Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, N.E., Mailstop G-11, Atlanta, Georgia 30333, USA
Received 17 June 2003; returned 1 August 2003; revised 10 October 2003; accepted 29 October 2003
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Abstract |
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Methods: Twelve C. albicans isolatesseven trailing and five susceptible dose dependent (SDD) or resistant (R)were screened for ERG11 mutations by DNA sequencing and quantification of ERG11, CDR1 and MDR1 expression by RT-PCR using the LightCycler high-speed PCR system.
Results: SDD and R isolates possessed more homozygous ERG11 mutations than did the trailing isolates. Two of these, V404I and V509M, have not been described previously and were found exclusively in fluconazole SDD and R isolates. Quantification of ERG11 expression revealed that both trailing and SDD and R isolates were capable of ERG11 up-regulation in response to fluconazole, although the SDD and R isolates showed maximal up-regulation at higher fluconazole concentrations. Quantification of CDR1 and MDR1 revealed that all isolates, regardless of in vitro fluconazole response, were capable of CDR1 and MDR1 up-regulation following fluconazole exposure. Furthermore, the SDD and R isolates expressed higher constitutive levels of CDR1 and MDR1 or CDR1, respectively, in the absence of drug compared with trailing isolates.
Conclusions: Trailing isolates, although susceptible to fluconazole, express the same molecular mechanisms as SDD and R isolates following fluconazole exposure but regulate them differently.
Keywords: C. albicans, azole drug resistance, molecular mechanisms
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Introduction |
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It has been suggested recently that trailing may be due, at least in part, to the ability of C. albicans isolates to up-regulate, in response to drug exposure, the transcription of genes encoding the azole drug target, lanosterol demethylase (ERG11), the terbinafine target, squalene epoxidase (ERG1) or the azole and terbinafine efflux transporters (CDR1, CDR2 and MDR1).5 These same mechanisms, as well as point mutations in ERG11, have been implicated in the development of azole drug resistance in C. albicans.6 However, potential differences in the expression of these mechanismsbetween susceptible isolates that display trailing growth and non-trailing isolates with reduced azole susceptibilitieshave not been investigated.
The purpose of the present study was to examine possible molecular mechanisms of azole resistance among fluconazole-susceptible bloodstream isolates of C. albicans that displayed the trailing growth phenomenon, and to compare these isolates with isolates that showed reduced susceptibilities to fluconazole. We used DNA sequencing to reveal any sequence variation in the azole drug target, ERG11, and reverse transcription coupled with LightCycler real-time PCR to quantify expression of ERG11 and the azole antifungal drug efflux genes, CDR1 and MDR1.
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Materials and methods |
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Twelve isolates of C. albicans (nine bloodstream and three mucosal) with varying in vitro fluconazole susceptibilities were selected. Bloodstream isolates were from a collection derived from active population-based surveillance for candidaemia conducted during 19982000.1 Mucosal isolates were from HIV-infected persons with oropharyngeal or vaginal candidiasis.79 Isolates were stored at 70°C as 30% glycerol stocks in sterile water. Prior to testing, isolates were subcultured on Sabouraud dextrose agar (SAB) plates (BBL, Cockeysville, MD, USA) at 35°C.
Broth microdilution susceptibility testing method
MICs of fluconazole, itraconazole and voriconazole were determined by the NCCLS M27-A broth dilution method.10 Standard powders of fluconazole and voriconazole were received as gifts from Pfizer Pharmaceuticals Group (Groton, CT, USA), and itraconazole drug powder was purchased from Research Diagnostics, Inc. (Flanders, NJ, USA). The final concentrations of the antifungal agents were in the range fluconazole 0.12564 mg/L and itraconazole and voriconazole 0.0158 mg/L. The MIC endpoints were read visually following 24 and 48 h of incubation and were defined as the lowest concentration that produced a prominent reduction in growth (50%) compared with that of the drug-free growth control. Interpretations of MICs were assigned according to the NCCLS criteria.10 Trailing growth was defined as a susceptible MIC after 24 h incubation and a resistant MIC after 48 h incubation.
Sterol quantification method
Total cellular ergosterol was quantified as described previously.1,3 The sterol quantification method MIC of fluconazole, itraconazole and voriconazole was defined as the concentration of drug that caused an 80% reduction in the total cellular ergosterol content compared with that in the drug-free control. MICs that fell between two drug concentrations (i.e. less than 80% reduction at one concentration but more than 80% reduction at the next-higher concentration) were mathematically extrapolated, based on the amount of reduction by the drug concentration that gave results closest to an 80% reduction endpoint.
PCR amplification and sequencing
Genomic DNA from C. albicans isolates grown overnight in SAB broth was extracted using a QIAGEN Genomic-tip 20/G and DNA buffer set (Qiagen, Valencia, CA, USA) in accordance with the manufacturers instructions, and was used as a template for amplification of ERG11. PCR was carried out with high-fidelity Pwo DNA polymerase (Roche Molecular Biochemicals, Indianapolis, IN, USA) and sequence-specific oligonucleotide primers (Table 1). PCR cycling conditions were one cycle at 95°C for 5 min, followed by 25 cycles at 95°C for 30 s, 60°C for 30 s, 72°C for 45 s, followed by one cycle at 72°C for 7 min. For direct sequencing, the PCR products were purified with the QIAquick PCR purification kit (Qiagen). Sequencing of the ERG11 open reading frame (ORF) was separated into five parts, each containing 400 nt and each amplified with a unique primer pair. DNA sequencing was performed using the same primers, purified PCR products as the template and the BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA, USA). After purification of cycle sequencing products using Centri-Sep spin columns (Princeton Separations, Adelphia, NJ, USA), sequence analysis was performed on an ABI 310 Genetic Analyzer (Applied Biosystems). Sequence data were assembled and compared with that of a previously reported ERG11 sequence from a standard, wild-type, fluconazole-susceptible C. albicans strain11 (accession number X13296) by using GCG sequence analysis software (NCD Software Co., Beaverton, OR, USA).
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RNA extraction. For each isolate, an overnight culture grown in 10 mL of SAB broth (Difco) was diluted 1:100 in fresh SAB broth and grown to mid-logarithmic phase (OD600=11.2). For detecting transcript levels in the presence or absence of fluconazole, overnight cultures of C. albicans isolates demonstrating fluconazole-SDD, -resistant, and trailing growth phenotypes were grown in SAB broth and diluted 1:100 in fresh SAB broth and in SAB broth containing fluconazole 8, 16, 32 or 64 mg/L. Cultures were incubated with shaking at 35°C until cells reached the mid-logarithmic phase of growth. Cells were harvested by centrifugation and washed once with sterile distilled water. Cell lysis was performed by re-suspending cells in sterile distilled water plus Lysis Binding solution (Bio 101 Systems, Vista, CA, USA), transferring them to Lysis Matrix C tubes (Qbiogene, Carlsbad, CA,USA) and vortexing twice for 45 s at speed level 6 in the FastPrep high speed vortexer (Qbiogene). Following the directions of the manufacturer of the RNAqueous-4PCR kit (Ambion, Austin,TX, USA), total RNA was extracted from C. albicans cells. To remove genomic DNA contamination, RNA samples were treated with two units of DNase I (Roche) per 100 µL of RNA at 37°C for 1 h.
Synthesis of cDNA. Reverse transcription was performed in a total volume of 40 µL with a 1st Strand cDNA Synthesis Kit for RT-PCR (Roche) using 2 µL of RNA, AMV reverse transcriptase and random primer p(dN)6, as recommended by the manufacturer. The cDNA was purified using the QIAquick PCR purification kit (Qiagen) and quantified by spectrophotometric measurement of A260 and A280 and standard calculations.
Quantitative real-time PCR with LightCycler. Primers and hybridization probes were designed using LightCycler Probe Design software (Roche) and are listed in Table 1. In order to optimize the real-time PCR conditions and verify the specificity of the designed primer pairs, the optimal MgCl2 concentration and annealing temperature were determined using FastStart DNA Master SYBR Green I (Roche) in the LightCycler (Roche) and melting curve analysis. Quantification of gene expression by LightCycler is based on a standard curve for each target gene and is included in each LightCycler real-time (RT)-PCR experiment. A template for the LightCycler standard curves was generated via conventional PCR using 15 ng of genomic DNA from the fluconazole-susceptible C. albicans isolate ATCC 32354, 0.2 µM of each PCR primer (sequence shown in Table 1), and 1.5 mM MgCl2 and 0.2 µM of each dNTP. Cycling conditions were one cycle at 95°C for 5 min, followed by 30 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, followed by one cycle at 72°C for 7 min. The samples were held at 4°C in the thermalcycler until retrieved.
For LightCycler relative quantification of ERG11, CDR1, MDR1 and ACT1 from C. albicans isolates with varying fluconazole susceptibility phenotypes, each specific standard curve was prepared by purification and 10-fold serial dilutions of a known concentration of amplicons (generated as described above) of each gene. Real-time LightCycler PCR reactions were performed using the FastStart DNA Master Hybridization Probe PCR mix (Roche). The reaction mixture consisted of 2.4 µL of 25 mM MgCl2, 0.4 µM of each PCR primer, 0.2 µM of each hybridization probe, 2 µL of LightCycler DNA FastStart Hybridization mix (Roche) and PCR-grade water up to a final volume of 18 µL. For all samples, a master mix was prepared and 18 µL was transferred into each glass capillary. Two microlitres of cDNA from the reverse transcription step (test samples) or PCR amplicons from each dilution (standard curve) were then pipetted into all but one of the LightCycler capillaries. PCR-grade water was added to the latter in place of cDNA template to serve as a negative control. After 10 min of denaturation at 95°C, 4050 PCR cycles were performed. Cycling conditions for ERG11 and CDR1 were 95°C for 10 s, 62°C for 15 s and 72°C for 10 s. MDR1 was amplified with an annealing temperature of 54°C because it has a lower primer melting temperature. The concentration of each gene (ERG11, CDR1, MDR1 and ACT1) was calculated by reference to the respective standard curve with the aid of the LightCycler software. Quantification of these genes was determined for each C. albicans isolate in three separate LightCycler PCR reactions using the same RNA preparation. Relative gene expression was calculated as a ratio of target gene (ERG11, CDR1 and MDR1) concentration to housekeeping gene (ACT1) concentration, and values reported represent the mean gene expression from three experiments ± the standard error of the mean (S.E.M.).
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Results |
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Table 2 summarizes the in vitro susceptibilities of the 12 isolates of C. albicans to fluconazole, as determined by the broth microdilution and sterol quantification methods. All fluconazole-SDD and -resistant isolates were classed as SDD and resistant by both methods. Seven bloodstream isolates (isolates 17) that showed trailing growth by the broth microdilution method were classed as susceptible to fluconazole when re-tested by the sterol quantification method. All five isolates that were classed as SDD or resistant to fluconazole also showed decreased susceptibilities to itraconazole and voriconazole (data not shown).
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The ERG11 ORF of all 12 C. albicans isolates was sequenced and compared with the previously reported standard sequence11 (Table 3). Trailing isolates 1 and 7, obtained from two different patients from different hospitals, had the same ERG11 sequence. No sequence variations leading to amino acid substitution were found in isolates 4 and 6, both of which showed trailing growth in the presence of azoles. ERG11 from the other 10 isolates contained one or more nucleotide changes that led to amino acid substitutions in the protein sequence. A total of nine different nucleotide changes in the ERG11 alleles investigated yielded amino acid substitutions. Five of these, K143R, V404I, F449V, R467K and V509M, were found exclusively in non-trailing isolates with reduced fluconazole susceptibilities (isolates 812), and all were due to homozygous nucleotide substitutions.
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Expression of ERG11, CDR1 and MDR1 in C. albicans bloodstream isolates
To investigate if distinct ERG11 expression patterns correlated with fluconazole resistance and/or azole trailing, ERG11 expression was quantified in all 12 C. albicans isolates by a LightCycler relative quantification method (Table 2). This method has been shown previously to correlate with traditional northern blotting methods for relative quantification of gene expression in C. albicans.12 Although the data in Table 2 represent the results of three independent LightCycler experiments using the same RNA preparation, we have observed that different RNA preparations from the same isolate show a consistent trend in gene expression (data not shown). In the absence of fluconazole, ERG11 was expressed in all isolates, regardless of fluconazole susceptibility. However, ERG11 expression levels showed no correlation with susceptibility. In fact, the mean ERG11 expression was higher in the seven trailing isolates (15.4 ± 2.2) than in the five isolates with reduced fluconazole susceptibilities (9.7 ± 2.6), as determined by Restricted Maximum Likelihood analysis for repeated measures with an unstructured correlation (P value = 0.127) (Table 2).
To evaluate the effect of fluconazole exposure on ERG11 expression among fluconazole-trailing, -SDD and -resistant isolates, four representative bloodstream isolates (isolates 1, 3, 9 and 10) were incubated with fluconazole 0, 8, 16, 32 and 64 mg/L, and ERG11 expression was measured by LightCycler relative quantification (Figure 1a). It is important to note that the fluconazole concentration added to the cells cultured in SAB broth does not correlate directly with the microbroth dilution fluconazole MIC of the isolates that were determined in RPMI 1640 medium. In SAB broth, adequate growth of all isolates in all drug concentrations was achieved. Under these conditions, both the trailing and less susceptible isolates were capable of up-regulating ERG11 in response to fluconazole exposure. However, the concentrations of fluconazole that elicited peak ERG11 expression differed. Specifically, the fluconazole-SDD and -resistant isolates (isolates 9 and 10) demonstrated peak levels of ERG11 expression at higher drug concentrations than the trailing isolates (isolates 1 and 3) (Figure 1a). In addition, the correlation between ERG11 expression and the concentration of fluconazole exposure was greater for the fluconazole-SDD and -resistant isolates (0.97) than for the trailing isolates (0.56) as determined by Pearsons r correlation coefficient.
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Discussion |
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No fewer than 35 different nucleotide substitutions leading to amino acid substitutions have been identified in ERG11 alleles from clinical isolates of C. albicans.1324 Twenty of these 35 amino acid substitutions have been identified in azole-resistant isolates, including F126L, G129A, Y132H, K143E, K143R, F145L, A149V, T229A, S279F, K287R, G307S, S405F, G448E, G448R, F449L, V452A, G464S, G465S, R467K and I471T.13,16,17,24 In this study, we found two of these mutations (K143R and R467K) in fluconazole-resistant C. albicans isolates derived from cases of oropharyngeal candidiasis (isolates 11 and 12). These mutations were not found in any of the nine bloodstream isolates in this study. We also found three ERG11 amino acid substitutions that have not been described previously. Two of these, V404I and V509M, were found only in bloodstream isolates with reduced fluconazole susceptibilities. Whereas the number of mucosal isolates included in this analysis is small, these findings suggest that further study of differences between bloodstream and mucosal isolates with regard to acquisition of azole drug resistance mechanisms may be warranted.
Marichal et al.22 have identified three hot spots within the amino acid sequence of the ERG11 gene based on a compilation of ERG11 mutations reported to be associated with azole resistance. These hot spots include amino acid regions 105165, 266287 and 405488. Two of the newly described amino acid substitutions, V404I and V509M, fall just outside the third hot spot. A possible explanation is that nucleotide sequence data used to delineate these three hot spots were from C. albicans mucosal isolates rather than bloodstream isolates. Further genetic analysis of substitutions V404I and V509M is required to determine their significance to the azole drug resistance phenotype. The third newly described substitution, F449V, was found in a fluconazole-SDD vaginal isolate (isolate 8). A similar substitution, F449L, has, however, been reported elsewhere.14
A comparison of trailing isolates of C. albicans with non-trailing isolates with reduced fluconazole susceptibilities showed that the trailers possessed a higher number, on average, of ERG11 nucleotide substitutions than the less susceptible isolates (10.7 versus 6.0, respectively) and that a larger proportion of these nucleotide substitutions were heterozygous (41% versus 13%, respectively). The observation that all of the ERG11 amino acid substitutions from isolates with reduced azole susceptibility phenotypes were homozygous supports the conclusion of a previous report that a mutation occurring on one allele may not appear as a phenotype unless it becomes dominant.15 In diploid cells, mutations usually occur randomly on each allele and result in heterozygosity. It is generally considered that meiosis, mating, frequent mitotic recombination and gene conversion are included in the formation of a homozygote for an altered gene. Mitotic recombination or gene conversion is a more likely explanation for ERG11 homozygosity in fluconazole-resistant C. albicans isolates. However, sexual generation may not be completely excluded.25,26 These results may suggest that as trailing isolates accumulate homozygous nucleotide changes leading to amino acid substitutions within critical sites of the lanosterol demethylase enzyme, susceptibility of the enzyme to azole agents decreases.
Increased mRNA levels for C. albicans ERG11, MDR1 and CDR genes have been shown previously to be associated with azole resistance.7,16,17,2730 In this study, ERG11 expression was variable among each isolate in the absence of fluconazole pressure. Although the number of fluconazole-resistant and -SDD isolates tested was limited, our data imply that, in the absence of fluconazole, constitutive ERG11 over-expression is not correlated with azole resistance. Other reports have also shown that ERG11 expression, in the absence of fluconazole, is variable and appears to have no correlation with resistance.16,27 This finding is logical in the sense that it is metabolically costly for an organism to over-express ERG11 in the absence of fluconazole pressure since the enzyme, lanosterol demethylase, is believed to be involved solely in ergosterol biosynthesis. Furthermore, over-expression of one sterol biosynthetic gene in a pathway that includes multiple genes would disrupt the flow of intermediates through the pathway and the overall regulation of ergosterol biosynthesis and storage.31 Therefore, it is appropriate to analyse ERG11 expression in the presence of fluconazole as well as in its absence.
When we studied whether trailing and less azole-susceptible isolates regulated ERG11 differently in response to fluconazole exposure, the results indicated that this was the case. Trailing isolates expressed more ERG11 in the absence of drug than did fluconazole-SDD or -resistant isolates. This may be because acquisition of mutations that give an organism a selective advantage in the presence of drug comes at a cost which could take the form of decreased fitness or decreased virulence.32 In the presence of low and intermediate concentrations of fluconazole, both groups of isolates were capable of ERG11 up-regulation. However, at high drug concentrations only the fluconazole-SDD and -resistant isolates maintained peak levels of ERG11 expression. With regard to the drug efflux genes, CDR1 and MDR1 expression patterns differed between the fluconazole-SDD and -resistant isolates as well as between the trailing and less susceptible isolates. These results support the hypothesis that azole drug resistance mechanisms evolve independently of each other and can be present alone or in combination in any given C. albicans isolate.17 A limitation of this study was that sequential isolates from individual cases were unavailable, and it was not therefore possible to determine if the evolution towards fluconazole resistance corresponded to increased constitutive expression of the drug efflux genes and/or ERG11 or the ability to up-regulate gene expression following drug exposure.
Smith & Edlind5 found that regulated expression of ERG11, CDR1, CDR2 and MDR1 contributed to azole trailing in C. albicans isolates. Interference with transcriptional regulation, by addition of the histone deacetylase inhibitor, trichostatin A, eliminated trailing growth and reduced ERG11, CDR1 and CDR2 up-regulation by 50%100%. Interestingly, the addition of trichostatin A had relatively little effect on the itraconazole MIC for two fluconazole-resistant C. albicans isolates. Other reports have shown that fluconazole-susceptible isolates without trailing growth up-regulated ERG11 in response to fluconazole exposure but did not up-regulate CDR1 or MDR1.18,28 In these experiments, investigators exposed cells to lower fluconazole concentrations (0.15 mg/L) compared with this work. The ability of these susceptible, non-trailing isolates to maintain increased levels of ERG11 expression at higher fluconazole concentrations is unknown. Taken together, our data and those of others imply that trailing and resistant or SDD isolates utilize known drug resistance genes, ERG11, CDR and MDR1, to yield their respective susceptibility phenotypes, but possess distinct mechanisms to regulate these genes in response to drug exposure.5,18,28 These recent findings emphasize the need to continue studying C. albicans isolates at the molecular level to understand the role that susceptible isolates with trailing growth may play in recurrent disease and/or the emergence of azole drug resistance following exposure to azole antifungals.
In conclusion, this study has shown that susceptible isolates of C. albicans with trailing growth possess more heterozygous ERG11 mutations and show different patterns of ERG11, CDR1 and MDR1 expression than non-trailing isolates with reduced fluconazole susceptibilities. Furthermore, trailing isolates possess a number of nucleotide substitutions that fall within the recently defined hot spots of the ERG11 amino acid sequence. In addition, fluconazole-SDD and -resistant bloodstream isolates of C. albicans possess ERG11 amino acid substitutions that have not been described previously. Finally, similar to fluconazole-SDD and -resistant isolates of C. albicans, ERG11, CDR1 and MDR1 up-regulation occurred in trailing growth isolates in response to fluconazole, but the pattern of expression was distinct.
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Acknowledgements |
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Footnotes |
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* Corresponding author. Tel: +1-404-639-4041; Fax: +1-404-639-3546; E-mail: BSkaggs{at}cdc.gov
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