MICROBIAL DRUG RESISTANCE Volume 11, Number 3, 2005 Mary Ann Liebert, Inc.
Establishing Surrogate Markers for Fluconazole
ABSTRACT Azole-resistant Candida can be a confounding factor for clinical management of opportunistic infections in immunocompromised patients, but rapid identification of such resistant organisms can improve patient out- come. New target-based molecular diagnostic strategies have the potential to identify resistant organisms faster than current culture-based assays. It was the objective of this study to determine whether target site muta- tions and/or drug pump over-expression are suitable surrogate markers of drug resistance that could aid new molecular-based diagnostic assays. A collection of 59 clinical isolates displaying a range of azole susceptibili- ties were assayed for mutations within the target gene Erg11 and for over-expression of drug-efflux pumps Cdr1, Cdr2, Flu1, and Mdr1, as well as drug target gene Erg11 by quantitative real-time PCR with molecu- lar beacons. A fluconazole-resistant (MIC Ն 64 g/ml) phenotype was closely associated with over-expression of Cdr1 (p ؍ 0.005), Cdr2 (p ؍ 0.01), and Mdr1 (p ؍ 0.03) along with four mutations in Erg11 (T229A, Y132F, S405F, G464S). Changes in expression levels for Erg11 and Flu1 were not statistically correlated with resis- tance (p ؍ 0.27 and p ؍ 0.86, respectively). Overall, these findings provide a statistical basis to establish Erg11 mutations and drug pump over-expression as surrogate markers for phenotypic fluconazole resistance. INTRODUCTION
dients as driving forces and have more limited substrate speci-ficity. In C. albicans, the ABC transporter genes Cdr1 and Cdr2TRIAZOLE ANTIFUNGAL AGENTS (e.g., fluconazole, itracona- and MFS gene Mdr1 have been linked to clinical drug resis-
zole, etc.) are commonly used to treat Candida infections
tance.4–6,11,16–18,20–22 A novel MFS transporter gene Flu1 has
because of their high therapeutic index. Resistance to these
been identified in C. albicans, but its contribution to clinical
agents, which were introduced in the early 1990s, is most no-
table among immunosuppressed patients.5,13 The molecular
In an effort to identify potential surrogate markers for clin-
mechanisms of triazole resistance in C. albicans have been well
ical drug resistance, the genes responsible for diverse resistance
characterized in recent years and include alteration of the tria-
mechanisms were evaluated in a collection of clinical isolates
zole drug target enzyme, lanosterol 14␣-demethylase encoded
displaying fluconazole susceptible (S), susceptible dose-depen-
by Erg11, and reduction in cellular drug accumulation.5,6,17,21,22
dent (SDD), and resistant (R) in vitro susceptibility phenotypes
Alterations in Erg11p include mutations that result in reduced
by DNA sequencing and molecular beacon-based transcrip-
affinity to triazole antifungal drugs, as well as those that lead
to over-expression of Erg11, which diminishes drug efficacyvia target titration.4–6,8,15,16,22 A reduction of cellular drug ac-cumulation has also been linked to over-expression of multidrug
MATERIALS AND METHODS
resistance (MDR) efflux transporter genes of the ATP-bindingcassette (ABC) and the major facilitator superfamily (MFS)
Strains and susceptibility testing
classes.4–6,11,14,16–18,20–22 ABC transporters require energy inthe form of ATP and can expel a multitude of substrates in con-
Candida albicans strains used in this study include a col-
trast to MFS transporters that use electrochemical proton gra-
lection of 59 fluconazole-susceptible (MIC Յ 8.0 g/ml) (n ϭ
Public Health Research Institute, Newark, NJ 07103. MARKERS FOR FLUCONAZOLE RESISTANCE IN C. albicans
14), susceptible dose-dependent (MIC 16–32 g/ml) (n ϭ 13),
coding region was performed using the DTCS Quick Start kit
and resistant clinical isolates (MIC Ն 64 g/ml) (n ϭ 32) as
(Beckman Coulter, Fullerton, CA) and a CEQ 2000 XL capil-
determined by the microdilution antifungal susceptibility test-
lary electrophoresis DNA sequencer (Beckman Coulter).
ing method outlined by the National Committee for ClinicalLaboratory Standards (NCCLS) document M27-A.9 These
Primer and molecular beacon design for
strains were obtained from separate patients from diverse geo-
graphic regions and were not epidemiologically linked. Flu-
PCR primers and molecular beacon probes were designed
conazole was obtained from Pfizer (New York).
to amplify and identify C. albicans genes Cdr1, Cdr2, Erg11,Flu1, Mdr1, and Pma1 as described by the molecular beacons
synthesis protocol (http//:www.molecular-beacons.org). Each
Candida albicans strains were grown in YPD medium
molecular beacon was labeled with tetrachloro-6-carboxylflu-
(Difco, Franklin Lakes, NJ) at 30°C until mid-log phase growth.
orescein (TET) (BioSearch Technologies, Inc., Novato, CA).
Total DNA-free RNA was isolated using the RNeasy Minikit
Primer and molecular beacon probe sequences are listed in
(Qiagen, Valencia, Calif.). For DNA extraction, an overnight
Table 1. For each probe, a linear relationship was observed
culture grown at 30°C was used and total genomic DNA was
between the cycle threshold (Ct) value and the logarithm of
extracted using the Wizard genomic DNA purification kit
known amount of initial template molecules (not shown). Fur-
thermore, each molecular beacon was shown to be specific toits target and did not show any cross-interaction or nonspe-
PCR and sequencing primers were designed to the coding
region of Erg11 (GenBank accession number X13296) usingOligo 4.04 primer design software (Molecular Biology Insights,
First-strand cDNA was synthesized using Omniscript RT
Inc., Cascade, CO). Primers used for PCR (5Ј-3Ј) were F52
PCR Kit (Qiagen) and 1 g of total RNA. Samples incubated
(GACAAAGAAAGGGAATTCAATCG) and R1763 (CACT-
in the absence of reverse transcriptase were used as controls.
GAATCGAAAGAAAGTTGCCG). PCR amplification was
Quantitative real-time PCR mixtures contained 0.2 M of each
carried out at 94°C for 1 min followed by 30 cycles of 94°C
molecular beacon, 10 pmol of each primer, 2 U of AmpliTaq
for 30 sec, 55°C for 30 sec, and 72°C for 2 min and a final ex-
Gold DNA polymerase (Applied Biosystems, Foster City, CA),
tension step of 72°C for 5 min. Primers used for DNA se-
0.25 mM dNTPs, 4 mM MgCl2, 1ϫ PCR buffer, and 250 ng
quencing included F478 (GATGTTTCTGCTGAAGATGC),
of cDNA. The thermal cycling program consisted of 10 min on
F947 (GTGGTGATATTGATCCAAATCG), F1363 (CCAG-
a spectrofluorometric thermal cycler (Applied Biosystems 7700
GTTATGCTCATACTAG), R498 (GCATCTTCAGCAGAA-
Prism) at 95°C followed by 40 cycles of 20 sec at 95°C, 30 sec
ACATC), R968 (GATTTGGATCAATATCACCAC), R1382
at 50°C, and 30 sec at 72°C, and real-time fluorescence was
(CTAGTATGAGCATAACCTGG), and the PCR primers as
monitored during the reaction and plotted against the number
described above. Automated DNA sequencing of the Erg11
of thermal cycles. All quantitative real-time PCR assays were
PRIMERS AND MOLECULAR BEACON DESIGN FOR QUANTITATIVE REAL-TIME PCR
aConstitutively expressed internal control gene.
SUMMARY OF CANDIDA albicans STRAINS USED IN THIS STUDY
FLU resistance gene over-expressionc
aOP, Oro-pharynx. bS, Susceptible (MIC Յ 8.0 g/ml); SDD, susceptible dose dependent (MIC 16–32 g/ml); R, resistant (MIC Ն 64 g/ml).
cOver-expression is defined as Ͼ 3 times the average FLU-susceptible strains values.
dRelative to GenBank Accession number X13296. MARKERS FOR FLUCONAZOLE RESISTANCE IN C. albicans
performed in triplicate and the essential gene Pma1 was used
ent in fluconazole-susceptible strains (Table 2). The mutation
as a highly-expressed internal normalization marker.
S405F was only found in SDD and resistant strains, while othermutations also occurred in strains with SDD phenotypes (Table
2). Nine isolates had multiple amino acid mutations in Erg11.
Association of individual mdr gene expression levels for
Cdr1, Cdr2, Mdr1, Erg11, and Flu1 relative to azole-suscepti-
Quantitative over-expression of Erg11 and
ble, susceptible dose-dependent and resistant phentoypes was
determined by one factor analysis of variance (ANOVA). p val-
Molecular beacon probes were used to quantify the level of
ues less than 0.05 were considered significant.
expression of azole target gene Erg11, and mdr pumps Cdr1,Cdr2, Flu1, and Mdr1. The 14 azole-susceptible strains wereused to establish baseline values of transcripts for expressionof the known resistance genes Cdr1, Cdr2, Erg11, Flu1, and
Mdr1. As expected, a majority of the susceptible strains dis-played low levels of expressed mdr genes, which remained
closely grouped as shown in the scatter plot diagram (Fig. 1).
An epidemiologically diverse collection of 59 clinical iso-
The susceptible dose-dependent isolates similarly displayed
lates of C. albicans encompassing fluconazole susceptible,
low-level expression of mdr genes, with the exception of Mdr1,
dose-dependent, and resistant phenotypes were evaluated by
which revealed moderate to high increases (2- to 10-fold) of
DNA sequence analysis of Erg11. Numerous mutations rela-
transcript. The azole-resistant isolates showed a 2- to 45-fold
tive to the published Erg11 sequence (GenBank accession num-
increase in Mdr1 transcript levels when compared to the sus-
ber X13296) were identified resulting in 15 different amino acid
ceptible and susceptible dose-dependent isolates. For Cdr1, all
substitutions, as follows: D116E, F126L, K128T, Y132F,
of the susceptible dose-dependent isolates had similar expres-
K143R, T229A, E266D, S405F, V437I, D446N, F449S,
sion profiles as the susceptible. However, Cdr1 over-expres-
G450E, G464S, R467K, and V488I (Table 2). The mutations
sion was found in ϳ34% (11/32) of the total R isolates tested
ranged in frequency from 2.2 to 8.8% in the 45 R or SDD strains
with 2- to 15-fold increases in transcript levels. A majority of
(Table 3) and clustered within four mutational “hot-spot” re-
the isolates also co-expressed Cdr2 (n ϭ 11), supporting the
gions previously identified, which were located between amino
previous notion that Cdr1 and Cdr2 are co-regulated.21 Over-
acid residues 72 to 467.8,12 However, six amino acid mutations,
all, the frequency of gene over-expression for Cdr1, Cdr2, and
D116E, K128T, G450E, V437I, D446N, and F449S, were not
Mdr1 was 24.4%, 24.4%, and 33.3%, respectively, for all R and
linked to triazole resistance as they were also found to be pres-
SDD isolates (n ϭ 45) examined (Table 3). Gene over-expres-
PREVALENCE AND ASSOCIATION OF SPECIFIC GENETIC MECHANISMS WITH RESISTANCE
aANOVA analyses were performed on the absolute expression levels values of the mdr genes. bOver-expression is defined as Ͼ3 times the average FLU susceptible strains values.
cND, Not determined because population size was too limited for statistical significance. PARK AND PERLIN
Relative expression levels of mdr genes. Quantitative real-time PCR with molecular beacons of known mdr genes was
performed on cDNA transcripts of a panel of C. albicans isolates (n ϭ 59). The collection included 14 azole-susceptible (ٗ), 13azole-susceptible dose-dependent (X), and 32 resistant (᭝) isolates. Variations in the initial amount of mRNA among the dif-ferent samples were normalized to the level of Pma1 mRNA, yielding a ratio between the specific mdr gene cDNA copy num-ber and Pma1 cDNA copy number.
sion was constitutively expressed in the strains, because there
level resistance largely results from incremental susceptibility
was no evidence for significant changes in expression due to
shifts mediated by resistance mechanisms induced in response
drug exposure. Erg11 over-expression was found in ϳ12% of
to short- or long-term drug exposure.4,7,21 Nevertheless, there
the resistant isolates tested (4/32) whereas Flu1 over-expres-
are subsets of the various resistance mechanisms that are more
sion was found in one isolate, but neither Erg11 nor Flu1 over-
frequently associated with in vitro resistance and therefore have
expression were statistically linked to clinical drug resistance
the potential to serve as diagnostic surrogates. Advances in mol-
(p ϭ 0.27 and p ϭ 0.87, for Erg11 and Flu1, respectively)
ecular diagnostic approaches have made it possible to rapidly
evaluate genetic changes (allele discrimination and gene over-
Five azole-susceptible dose-dependent strains had single
expression) in an organism that result in a prominent pheno-
mechanisms of resistance in contrast to 20 triazole-resistant iso-
type, such as drug resistance.1,3,10 Most recently, it was shown
lates that displayed multiple mechanisms of resistance (ϳ62%).
that gene over-expression of multiple resistance targets could
A majority of strains showed multiple types (target site or drug
be profiled in a simple real-time assay,3 and at least seven dif-
efflux pump) of resistant mechanisms (Table 2). Of 10 isolates
ferent alleles associated with itraconazole resistance in As-
with a single resistance mechanism, five had target-based mu-
pergillus fumigatus could be readily detected in a single mul-
tations and five showed efflux pump over-expression. Of the
strains with target site mutations linked to resistance, a major-
In this report, the most commonly associated resistance
ity also showed over-expression of mdr pumps.
mechanisms include Erg11 target site mutations T229A,Y132F, K143R, E266D, S405F, G464S, and V488I (Table 2)and over-expression of drug efflux transporter genes Cdr1,
DISCUSSION Cdr2, and Mdr1, above a baseline threshold value of 3-foldgreater than the average expression in susceptible strains (Fig.
It is now well established that traizole resistance in C. albi-
1). In each case, the manifestation of either a given Erg11 mu-
cans is multifactorial involving several different resistance
tation and/or gene over-expression was correlated with resis-
mechanisms operating individually or collectively in a given
tance. All the Erg11 mutations have been identified previ-
strain.4,7,11,16,21,22 Such complexity implies that no one resis-
ously.4,6–8,15,16 Y132F is one of the most frequent Erg11
tance mechanism plays a dominant role. Furthermore, high-
mutations encountered (Table 3). This residue is believed to re-
MARKERS FOR FLUCONAZOLE RESISTANCE IN C. albicans
side close to the access channel for azoles in the demethylase
Overall, Ͼ90% of the 32 resistant strains could be accounted
enzyme, which may alter entry of the inhibitor to its binding
for by target site mutations and/or up-regulation of drug efflux
site.8,12 Mutation T229A was found unlinked to other resistance
transporters. Only three strains, 31, 32, and 57 (containing sta-
mechanisms. It is believed to reside on the F helix, which may
tistically unlinked D116E, E266D mutations) had unknown
contribute dynamically to the access channel for drug. Al-
mechanisms. These results suggest that existing mechanisms
though, independent confirmation of the contribution of this
provide excellent coverage for non-culture-based assays of flu-
mutation to resistance has not been reported. Amino acid sub-
conazole resistance. As new targets emerge and are validated,
stitutions involving E266D and V488I mutation are not likely
they can be incorporated into the new generation of nucleic acid
to be important for high-level resistance because they have been
amplification systems with self-reporting probes capable of al-
observed in susceptible strains.8 Residue G464, which is highly
lele discrimination and quantitative assessment of target num-
conserved across all known species, appears to lie directly be-
hind the plane of the heme prosthetic. The G464S mutation mayinterfere with coordination of the azole molecule. Finally, theS405F substitution lies immediately beyond the K helix near
the substrate- and azole-binding pocket.8 The dynamic natureof the demethylase enzymes helps account for the numerous
The authors acknowledge the following groups for provid-
mutations that can confer varying levels of resistance and may
ing clinical isolates: Phyllis Della-Latta (Columbia-Presbyter-
result in either resistant or intermediate-level resistance such as
ian Medical Center, New York), Brahm H. Segal (Roswell Park
S405F or can result in common mutations such as D116E,
Cancer Institute, New York), Theodore C. White (Seattle Bio-
which have a weak or no apparent phenotype. Mutations F126L
medical Research Institute). Joachim Morschhäuser (Univer-
and K143R are often found together along with up-regulation
sität Würzburg, Germany), Frank Odds (University of Ab-
of an efflux transporter, suggesting that their effect is weak.
erdeen, UK), Spencer Redding, Jose Lopez-Ribot, and Thomas
Overall, it appears that mutations T229A, Y132F, S405F, and
F. Patterson (University of Texas Health Science Center at San
G464S are sufficient to confer fluconazole resistance. Their fre-
Antonio, Texas). The authors are also grateful to Salvatore
quency in the clinical resistant population is difficult to assess
A. E. Marras and Barun Mathema for helpful discussions of
because the sample size in this study was too small to deduce
molecular beacon design and statistical analyses.
statistic significance. Nevertheless, these alleles are likely to bereliable indicators that could serve as useful clinical markers inscreening for triazole resistance.
The over-expression of multidrug efflux transporters repre-
sents the second major mechanism associated with resis-tance.11,16,17,20–22 The C. albicans genome contains in excess
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The discovery of ribonucleic acid interference has sparked a wave of enthusiasm in the scientific community. Since the discovery of RNA interference scientists have used it to identify the molecular processes of different diseases. RNA interference has the potential to be used in a wide range of applications one of which is cancer therapies. The unique key to RNA interference as it does have an
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