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Antimicrobial Agents and Chemotherapy, December 2001, p. 3674-3676, Vol. 45, No. 12
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.12.3674-3676.2001
LETTERS TO THE EDITOR
Identification of Azole-Responsive Genes by Microarray
Technology: Why Are We Missing the Efflux Transporter Genes?
 |
LETTER |
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.
| |
FOOTNOTES |
*
Phone: (713) 792-6237 Fax: (713)
745-6839 E-mail: dkontoyi{at}mdanderson.org
| |
REFERENCES |
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Bammert, G. F., and J. M. Fostel.
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Genome-wide expression patterns in Saccharomyces cerevisiae: comparison of drug treatments and genetic alterations affecting biosynthesis of ergosterol.
Antimicrob. Agents Chemother.
44:1225-1263.
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De Backer, M.,
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S. Vandoninck,
W. Luyten, and H. Vanden Bossche.
2001.
Genomic profiling of the response of Candida albicans to itraconazole treatment using a DNA microarray.
Antimicrob. Agents Chemother.
45:1660-1670[Abstract/Free Full Text].
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Kontoyiannis, D. P.
1999.
Genetic analysis of azole resistance by transposon mutagenesis in Saccharomyces cerevisiae.
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43:2731-2735[Abstract/Free Full Text].
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Sanglard, D.,
K. Kuchler,
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M. Monod, and J. Bille.
1995.
Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters.
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39:2378-2386[Abstract].
|
| | | | |
D. P. Kontoyiannis*
Gregory S. May
Department of Infectious Diseases
M. D. Anderson Cancer Center
University of Texas
1515 Holcombe Blvd., Box 402
Houston, Texas 77030
|
| |
AUTHORS' REPLY |
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 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 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 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. (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 
-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 (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, 2, 4, 6, 8, and
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 4, 5, and 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
(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.
(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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| | | | |
Marianne D. De Backer
Walter H. M. L. Luyten
Janssen Research Foundation
B-2340 Beerse, Belgium
|
| | | | |
Hugo Vanden Bossche
Turnhout, Belgium
|
Antimicrobial Agents and Chemotherapy, December 2001, p. 3674-3676, Vol. 45, No. 12
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.12.3674-3676.2001
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