLO8 group were significantly
248
higher than those in high-FLO8 group(P < 0.05). In low-FLO8 group, 8 isolates were
249
fluconazole resistant, and 7 isolates were susceptible-dose dependent. And, all the 16 isolates
250
in high-FLO8 group were fluconazole sensitive.
251
3.5. CDR1, CDR2 and TAC1 expression levels in low-FLO8 group and high-FLO8 group
252
To verify the association of FLO8 expression with CDR1, CDR2, and TAC1 in clinical
253
strains, and that the expression relationships were consistent with those in the engineered
254
isolates, we performed real-time quantitative RT-PCR analysis of a panel of 32 clinical
255
isolates, comprising 16 (C1–C16) low-FLO8 strains and 16 (C17–C32) high-FLO8 strains.
256
Expression levels of CDR1, CDR2, and TAC1 in the 32 clinical isolates relative to
257
SC5314 are shown in Figure 7A(CDR1), B(CDR2), and C(TAC1). CDR1 expression levels
258
ranged from 0.83-fold to 6.0-fold(in low-FLO8 group), and from 0.52-fold to 1.98-fold(in
259
high-FLO8 group). CDR2 expression levels ranged from 1.3-fold to 8.18-fold(in low-FLO8
260
group), and from 0.58-fold to 4.75-fold(in high-FLO8 group). TAC1 expression levels ranged
261
from 0.87-fold to 1.93-fold(in low-FLO8 group), and from 0.67-fold to 1.23-fold(in
262
high-FLO8 group).The expression levels of CDR1, CDR2, and TAC1 in low-FLO8 group
263
were higher than those in high-FLO8 group(P<0.05).
264 265
13
266
4. Discussion
267
In the present study, we confirmed that Flo8 is associated with the antifungal azole
268
susceptibility of C. albicans, with FLO8 deletion leading to constitutive overexpression of the
269
multidrug efflux transporters CDR1 and CDR2 and resistance to antifungal azoles.
270
To confirm the hypothesis that FLO8 expression is involved in regulation of the azole
271
susceptibility of C. albicans via expression of drug resistance related genes, we constructed
272
the FLO8/flo8, flo8/flo8 and flo8/flo8::FLO8 strains and evaluated their antimicrobial
273
susceptibility using three different methods. Using the agar dilution method, flo8/flo8 showed
274
superior resistance to fluconazole, voriconazole and itraconazole compared with that of
275
FLO8/flo8, while flo8/flo8::FLO8 was the least resistant strain. A similar pattern of drug
276
resistance was identified using the broth microdilution method. Furthermore, the MIC values
277
of the three antifungal drugs for flo8/flo8 were >128 µg/mL for fluconazole, 16 µg/mL for
278
itraconazole and 16 µg/mL for voriconazole, which reflected the drug-resistant phenotype of
279
flo8/flo8. The consistency of the phenotypes of FLO8/flo8, flo8/flo8 and flo8/flo8::FLO8 in
280
these tests indicates the involvement of FLO8 expression in the mechanism of susceptibility
281
to antifungal azoles in C. albicans, with FLO8 deletion associated with a marked increase the
282
resistance of C. albicans to antifungal azoles. This resistance was reversed by FLO8
283
overexpression.
284
To explore the mechanism underlying the resistance to antifungal azoles caused by FLO8
285
deletion in C. albicans, we analyzed the expression levels of nine drug resistance-related
286
genes (CDR1, CDR2, TAC1, MDR1, MRR1, FLU1, ERG11, UPC2 and FCR1) in FLO8/FLO8,
287
FLO8/flo8, flo8/flo8 and flo8/flo8::FLO8. As shown in Figure 3, the expression of
14
288
CDR1,CDR2 and TAC1 was increased in flo8/flo8, with 1.75-fold, 5.44-fold and 1.85-fold
289
compared with that in FLO8/FLO8, while only the expression of CDR1 was decreased in
290
flo8/flo8::FLO8, with 0.64-fold compared with that in FLO8/FLO8. The changes in the
291
expression of CDR1,CDR2 and TAC1 in flo8/flo8 and flo8/flo8::FLO8 could explain the
292
changes in the susceptibility of these strains to antifungal azoles. These results indicate that
293
FLO8 deletion results in activation of the transcription of TAC1, CDR1 and especially CDR2,
294
while FLO8 overexpression just decreases CDR1 expression. Because TAC1 controls the
295
upregulation of CDR1 and CDR2 at the transcriptional level[18], we guess FLO8 deletion
296
increases the expression of TAC1, and then TAC1 activates the transcription of CDR1 and
297
CDR2. CDR1 is regulated not only by TAC1 but also by MRR2 and NDT80[19], other
298
regulation mechanisms between FLO8 and CDR1 may exist.
299
The function of the changes in expression of CDR1,CDR2 and TAC1 caused by FLO8
300
deletion or overexpression were then confirmed by the R6G efflux assay. We set the time of
301
efflux with glucose at 1h and 2h. As is shown in Figure 4, there were no significant
302
differences in R6G uptake by FLO8/FLO8, FLO8 /flo8, flo8/flo8 and flo8/flo8::FLO8, and for
303
each of them, the 2h-efflux rate was higher than the corresponding 1h-efflux rate. In flo8/flo8,
304
the 1h-efflux rate was 40%, and 2h-efflux-rate 72%, significantly higher than the others(P <
305
0.05). And, flo8/flo8::FLO8 exhibited the lowest 1h-efflux and 2h-efflux rates (P < 0.05).
306
Similarly, the results of the R6G efflux assay were consistent with those of the antifungal drug
307
sensitivity tests, and also corresponded with the drug resistance-related genes expression
308
levels.
309
Based on our findings, we hypothesized that the removal or low expression of FLO8
15
310
results in upregulations of TAC1, CDR1 and especially CDR2, and finally leads to the
311
decreased sensitivity to antifungal azoles. While FLO8 overexpression can reversed this
312
phenotype.To verify that the relationship between FLO8 expression and fluconazole
313
susceptibility, and the correlations of FLO8 expression with that of CDR1, CDR2 and TAC1
314
in C. albicans clinical isolates were consistent with those of the engineered isolates, we
315
collected and analyzed FLO8 expression levels in 200 C. albicans clinical isolates. As is
316
shown in Figure 5, C1-C16 belong to low-FLO8 group, and C17-C32 high-FLO8 group.And
317
fluconazole susceptibility is shown in Figure 6. The MICs in low-FLO8 group(with 8
318
fluconazole resistant, and 7 susceptible-dose dependent) were significantly higher than those
319
in high-FLO8 group(all fluconazole sensitive)(P < 0.05). Based on this result, it can be
320
considered that low expression of FLO8 could reduce the susceptibility of C.albicans clinical
321
strains to fluconazole, which is consistent with that in the engineered isolates. The
322
correlations of FLO8 expression with that of CDR1, CDR2 and TAC1 in C. albicans clinical
323
isolates are shown in Figure 7. The expression levels of CDR1, CDR2, and TAC1 in
324
low-FLO8 group were higher than those in high-FLO8 group(P<0.05). Based on the results in
325
clinical isolates, it can be considered Flo8 is associated with the antifungal azole susceptibility
326
of C. albicans. The low expression of FLO8 increases the expression of TAC1, CDR1 and
327
CDR2, and can finally decrease azoles susceptibility in C.albicans.
328
There are numerous reports describing the function of FLO8 and Flo8 in C. albicans
329
[9-11,20]. Cao et al. found that FLO8 deletion in C. albicans blocked hypha-specific gene
330
expression and hyphal development, and the flo8/flo8 mutant was avirulent in a mouse
331
systemic infection model[10]. Liu et al. showed that the T751D and G723R Flo8 mutants
16
332
enhanced the Flo8 C-terminus activation, thereby increasing virulence and promoting
333
filamentous growth[13]. In C.albicans clinical isolates, the overexpression of Cdr1 and Cdr2
334
is responsible for azole resistance. Cdr1 and Cdr2 upregulation are primarily controlled by
335
TAC1 at the transcriptional level, and are coordinately regulated[18,21]. TAC1 binds to the
336
drug-responsive element(DRE) of CDR1 and CDR2 and is localized in the nucleus. In
337
C.albicans clinical strains, the gain-of-function mutations in TAC1 can result in high
338
expression of CDR1 and CDR2[22], and then azole resistance. As compared with CDR2,
339
CDR1 is a prime contributor of azole resistance in clinical strains[23]. In C.albicans clinical
340
strains resistant to azoles, the removal of TAC1 can abolish CDR1/CDR2 expression and
341
therefore drug resistance, and this phenotype can be reversed when the TAC1 allele
342
reintroduced[18]. A TAC1 allele from an azole-resistant clinical strain can confer the
343
phenotype to a susceptible strain[18].
344
It can be considered that the removal or low expression of FLO8 can increase the
345
transcriptional regulation of TAC1, CDR1 and CDR2, and then decreases azoles susceptibility
346
in C.albicans. And this phenotype can be reversed when FLO8 allele reintroduced.
347
Approximately 50%–60% of candidiasis cases are caused by the opportunistic C. albicans
348
infections[24]. And treatment failure caused by the emergence of azole-resistant clinical
349
isolates has become a serious problem[25,26]. Several mechanisms that account for azole
350
resistance in C. albicans clinical isolates have been elucidated, including upregulation of
351
CDR1 and CDR2. Our discoveries provide new insights into the mechanisms of azole
352
resistance and the functions of Flo8 in C. albicans. Treatment failure caused by azole
353
resistance has become an emergence. And, our discoveries represent the basis of further
17
354
research and development of new antifungal agents for the treatment of C. albicans. The
355
researchers can develop drugs especially according to the regulation mechanisms between
356
Flo8 and CDR1 and CDR2 for the patients infected by azole resistance C.albicans, in which
357
the resistance comes from the overexpressions of CDR1 and CDR2.
358
Some limitations of our study should be noted. On the one hand, the overexpression of
359
FLO8 in flo8/flo8::FLO8 decreases the expression of CDR1(0.64-fold compared with that in
360
FLO8/FLO8) , while it has no significant effects on the expression of CDR2(0.97-fold) and
361
TAC1(0.94-fold). CDR1 is regulated not only by TAC1 but also by MRR2 and NDT80[19].
362
The regulation of CDR1 may be not all attributed to TAC1, and additional mechanisms
363
between FLO8 and CDR1 can be expected. On the other hand, it can be determined that the
364
removal or low expression of FLO8 can increase the transcriptional regulation of TAC1,
365
CDR1 and CDR2, and then decrease azoles susceptibility in C.albicans. Whether the
366
upregulation of CDR1 and CDR2 comes from the increased TAC1 or by other mechanisms,
367
needs further research.
368
To sum up, we confirmed that Flo8 is associated with the antifungal azole susceptibility
369
of C. albicans, with the removal or low expression of FLO8 leading to constitutive
370
overexpression of TAC1, CDR1 and CDR2, and azoles resistance or decreased azoles
371
susceptibility. And the reintroduced FLO8 allele can reverse this phenotype. Of course, further
372
studies are required to fully clarify the regulation mechanisms between FLO8 and TAC1,
373
CDR1 and CDR2 expression in C. albicans.
374 375
18
376 377
Conflict of interest The authors declare no conflicts of interest.
378 379
Acknowledgements
380
This work was financed by grants from the Program of Shanghai Natural Science
381
Foundation [#15ZR1426900],the Program of Shanghai Key Specialty [#ZK2012A21] and
382
Excellent Youth of HuangPu District of Shanghai [#RCPY1407].
383 384 385 386 387 388
Ethical approval Not required.
19
389
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390
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452 453 454
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Legends to figures
456
Fig. 1. Susceptibilities of five C. albicans strains [FLO8/FLO8, FLO8/flo8, flo8/flo8, flo8/flo8::FLO8 and
457
EV] to fluconazole, voriconazole and itraconazole were determined using the agar dilution assay. Five
458
10-fold serial dilutions (0.5×108–0.5×104 cells/mL) of each sample grown on YPD plates overnight were
459
spotted (2 µL per spot) onto YPD plates with or without fluconazole (10 mg/L), voriconazole (5 mg/L) or
460
itraconazole (2.5 mg/L). (Left panel) YPD medium without antifungal drugs was used as a control. The
461
plates were incubated for 2 days at 30°C.
462 463
Fig. 2. Susceptibilities of three C. albicans strains (FLO8/FLO8, flo8/flo8 and flo8/flo8::FLO8) to
464
fluconazole (A), voriconazole (B) and itraconazole (C) were determined by the broth microdilution assay.
465
Cells were incubated for 2 days at 30°C, The relative growth of cells was normalized according to cell
466
growth in the absence of drug (defined arbitrarily as 100%).
467 468
Fig. 3. Fold changes in the expression levels of CDR1, CDR2 and TAC1 in FLO8/flo8, flo8/flo8 and
469
flo8/flo8::FLO8. In flo8/flo8, the disruption of FLO8 gene increased the expression of CDR1, CDR2, and
470
TAC1 by 1.75-fold, 5.44-fold and 1.85-fold, respectively, compared with that in FLO8/FLO8 (P<0.05) . In
471
flo8/flo8::FLO8, only the expression of CDR1 was decreased (0.64-fold) compared with
472
FLO8/FLO8(P<0.05).
473 474
Fig. 4. Flow cytometric analysis of R6G uptake and efflux. There was no significant difference in the R6G
475
uptake of FLO8/FLO8, FLO8/flo8, flo8/flo8 and flo8/flo8::FLO8 after glucose exhaustion. After incubation
476
in the presence of glucose for 1 h and 2h, the R6G efflux of flo8/flo8 was significantly higher than that of
23
477
FLO8/FLO8, FLO8/flo8 and flo8/flo8::FLO8 (P < 0.05).
478 479
Fig. 5. Fold changes in the expression levels of FLO8 in low-FLO8 group (C1–C16) and high-FLO8 group
480
(C17–C32) of C. albicans relative to the value of SC5314.
481 482
Fig. 6. The MICs of fluconazole against 32 C.albicans clinical isolates. The MICs in low-FLO8 group
483
(C1–C16) were significantly higher than those in high-FLO8(C17–C32) group(P < 0.05).
484 485
Fig. 7. Fold changes in the expression levels of CDR1(A), CDR2(B) and TAC1(C) in 32 C. albicans clinical
486
isolates relative to SC5314. The expression levels of CDR1, CDR2, and TAC1 in low-FLO8 group were
487
higher than those in high-FLO8 group(P<0.05).
488 489 490 491 492
Table 1. Candida albicans strains used in this study Strain
Genotype or description
Source or reference
SC5314
Wild-type
(Liu et al., 2015)
ATCC90028
Candida albicans ATCC strain
ATCC
ATCC6258
Candida krusei ATCC strain
ATCC
ura3::imm434::URA3/ura3::imm434 iro1::IRO1/iro1::imm434 his1::hisG/his1::hisG leu2/leu2
(Noble and Johnson,
FLO8/FLO8
arg4/arg4
2005)
FLO8/flo8
ura3::imm434::URA3/ura3::imm434 iro1::IRO1/iro1::imm434 his1::hisG/his1::hisG leu2/leu2
This study
arg4/arg4 flo8::CdHIS1 flo8/flo8
ura3::imm434::URA3/ura3::imm434 iro1::IRO1/iro1::imm434 his1::hisG/his1::hisG leu2/leu2
This study
arg4/arg4 flo8::CdHIS1/flo8::CmLEU2 flo8/flo8::FLO8
ura3::imm434::URA3/ura3::imm434 iro1::IRO1/iro1::imm434 his1::hisG/his1::hisG leu2/leu2
This study
arg4/arg4 flo8::CdHIS1/flo8::CmLEU2 ADE2/ade2::ADH1p-FLO8-PAP EV
ura3::imm434::URA3/ura3::imm434 iro1::IRO1/iro1::imm434 his1::hisG/his1::hisG leu2/leu2 arg4/arg4 flo8::CdHIS1/flo8::CmLEU2 ADE2/ade2::ADH1p-PAP
Table 2. Primers used in this study Primers
Sequence (5ʹ-3ʹ)
18SRNA-F
TCTTGTACGGCACATATCTCGT
18SRNA-R
GTTTCGAGAGGACCTTCAGTC
MDR1-QF
GTTAAACATTTCACCCTCG
MDR1-QR
AACAAGATACCACCGACA
MRR1-QF
AACGCTGGTTATGGGTGA
MRR1-QR
TTTGCTGTTGGGCTTCTT
ERG11-QF
TTGGTGGTGGTAGACATA
ERG11-QR
TCTGCTGGTTCAGTAGGT
FCR1-QF
CCACTGGTCCAATCCTTA
FCR1-QR
CTGTAGACTCCATTCGTT
UPC2-QF
AATGCCTCGTTTCCTTCG
UPC2-QR
CACGGTCAATTCCTTTCG
FLU1 -QF
AGAAGAGCCACCAGAAGT
FLU1 -QR
CGATAAGGCAGCAAGACC
CDR1-QF
GGTCAACTTGTAATGGGTC
CDR1-QR
AGGACGATAAAGGGCATA
CDR2-QF
GCCAATGCTGAACCGACA
This study
CDR2-QR
ACCAGCCAATACCCCACA
TAC1-QF
GTCAATTAGGCGAGACAC
TAC1-QR
CTGCCACCAATACAAGATA
Table 3. Minimum inhibitory concentrations of fluconazole, itraconazole and voriconazole for the C. albicans isolates Strain
fluconazole (µg/mL)
itraconazole (µg/mL)
voriconazole (µg/mL)
ATCC90028 FLO8/FLO8 FLO8/flo8 flo8/flo8
0.25 0.25 32 >128 0.25 >128
≤0.125 ≤0.125 0.5 >16 ≤0.125 >16
≤0.125 ≤0.125 2 >16 ≤0.125 >16
flo8/flo8::FLO8 EV