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editorial
. 2001 Dec;45(12):3674–3676. doi: 10.1128/AAC.45.12.3674-3676.2001

Identification of Azole-Responsive Genes by Microarray Technology: Why Are We Missing the Efflux Transporter Genes?

D P Kontoyiannis 1,*, Gregory S May 1
PMCID: PMC90898  PMID: 11724035

We read with interest the recent important study by Backer et al. (2). The authors reported for the first time the genome-wide gene expression pattern in Candida albicans in response to subinhibitory concentrations of an azole (itraconazole). The duration of exposure to itraconazole in the study was 24 h. There are some remarkable similarities between the findings of that study and one previously published in this journal in which, for the first time, a genome-wide gene expression profile in response to (subinhibitory) concentrations of azoles was reported in the model yeast Saccharomyces cerevisiae (1). Even though Bammert and Fostel (1) used different experimental conditions characterized by a shorter duration of exposure to azoles (only 90 min), both studies had the following in common: (i) a convergent pattern of upregulation of various ergosterol biosynthetic genes; (ii) upregulation of genes involved in a variety of cell functions, such as stress response, the cell cycle, and protein synthesis; and most remarkably, (iii) the absence of significant upregulation of the efflux transporters. Upregulation of efflux transporters in response to azoles has been shown in both laboratory (4) and clinical (7) C. albicans isolates and in S. cerevisiae (6).

The lack of discovery of transporters as azole-responsive genes using the microarray technology could imply that critical elements of the study conditions (such as the concentration of the drug and exposure time) or the microarray preparation, hybridization, and scanning may not have been fully representative of the real-time transporter-inducing conditions. Northern blot analysis using probes of the transporter genes (CaMDR1, CDR1, and CDR2 for C. albicans and PDR5 for S. cerevisiae) could be helpful in testing whether the study conditions used in the microarray method upregulate these genes. Additional controls could also be of help in interpreting the specificity and validity of all of these gene catalogues. For example, Northern blot analysis using probes of the genes found to be upregulated under those specific experimental conditions using microarray technology could be an important backup mechanism (3). Also, performing the experiments multiple times could be of importance with an assay that is used to monitor multiple cellular functions in parallel. For example, subtle variations in gene expression profiles in a pattern unrelated to the condition tested have been shown (5). Lastly, it would be helpful if the specificity of the genome-wide upregulation of a battery of genes in response to the drug of interest is contrasted with nonspecific upregulation of genes in response to the presence of nonspecific growth inhibitors (e.g., oxidizing agents).

Unquestionably, this approach is the most vigorous and promising for the future. However, further work is needed to define the most relevant experimental conditions for evaluating the validity of the data provided using microarray technology.

REFERENCES

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Antimicrob Agents Chemother. 2001 Dec;45(12):3674–3676. doi: 10.1128/AAC.45.12.3674-3676.2001

AUTHORS' REPLY

Marianne D De Backer 1,2, Walter H M L Luyten 1,2, Hugo Vanden Bossche 1,2

We appreciate the interest of Kontoyiannis and May (K&M) in our recently published study on itraconazole-responsive genes in C. albicans. K&M are correct in pointing out that neither our DNA microarray study nor the previous one by Bammert and Fostel on S. cerevisiae showed an upregulation of the drug transporter genes MDR1 (CaMDR1), CDR1, and CDR2 in response to azole treatment, whereas others have reported such up-regulation using different techniques. They are concerned that this casts doubt on the validity of the microarray findings.

The studies cited by K&M as showing induction of drug transporter expression differ in several important aspects from ours. One (reference 1-6 in the Letter) used S. cerevisiae, which is in many aspects quite different from C. albicans. In addition that study used a subinhibitory concentration of fluconazole, whereas we used a high (10 μM) (not subinhibitory, as K&M state) concentration of itraconazole. Moreover, in that study Kontoyiannis did not investigate the mRNA level of drug transporters but the enzymatic activity of a LacZ fusion construct generated by transposon mutagenesis. It is conceivable that the transposon insertion altered the regulation of the disrupted drug transporter gene; the author did not verify this by the appropriate Northern blots. Hernaez et al. (reference 1-4 in the Letter) studied C. albicans, but also using an indirect assay of a reporter gene (a green fluorescent protein variant) fused to the CDR1 multidrug transporter. The same caveat obtains: this fusion construct may have an altered expression compared to the wild-type CDR1. In their genetically engineered strain they showed a dose-dependent increase in fluorescence by fluconazole, as well as (lesser) increases by other azoles, but they did not test itraconazole. Sanglard et al. (reference 1-7 in the Letter) studied clinical isolates of C. albicans from patients who had become resistant to fluconazole treatment after many weeks or even months of therapy. Many (but not all) of these isolates had developed cross-resistance to itraconazole. The fluconazole resistance phenotype was both acquired and stable, pointing to a genetic alteration and not a reversible induction of gene expression by the azoles. The authors indeed propose that the reason for increased mRNA levels of CDR1 or MDR1 may be gene amplification, promoter mutations, or mutations leading to increased mRNA stability. Moreover, they point out that in contrast to fluconazole, itraconazole and ketoconazole are not substrates for MDR1. Therefore, itraconazole (or ketoconazole) is not expected to exert a selective pressure favoring mutants overexpressing MDR1.

Krishnamurthy et al. (1-5) demonstrated using Northern blots that CDR1 mRNA is induced by a 60-min exposure to various agents (including miconazole, nystatin, and vinblastin) but also to heat shock and some human steroid hormones. Induction of CDR1 expression by fluconazole was very modest, and itraconazole was not tested. The inducing effect of some compounds (like cycloheximide and β-estradiol) appears transient. These authors also showed that (in the absence of any drug) CDR1 expression levels increase significantly during the early logarithmic growth phase, decrease in the mid-exponential phase, and increase once more during the late exponential and stationary phase. In a subsequent publication the same group (1-7) dissected the regulatory domains of the CDR1 gene responsible for transcriptional induction by azoles. Multiple positive as well as negative cis-regulatory regions were identified in the promoter region of CDR1, one of which seemed particularly important for induction of miconazole (but not for induction by, e.g., steroid hormones) (itraconazole was not tested).

In short, we are not convinced that there is at this stage a discrepancy to be explained, especially given the differences in strains (or even species), growth conditions, treatment regimens, compounds tested, etcetera, between the various published reports.

Our study is just a one-time snapshot of a response in one Candida strain treated with one specific drug at a single dose. Despite this, the expression profile clearly allowed us to surmise the mechanism of action of the compound used: more than 15 genes involved in ergosterol biosynthesis were clearly up-regulated in response to a drug, itraconazole, known to target this pathway specifically. There is no doubt, however, that some responses could and presumably have been missed if one limits oneself to just one strain, one treatment, and one time point. More in-depth analysis would involve collection of cells after different incubation times upon treatment with different concentrations of drug. In addition, different azoles as well as different C. albicans strains (both azole-sensitive and azole-resistant ones) would have to be tested.

K&M suggest that we perform Northern blot experiments using probes of the transporter genes found to be up-regulated by others, but under our experimental conditions. All experimental methods have their shortcomings, including Northern blots, and we have no reason to believe that they are somehow more accurate or trustworthy than DNA microarrays for quantitation of gene expression (where quantitative PCR most likely has an edge). Clearly, the great advantage of DNA microarrays is that they allow one to monitor the expression of an entire genome in parallel. The technique has proven trustworthy (selected examples are references 1-1, 1-2, 1-4, 1-6, 1-8, and 1-9), and even Galitski et al., who were cited, found that “effects observed in microarray experiments were in excellent agreement with quantitative phosphorimager analysis of Northern blots.” It is neither feasible nor logical to go back and check by Northern blot analysis the expression levels of every single gene that was not up-regulated in our study to make sure that one has not missed any changes. Bammert and Fostel have verified by quantitative PCR a number of the changes in mRNA levels they observed using DNA microarrays and found the correlation between the two methods to be excellent, as have many others. Carrying out identical experiments multiple times will undoubtedly increase the confidence level with which one can detect modest changes, which is why we use a conservative cutoff of a larger than 2.5-fold change to avoid being confounded by small random fluctuations. Moreover, for some genes multiple cDNA fragments are present on the DNA microarray, which provides a nice internal control. In our study CDR1 was 1.6-fold down-regulated, CDR2 was present twice on the microarray and was down-regulated 1.9 and 2.0-fold, CDR3 was not present on the microarray, CDR4 was present in four copies (−2.3, −2.6, −2.8, −2.7), and MDR1 was not present on the microarray.

K&M are concerned that our experimental conditions are not “fully representative of the real-time transporter-inducing conditions.” Clearly, all in vitro studies (including those reported in, e.g., references 1-4, 1-5, and 1-7 as well as the studies by Hernaez et al., Kontoyiannis, and our group cited by K&M) are artificial compared to the clinical situation, but we see no reason to consider any of these in vitro conditions to be more representative than the others. In clinical isolates of C. albicans increased mRNA levels for ERG11, MDR1, or CDR genes have been causally implicated in resistance to azoles after long-term treatment. It is clear that neither we nor Bammert and Fostel have observed up-regulation of these efflux drug transporters in response to much shorter and in vitro exposure to azoles (more specifically itraconazole in our study and various other azoles in the case of Bammert and Fostel). It seems best to take these data at face value. Moreover, the mechanisms involved in up-regulation are almost certainly completely different. The clinical isolates have undergone stable genetic changes leading to overexpression of one or more efflux transporter genes; chronic selective pressure by azole treatment permits these strains to survive. Mechanistically, this is totally different from the transient and reversible increase in the expression of the same efflux transporters upon acute exposure to azole antifungals, which is most likely due to transcriptional activation by transcription factors such as AP-1 (1-7). The fact that different efflux transporters are up-regulated in different resistant isolates is clear evidence that such up-regulation is by no means a universal response to azoles. Differences with other studies that used acute exposure to antifungals may also be due to the type of azole used; Dimster-Denk et al. (1-3), for instance, reported very different effects on gene expression dependent on the type of azole that was used against S. cerevisiae. Secondly, we doubt that, e.g., itraconazole-induced CDR1 and/or CDR2 up-regulation would be found in every C. albicans strain even under real-time transporter-inducing conditions. If this were the case, then all C. albicans strains would become itraconazole resistant, which is (fortunately) not the case. There is no doubt that efflux transporters play a critical role in the clinical resistance to, e.g., fluconazole, but even under relevant potentially transporter-inducing conditions one cannot expect to find CDR1 and/or CDR2 upregulation in every strain or isolate tested. Maybe one should not even expect this up-regulation to be apparent in the once-treated, fully azole-susceptible laboratory strain used in our study.

In conclusion, we agree with K&M that further work is needed, not however to validate DNA microarrays as a technique, but to use them for exploring in greater detail the effects of antifungals on gene expression under a variety of conditions.

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