Transcriptional Analyses of Antifungal Drug Resistance in Candida Albicans

Transcriptional Analyses of Antifungal Drug Resistance in Candida albicans

CHRIS N. LYONS AND THEODORE C. WHITE* Department of Pathobiology, School of Public Health and Community Medicine, University of Washington and the Seattle Biomedical Research Institute

Received 16 November 1999/Returned for modiﬁcation 22 March 2000/Accepted 7 June 2000

Oral infections with the pathogenic yeast Candida albicans are one of the most frequent and earliest opportunistic infections in human immunodeficiency virus-infected patients. The widespread use of azole antifungal drugs has led to the development of drug-resistant isolates. Several molecular mechanisms that contribute to drug resistance have been identified, including increased mRNA levels for two types of efflux pump genes: the ATP binding cassette transporter CDRs (CDR1 and CDR2) and the major facilitator MDR1. Using Northern blot analyses, the expression patterns of these genes have been determined during logarithmic and stationary phases of cell growth and during growth in different carbon sources in a set of matched susceptible and fluconazole-resistant isolates that have been characterized previously. MDR1, CDR1, and CDR2 are expressed early during logarithmic growth, CDR4 is expressed late during logarithmic growth, and CDR1 is preferentially expressed in stationary-phase cells. There is a small decrease in expression of these Downloaded from genes when the cells are grown in carbon sources other than glucose. While increased mRNA levels of efflux pump genes are commonly associated with azole resistance, the causes of increased mRNA levels have not yet been resolved. Southern blot analysis demonstrates that the increased mRNA levels in these isolates are not the result of gene amplification. Nuclear run-on assays show that MDR1 and CDR mRNAs are transcriptionally overexpressed in the resistant isolate, suggesting that the antifungal drug resistance in this series is associated with the promoter and trans-acting factors of the CDR1, CDR2, and MDR1 genes. aac.asm.org

Candida albicans is a pathogenic yeast that causes oral, vag- tor) have correlated with increased resistance. At least seven inal, and systemic infections (reviewed in reference 28). These CDR genes have been identified (CDR1 to CDR7)(C. albicans infections are usually treated with antifungal drugs, including information web page [http://alces.med.umn.edu/Candia.html]) the polyene amphotericin B and the azoles, such as flucon- although to date only CDR1 and CDR2 have been associated by on June 25, 2007 azole. Azole-resistant strains of C. albicans are an increasing with azole resistance (2, 7, 32, 33). A series of 17 isolates from problem in human immunodeficiency virus-infected patients a human immunodeficiency virus-infected patient has previ- and other immunosuppressed individuals (37). One recent ously been shown to exhibit many of the resistance mechanisms study estimates that up to a third of all AIDS patients retain an described above (35–38). Azole resistance developed gradually azole-resistant C. albicans isolate orally (17). Recently, there in this series. Several resistance mechanisms were identified in have been reports of azole-resistant Candida infections in the series. The timing of the occurrence of each of these other immunosuppressed patients (21, 22, 24, 27). Factors that resistance mechanisms correlated with an incremental increase contribute to the development of clinical resistance in patients in the MIC, a standard measure of the resistant phenotype of are numerous and include the extent of immunosuppression, the cells (25). the level of exposure to azole drugs, and intrinsic properties of The correlation between resistance and increased mRNA the fungus, including drug susceptibility (37). levels of efflux pumps and genetic alterations of ERG11 has Several molecular mechanisms that contribute to C. albicans been well-documented in several different C. albicans series. azole resistance have been identified (reviewed in reference These increases have usually been investigated during mid- 37). The fungistatic azoles, such as fluconazole, work by com- logarithmic growth of the culture in media containing glucose petitive inhibition of lanosterol demethylase, the product of (as in references 35 and 36). Recently, one study has demon- ERG11 and an important enzyme in the ergosterol biosynthetic strated changes in CDR1 mRNA levels during cell growth (15). pathway. Ergosterol is an essential component and the major Under standard growth conditions (i.e., 30°C in rich or mini- sterol of the fungal cell membrane. Alterations in this pathway mal medium with glucose as a carbon source), yeast cells such that contribute to resistance include point mutations and in- as Saccharomyces cerevisiae or C. albicans undergo several creased expression of ERG11 and possible genetic alterations phases of growth (reviewed in references 14 and 34). After an in other genes in the biosynthetic pathway for ergosterol. initial lag phase, the cells begin a rapid growth phase (loga- Azole resistance has also been correlated with increased export rithmic growth) in which glucose fermentation is the major of azoles from the cell, usually associated with the increased source of ATP production and cells divide exponentially. As expression of efflux pumps. Increased mRNA levels of the cells exhaust the glucose in the medium, they undergo a diauxic efflux pump gene family CDR (members of the ATP binding shift and begin preparation for the use of other carbon sources cassette transporter superfamily) and MDR1 (a major facilita- (e.g., ethanol). Finally, cell growth slows as the culture reaches stationary phase, in which cell growth arrests due to depletion * Corresponding author. Mailing address: Seattle Biomedical Re- of available carbon sources. search Institute, 4 Nickerson St., Suite 200, Seattle, WA 98109-1651. The mRNA levels of genes linked with azole resistance have Phone: (206) 284-8846, ext. 344. Fax: (206) 284-0313. E-mail: tedwhite not been defined throughout these phases of growth. The di- @u.washington.edu. auxic shift of C. albicans has only been investigated as it relates

2296 VOL. 44, 2000 ANALYSES OF ANTIFUNGAL DRUG RESISTANCE IN C. ALBICANS 2297

to mannitol catabolism (26). If gene expression associated with was prepared when the culture reached an OD600 of 6.4 and at 3 and 8 days of azole resistance is variable during particular growth phases, growth. (iii) Carbon source. Cells from 24-h stationary-phase YEPD cultures were this may have a large impact on azole susceptibility in the transferred to three different media: YEP (10 g of yeast extract, 20 g of peptone distinct growth environments of oral, vaginal, and systemic per liter)–2% glucose (equivalent to YEPD), YEP–3% glycerol, and YEP–3% candidiasis during both colonization and infection. sodium acetate at an initial concentration of 5 ϫ 106 cells/ml. The cultures were As mentioned above, increased mRNA levels of ERG11, grown at 30°C with agitation, and RNAs were prepared when the culture reached CDR1, and MDR1 have been correlated with azole resistance. an OD600 of approximately 1.0. Total RNA preparation, gel electrophoresis, Northern blotting, oligonucleo- However, it has not been determined by what mechanism(s) tide labeling with polynucleotide kinase, and random priming for radioactive these mRNAs are increased. Eukaryotic cells generally employ probe preparation were performed according to standard published methods (19, several techniques to increase mRNA levels. One common 31). method is gene ampliﬁcation, whereby a gene is copied several Nuclear run-on analysis. The nuclear run-on was performed using previously times. Normal transcription rates of each gene copy result in a described methods (5) with the following modiﬁcations. Cells were grown at 30°C in YEPD with agitation until the culture reached an OD600 of 1.0. An aliquot of greater total mRNA product. Alternatively, the transcription 3 ϫ 107 cells was mixed with a smaller volume of crushed ice in a prechilled of a gene can be increased by altering the levels of trans-acting round-bottom 15-ml polypropylene tube. The cells were centrifuged for 5 min at factors that interact with the gene promoter or by mutations in 4,000 ϫ g and resuspended in 5 ml of cold TMN (10 mM Tris, 100 mM NaCl, 5 ϫ the promoter. mRNA levels can also be increased by transcrib- mM MgCl2, pH 7.4). The cells were again centrifuged for 5 min at 4,000 g and ing a gene at a normal rate but increasing the half-life of the resuspended in 0.95 ml of ice-cold water. Fifty microliters of 10% N-lauroyl Ј sarcosine (Sigma) was added and the mixture was incubated at 4°C for 15 min to mRNA, generally accomplished by a mutation in the 3 end of permeabilize the cells. The cells were transferred to an Eppendorf tube and the gene that affects the degradation of the mRNA. Further, centrifuged at 4°C and 6,000 rpm for 2 min (Eppendorf centrifuge model 54 15C; nuclear export, 5Ј capping, polyadenylation, and RNA splicing Brinkman Instruments, Westbury, N.Y.). The cells were resuspended in 60 ␮lof can all affect mRNA levels. The standard method for detecting the following freshly made ice-cold reaction mixture: 50 mM Tris (pH 7.9), 100 mM KCl, 5 mM MgCl2, 1 mM MnCl2, 2 mM dithiothreitol, 0.5 mM rATP, 0.25 increased mRNA transcription is a nuclear run-on assay (8, 9, mM rGTP, 0.25 mM rCTP,1UofRNase inhibitor (Boehringer Mannheim,

23). In this assay, cells are permeabilized and radioactively Indianapolis, Ind.) per ␮l, 10 mM phosphocreatine, 1.2 ␮g of creatine phos- Downloaded from labeled UTP is added. The labeled UTP then enters the nu- phokinase per ␮l and 2 ␮Ci of UTP per ␮l (3,000 Ci/mM). The mixture was cleus, where polymerases that are actively transcribing ex- incubated at room temperature for 12 min. To stop the reaction, 5 ␮lofEsch- erichia coli tRNA (50 mg/ml for carrier RNA) (Boehringer Mannheim), 6 ␮lof pressed genes incorporate the labeled nucleotide into nascent ␮ 1 mM CaCl2, and 1 l of RQ1 DNase (Promega, Madison, Wis.) were added and RNA chains. Total RNA, which includes the labeled nascent the mixture was incubated at 37°C for 15 min. (Unlike in the published protocol, transcripts, is prepared and hybridized to specific gene probes. ␣-amanitin was not used to stop the reaction.) One hundred and thirty microli- The radioactive signal detected is a measure of the level of ters of RNA buffer (0.05 M Tris, 0.1 M EDTA, 0.1 M NaCl, pH 8.0), 10 ␮lof active transcription of the gene. This technique has been suc- 10% sodium dodecyl sulfate, and 4 ␮l of proteinase K (10 mg/ml) were added, aac.asm.org and the mixture was incubated at 37°C for 30 min. To prepare RNA, 200 ␮lof cessfully performed in a variety of eukaryotic cells, including S. buffered phenol (pH 8.0) and 200 ␮g of acid-washed glass beads (Sigma) were cerevisiae (3, 5), and we have adapted the procedure for use in added and the tube was vortexed for 5 min. The resulting slurry was centrifuged C. albicans. at 14,000 rpm for 5 min in the microcentrifuge. The aqueous phase was trans- ␮ In this study, levels of the ERG11, CDR, and MDR1 mRNAs ferred to a new tube on ice at 4°C. Another 200 l of RNA buffer was added to by on June 25, 2007 were determined in a susceptible isolate and a fluconazole- the remaining slurry, and the tube was again vortexed and centrifuged to extract any remaining RNA. The aqueous phases were pooled, and 400 ␮l of buffered resistant isolate from a single strain taken from an AIDS pa- phenol (pH 8.0) was added. The mixture was vortexed for 1 min and centrifuged tient. The levels of expression were monitored throughout the at 14,000 rpm for 5 min in the microcentrifuge. The aqueous phase was trans- course of cell growth (logarithmic, diauxic, and stationary) and ferred to a new tube, and the RNA was precipitated with 2.5 M NH4-acetate and during growth in different carbon sources. In addition, South- an equal volume of isopropanol. The mixture was stored overnight at Ϫ20°C. To ern blot analyses were used to rule out gene amplification of pellet the RNA, the mixture was centrifuged at 14,000 rpm for 10 min in the microcentrifuge. The isopropanol was removed, and the pellet was resuspended the efflux pumps and nuclear run-on analysis was used to de- in 0.75 ml of Trizol (GIBCO BRL, Grand Island, N.Y.). The total RNA was then termine if increased transcription is a cause of the increased prepared according to the manufacturer’s specifications. This double extraction mRNA levels of ERG11, CDR, and MDR1 observed in this of RNA was used to ensure that there was a minimum of DNA contamination. series. Gene fragments and gene probes. Plasmids containing gene inserts (all within the coding regions) of ERG11, CDR1, MDR1, and ACT1 were used as gene targets against the labeled nuclear run-on probe. The gene fragments were MATERIALS AND METHODS prepared by PCR amplification of a section of the coding region of the gene. The sections that were amplified and cloned were as follows (numbers represent Strains and growth of cultures. The C. albicans isolates used in this study positions in the GenBank sequences; GenBank accession numbers and refer- include a susceptible isolate (isolate 1, designated 2-76) and a resistant isolate ences are given in parentheses): ERG11: position 164 to 1589 (X13296 [16]), (isolate 17, designated 12-99) from a series of 17 oral isolates from a single AIDS MDR1: position 2885 to 3754 (X53823 [6]), CDR1: position 1210 to 2016 patient (30). Cultures were routinely inoculated from single colonies. The iso- (X77589 [29]), and ACT1: position 1714 to 2515 (X16377, [18]). ACT1 is the gene lates were grown at 30°C in YEPD (10 g of yeast extract, 20 g of peptone, and encoding the actin gene and is used as a control in most of the experiments in this 20 g of dextrose per liter) or on YEPD agar plates (10 g of yeast extract, 20 g of study. In addition to these gene fragments, a 1,045-bp PvuII fragment of the peptone, 20 g of dextrose, and 15 g of agar per liter), stored at 4°C, and pCR-Script Amp SK(ϩ) plasmid (position 2416 to 550 [including position 1]; subcultured weekly or stored at Ϫ80°C in YEPD containing 10% glycerol. MICs Stratagene, La Jolla, Calif.) was used to control for binding of labeled nuclear of fluconazole were determined using the NCCLS broth microdilution method (25). All reagents were purchased from Fisher Scientific (Pittsburgh, Pa.) or RNA to nonspecific DNA targets. These plasmids were digested so that the gene Sigma Chemical Co. (St. Louis, Mo.) unless otherwise specified. insert was separated from the vector DNA and electrophoresed on a 0.8% DNA extraction and Southern analysis. Genomic DNAs from the susceptible agarose gel at 80 V for 3 h. Southern blotting, hybridization, and washing were and resistant isolates were prepared as described (13). Restriction enzyme di- performed at 60°C using previously described methods (19, 31). gestions and Southern blot analyses were performed using standard techniques Gene fragments and oligonucleotides were used as probes for Northern blots. (19, 31). Oligonucleotides were prepared to be complementary to the mRNA for each of RNA manipulations for Northern analysis. (i) Logarithmic growth. Cells from the genes. The probe for ACT1 was a 50-mer, positions 2478 to 2527 (X16377 24-h cultures grown in YEPD were inoculated in 200 ml of YEPD to a starting [18]). The probes for ERG11 and MDR1 were the gene fragments listed in the concentration of 2 ϫ 104 cells/ml. The cultures were grown overnight at 30°C previous paragraph. The oligonucleotides for the CDR genes are as follows: with agitation. Total RNAs were prepared from cultures of the susceptible and CDR1: short, position 1211 to 1229, and long, position 1211 to 1260 (X77589 resistant isolates at an optical density at 600 nm (OD600) of 0.1 and at each [29]); CDR2: short, position 902 to 920, and long, position 902 to 951 (U63812 subsequent doubling time (roughly every 90 min) up to an OD600 of 6.4. [32]); CDR3: position 651 to 680 (U89714 [2]); and CDR4: position 501 to 530 (ii) Late-logarithmic phase, diauxic shift, and stationary phase. Cells from (AF044921 [7]). The short CDR1 and CDR2 oligonucleotides were used as 24-h YEPD cultures were inoculated into 50 ml of YEPD to a starting concen- probes for Fig. 1. The long CDR1 and CDR2 oligonucleotides were used as tration of 2 ϫ 104 cells/ml. The cultures were grown at 30°C with agitation. RNA probes for Fig. 2 to 4. 2298 LYONS AND WHITE ANTIMICROB.AGENTS CHEMOTHER.

RESULTS

Time course Northern blot analysis. A time course Northern blot analysis was performed to understand mRNA expression of genes associated with azole resistance throughout cell growth. A susceptible isolate (isolate 1) (MIC ϭ 1.0 ␮g/ml) and a resistant isolate (isolate 17) (MIC Ն64 ␮g/ml) were grown at 30°C in YEPD, and total RNA was prepared from the culture at an OD600 of 0.1 and each subsequent doubling time to an OD600 of 6.4. Total RNA was also prepared from the isolates at 3 days and 8 days of growth. Using these RNAs, Northern blot analysis was performed to examine mRNA levels throughout the course of cell growth for the efﬂux pumps CDR1 to CDR4 and MDR1 and also the target gene for ﬂu- conazole ERG11. For all Northern blot analyses, RNAs were loaded onto the agarose gel so that the visible rRNA bands were equivalent in amount in all lanes, which ensures equivalent loading of RNAs. Loading based on total RNA concentration can give uneven amounts of RNA because of unequal recovery of small RNAs, including small rRNAs and degraded RNAs, as well as aggregation of RNAs in aqueous solution. We have found that loading based on visible rRNAs is the most

accurate. The ACT1 gene was used as a control, as ACT1 is Downloaded from expected to be constitutively expressed under most of the growth conditions. mRNA levels of ERG11 and MDR1 during logarithmic growth. As shown in Fig. 1A, ACT1 mRNA levels are constant throughout logarithmic growth (usually to an OD600 of 6.4 [see below]), consistent with the equivalent loading of all gel lanes based on amounts of rRNA. Figure 1A also shows the levels of aac.asm.org ERG11 (the target enzyme of the azole drugs) during this time course. ERG11 mRNA expression consistently shows a small increase in the resistant isolate compared to the susceptible isolate after standardization for ACT1 (Fig. 1B). The ERG11 by on June 25, 2007 overexpression in the resistant isolate varied from 1.2- to 2.3- fold, which is consistent with our previous report of overexpression at mid-log growth (35). The mRNA levels observed in both growth series (susceptible and resistant isolates) re- mained roughly equivalent from early to late logarithmic growth. mRNA levels for the major facilitator efflux pump MDR1 (Fig. 1) were measured from early to late logarithmic growth. In the susceptible isolate, an MDR1 mRNA signal was only detectable early in logarithmic growth at an OD600 of 0.1. In the resistant isolate expression of MDR1 varied considerably. This variation was observed in repeated RNA preparations (data not shown), suggesting that MDR1 mRNA may have a short half-life. At an OD600 of 0.1, the overexpression of MDR1 in the resistant isolate was approximately threefold higher than that in the susceptible isolate. Overexpression of MDR1 in the resistant isolate was at least 30-fold higher than that in the FIG. 1. (A) Northern analysis of gene expression during logarithmic growth. Total RNA was prepared from the susceptible and resistant isolates at an initial susceptible isolate for the rest of the time points, again con- OD600 of 0.1 and at each doubling time (roughly every 90 min) until the cells sistent with our previous report for mid-logarithmic growth reached an OD600 of 3.2. Northern blots of these RNAs were hybridized with (35). gene probes for ERG11, MDR1, CDR1, CDR2, and CDR4 (see Materials and mRNA levels of the CDR genes during logarithmic growth. Methods). Each of these blots was simultaneously probed with an oligonucleotide for ACT1. Only the ERG11 actin control is shown here. (B) Relative mRNA levels of the ATP binding cassette transporter genes intensities of gene expression during logarithmic growth. The intensities of CDR1 to CDR4 were measured from early to late logarithmic mRNA levels from panel A were quantified using a Storm phosphorimager. The intensities were standardized for each RNA in the series using the ACT1 control growth (OD600s of 0.1 to 3.2) in both the susceptible and resistant isolates using gene-specific oligonucleotides (Fig. 1). for each blot. These standardized levels were then normalized to the standardized level of the susceptible isolate at the start of growth, at an OD600 of 0.1. The mRNA for the CDR1 gene was detected throughout the time exceptions were the CDR4 signals, which were not apparent until later in the course for both the susceptible and resistant isolates. CDR1 growth of the culture and which were normalized to the standardized level of the was overexpressed in the resistant isolate compared to the susceptible isolate at an OD600 of 3.2. For each gene, the resistant isolate is susceptible isolate at every time point, with levels of overex- represented by filled symbols and the susceptible isolate is represented by open symbols. The relative intensities for each gene are presented on a logarithmic pression varying from 2.5 to 7.6. There was an increase in scale with a range of 0.1 to 10 (y axis, labeled on right side of graph). CDR1 expression in the susceptible isolate at the start of the series—an OD600 of 0.1 (Fig. 1). There was also a small de- VOL. 44, 2000 ANALYSES OF ANTIFUNGAL DRUG RESISTANCE IN C. ALBICANS 2299

but not sufﬁcient to present quantitative results. rRNA bands were equivalent in each lane of the gels analyzed. For all genes tested, the hybridization patterns of the RNAs prepared at an OD600 of 6.4 (0.4 day) were similar to the patterns from RNAs prepared at an OD600 of 3.2 (compare Fig. 1A, OD600 3.2, with Fig. 2, day 0.4). For CDR2, CDR4, MDR1, ERG11, and ACT1, mRNA levels were greatly reduced at 3 and 8 days. In Fig. 2, these mRNAs were not detectable at 3 and 8 days. In comparable experiments with higher levels of sensitivity, low levels of the mRNAs are detected at these times (data not shown). The exact timing of this reduction in the mRNA signal for these genes is variable. It has been observed to occur at day 3 or day 4 depending on the experiment (data not shown). This reduction in expression most likely corre- sponds to the diauxic shift, where the glucose in the culture is exhausted and cells shift to alternative carbon sources. mRNA for the CDR1 gene was detectable at 3 and 8 days at levels that are comparable to the levels seen at an OD600 of 6.4, suggesting that CDR1 is expressed preferentially in stationary phase cells or not degraded as rapidly or as thoroughly as the other mRNAs. FIG. 2. Northern analysis of gene expression in stationary phase. Total RNA mRNA levels during growth in different carbon sources. was prepared from the susceptible and resistant isolates at a cell concentration of

Since the diauxic shift is a shift from the utilization of one Downloaded from 6.4 OD600 and at 3 and 8 days. Northern blots of these RNAs were hybridized with gene probes for ERG11, MDR1, CDR1, CDR2, and CDR4 (see Materials carbon source to another, it was of interest to determine the and Methods). RNAs were loaded so that the visible rRNA bands were approx- expression patterns of these genes during growth in different imately equivalent. A control hybridization using housekeeping genes ACT1 or carbon sources. Glycerol and acetate were chosen as nonfer- TEF3 was not possible using these RNAs (see Results). mentable carbon sources. The susceptible and resistant isolates were grown to an OD600 of 1.0 in glucose, glycerol, or acetate. Northern blots of total RNA were probed with the individual genes CDR1, CDR2, MDR1, and ERG11. The signals from the aac.asm.org crease in CDR1 expression in late log phase in both the sus- Northern blots were quantified, standardized using ACT1 ceptible and resistant isolates. mRNA expression as a control, and normalized to the signal mRNA for CDR2 was detected in the susceptible isolate at for the gene in the resistant isolate grown in glucose (Fig. 3). In an OD600 of 0.1 but was not detected at later time points, by on June 25, 2007 similar to the MDR1 pattern of expression. In the resistant isolate, CDR2 expression was consistent from early to mid-log growth but decreased as the cells reached late log phase. Ex- pression of CDR3 was not detected at any time point for either the susceptible or resistant isolates (data not shown). CDR4 mRNA was observed only during late log-phase growth. The mRNA levels of CDR4 were repeatedly higher in the susceptible isolate than in the resistant isolate. At OD600s of 1.6 and 3.2, the CDR4 mRNA levels of the susceptible isolate were 1.6- to 1.7-fold higher than the CDR4 mRNA levels of the resistant isolate. mRNA levels during late log phase and stationary phase. mRNA levels were also studied during late log phase (an OD600 of 6.4), after 3 days of growth (post-diauxic shift phase), and after 8 days of growth (stationary phase) using Northern analysis (Fig. 2). Post-diauxic shift phase and stationary phase were determined by repeated monitoring of the growth of the culture and assessing shifts to slower growth (diauxic shift) and eventually no growth (stationary phase). The results cannot be quantified as mRNA is partially degraded when prepared at these later time points, due to the nature of the cells. Low- FIG. 3. Relative intensities of gene expression during growth in different molecular-weight RNA (less than 200 bp) is consistently ob- carbon sources. Northern blots were prepared with total RNA from the susceptible and resistant isolates at an OD600 of 1.0 in YEP medium containing glucose served in ethidium bromide-stained gels of RNA prepared at (Glu), glycerol (Gly) or acetate (Ace). The blots of these RNAs were hybridized these later time points (data not shown). In addition, rRNA with gene probes for CDR1, CDR2, MDR1, ERG11, and ACT1 (see Materials levels and mRNA levels do not always correlate in these and Methods). The signals from each blot were quantified using a Storm phosphorimager. The intensities were standardized for each RNA in the series using growth phases (reference 34 and data not shown). An ade- the corresponding ACT1 signal for the same lane. The standardized levels were quate control for mRNA levels between samples is not possible then normalized to the standardized level of the resistant isolate grown in because the housekeeping genes ACT1 and TEF3 are degraded glucose, which was assigned a value of 1. For each gene (labeled on the right side of the graph), the resistant isolate is represented by filled symbols and the or down-regulated after cell growth reaches an OD600 of 6.4 susceptible isolate is represented by open symbols. The relative intensities for (Fig. 2 and data not shown). RNA levels were monitored by each gene are presented on a linear scale with a range of 0 to 1.2 (y axis, labeled ethidium bromide staining of the rRNA bands in agarose gels, on left side of graph). The lines connecting similar data points are presented for sufficient to obtain qualitative comparisons of the time points interpretation of the figure, not to imply a temporal or stepwise progression. 2300 LYONS AND WHITE ANTIMICROB.AGENTS CHEMOTHER.

single gene fragment from CDR1 that cross-hybridizes with CDR2-4 (data not shown) was used in this analysis. As seen in Fig. 5, the resistant isolate demonstrated increased transcription rates for the efflux pumps CDR and MDR1 relative to the susceptible isolate. The CDR transcription rate was increased by 2.7-fold and the MDR1 rate was increased by at least 9.3-fold in the resistant isolate compared to the susceptible isolate. The MDR1 signal for the susceptible isolate is indistinguishable from background. Therefore, the increased transcription seen for MDR1 in the resistant isolate (9.3-fold) is a minimum estimate of the contribution due to transcription. No difference was detected for ERG11 between FIG. 4. Southern analysis of gene amplification for CDR1, CDR2, and the susceptible and resistant isolates. These results demon- MDR1. Southern blots were prepared for total genomic DNA from the susceptible (S) and resistant (R) isolates, which were digested with HincII and hybrid- strate that at least part of the resistance phenotype of isolate 17 ized with gene-specific probes. Sizes of the hybridizing bands are presented in is due to increased transcription of the efflux pump genes. kilobases. The MDR1 probe recognizes two bands, 2.0 and 1.1 kb, because a HincII restriction site is located near one end of the MDR1 probe. Each blot was also hybridized with ACT1 as a loading control. The MDR1, CDR2, and ACT1 DISCUSSION restriction fragments are similar but not identical in size and could easily be distinguished from each other on the blots. This series of experiments has documented altered mRNA levels for several genes associated with azole resistance (ERG11, MDR1, CDR1, CDR2, and CDR4) during logarithmic, diauxic, and stationary phase growth in this series of isolates the resistant isolate, there was a consistent reduction in signal (summarized in Fig. 6). mRNA levels for ERG11, MDR1, Downloaded from for each of the genes when the cells were grown in glycerol or CDR1, and CDR2 are consistently higher in the resistant iso- acetate relative to growth in glucose. However, the resistant late compared to the susceptible isolate at each time point isolate consistently overexpresses these genes compared to the during cell growth, while overall mRNA levels vary depending susceptible isolate. The largest change in gene expression was upon the stage of cell growth. Two experiments address the a 60% reduction in the MDR1 signal for the resistant isolate possible molecular mechanisms that result in these increased

when the isolate was grown in glycerol or acetate. The expres- mRNA levels in the resistant isolate: a genomic Southern blot aac.asm.org analysis revealed that the genes for CDR1, CDR2, and MDR1 sion patterns seen with different carbon sources at an OD600 of 1 do not explain the expression patterns seen during 3 and 8 are not ampliﬁed, and nuclear run-on analysis demonstrated days of growth of the cells (Fig. 2). Despite the reduction in

gene expression in these different carbon sources (Fig. 3), no by on June 25, 2007 significant changes in MICs were observed when cells were grown in defined media containing these different carbon sources (data not shown). Lack of gene amplification for efflux pumps. In drug-resistant eukaryotes overexpression of a resistance gene is often associated with gene amplification (4). For this series it has been previously documented that there is no gene amplification of ERG11 (35). To test for gene amplification of the efflux pumps, Southern blots of genomic DNA from the susceptible isolate and the resistant isolate were hybridized with MDR1, CDR1 and CDR2 (Fig. 4). The blots were also hybridized with ACT1 as a control for DNA loading. In this series, the MDR1, CDR1, and CDR2 genes were not amplified in the resistant isolate compared to the susceptible isolate. This eliminates gene amplification as an explanation for increased mRNA levels of these genes. Nuclear run-on analysis of susceptible and resistant isolates. Levels of cellular mRNA can be altered by several methods including, but not limited to, increased transcription of a gene, mRNA transport from the nucleus, and mRNA degradation. To test for elevated transcription levels of CDR, MDR1, FIG. 5. Nuclear run-on analysis for the susceptible and resistant isolates. ␮ and ERG11 a nuclear run-on assay was performed. Susceptible Southern blots were prepared for DNA (10 g/sample) from within the coding regions of the gene targets ERG11, MDR1, CDR, ACT1, and a DNA plasmid and resistant cells at an OD600 of 1.0 were permeabilized with control (see Materials and Methods). The blots were probed with labeled nuclear a detergent, and radioactively labeled UTP was added. The run-on RNA (see Materials and Methods). Signal intensities of nuclear RNA labeled UTP enters the cell and the nucleus, where it is incor- were quantified using a Storm phosphorimager and standardized to the actin porated by transcriptionally active polymerases into nascent intensities. DNA from a pBluescript SK plasmid was used to control for the nonspecific binding of nuclear RNA to random DNA fragments. The standard- RNAs. These labeled nascent RNAs were then used as a probe ized intensities for the susceptible isolate are 0.64, 0.15, 1.38, and 1 for ERG11, against Southern blots with MDR1, CDR, and ERG11 gene MDR1, CDR, and ACT1, respectively. The standardized intensities for the resis- fragments as targets. The size of gene fragment required for tant isolate are 0.49, 1.42, 3.64, and 1. The ratio between resistant and susceptible this analysis (greater than 300 bp) precludes the use of gene- nuclear RNA standardized intensities is given at the bottom of the figure for each gene. Background levels were observed for the DNA controls and for the MDR1 specific fragments for the CDR gene family, since the frag- signal from the susceptible isolate. Since the MDR1 signal for the susceptible ments can cross-hybridize to several CDR genes. Therefore, a isolate is indistinguishable from background, the MDR1 ratio is a minimal estimate. VOL. 44, 2000 ANALYSES OF ANTIFUNGAL DRUG RESISTANCE IN C. ALBICANS 2301 Downloaded from

FIG. 6. Schematic of gene expression during cell growth. The expression patterns of six genes are shown during four different phases of cell growth. The timing of aac.asm.org expression is shown horizontally in the figure. S represents gene expression in susceptible cells, R (shaded area) represents gene expression in resistant cells, and S/R represents equivalent expression in both cell types. The height of the arrow is a qualitative representation of the amount of expression at each time point for each gene. Approximate OD600s or length for each phase would be as follows: exponential growth, OD600 of 0.1 to 3.2; diauxic shift, OD600 of approximately 6.4; post-diauxic shift, 3 to 6 days; and stationary phase, 8 days. The format for this figure was adapted from a review of stationary phase growth in S. cerevisiae by Werner-Washburne et al. (34). by on June 25, 2007 that one mechanism for increased mRNA levels of MDR1 and CDR3 was not detected, which is consistent with its description CDR is increased mRNA transcription. as a phase-specific gene (2). The expression of efflux pumps The Northern analysis shown in Fig. 1 revealed that the early in logarithmic growth occurs in cells that have been levels of ERG11 mRNA were slightly increased in the resistant grown for 16 h from a very small inoculum (Fig. 1). Thus, pump isolate throughout logarithmic growth, and expression in both expression is not residual from stationary phase growth or a the susceptible and resistant isolates was constant during cellular response to fresh medium, since the cells have been growth, correlating with actin mRNA levels. MDR1 mRNA growing for 16 h. Expression may be related to quorum sensing levels in the susceptible isolate were only detectable in early in bacteria (reviewed in reference 11, to different metabolic log phase growth, while MDR1 mRNA in the resistant isolate needs during different stages of growth, or small regulators was detected at high levels throughout growth. However, the such as MARS (morphogenic autoregulatory substance) of C. level of MDR1 mRNA in the resistant isolate varies widely albicans that represses hyphal growth at high cell concentra- during growth. This variability is likely due to a short half-life tions (12). of the mRNA, such that variations in the preparation of total In this series, increased mRNA levels of CDR are present in RNA from the cells can result in different levels of MDR1 the resistant isolate at mid-log growth. These mRNA levels at mRNA despite constant levels of ACT1, which is expected to mid-log growth have been previously described (35). The in- have a relatively long half-life. This variability during growth creased mRNA levels can be attributed to the cumulative ex- has been observed in several independent time courses (data pression of CDR1 and CDR2. In the resistant isolate, CDR1 not shown). Recently, several mutant alleles of MDR1 were and CDR2 showed increased mRNA levels throughout cell shown to express mRNA at varying levels and were shown to growth, with a slight decrease as the cells reached late log be inducible under several different growth conditions (10). phase. CDR4 mRNA is again expressed only in late log phase Figure 1 also demonstrates the differential expression of cells, but the mRNA levels are reduced in the resistant isolate CDR1, CDR2, and CDR4 between the susceptible isolate and compared to those in the susceptible isolate (Fig. 1 and 2). This resistant isolate, as detected by oligonucleotide probes specific is surprising, as it is the first description of an efflux pump that for each of the genes. In the susceptible isolate, both CDR1 is down-regulated as cells develop a resistant phenotype. The and CDR2 show expression at early log growth. While CDR2 data suggest that both CDR1 and CDR2 may contribute to the expression is only detected in early log growth, CDR1 shows final azole-resistant phenotype of this series but that CDR3 and expression throughout the logarithmic growth phase. This is CDR4 do not contribute to resistance, at least in vitro. Expres- consistent with a previous publication that described the ex- sion patterns for CDR5 to CDR7 have not been tested, so it is pression of CDR1 during growth of the cells (15). mRNA for possible that these and other CDR genes may also contribute CDR4 is only detected in late log-phase cells, and mRNA for to the drug-resistant phenotype seen in the resistant isolate. 2302 LYONS AND WHITE ANTIMICROB.AGENTS CHEMOTHER.

These observations are consistent with other studies (2, 7, 32, The transcription rates of the genes are consistent with the 33), which find that CDR1 and CDR2 are the only members of Northern blot analysis. The level of sensitivity of the nuclear the CDR gene family to date that correlate with azole drug run-ons is lower than that of Northern analysis, since the run- resistance. ons have not been able to consistently detect genes with a low In Fig. 1, the levels of overexpression of ERG11, MDR1, and level of expression, such as TEF3, a housekeeping gene. There- CDR in the resistant isolate in mid-logarithmic growth are fore, the nuclear run-ons underestimate the contribution of substantial but are not as large as previously reported (35). transcription to overexpression (data not shown). This may There are several possibilities for this. The RNAs for the explain the differences between Northern blot quantification of previous publication were prepared at an OD of 1, while the mRNA and transcription rates as determined by nuclear RNAs in the present study were prepared at ODs of 0.8 and run-on experiments. A generalized conclusion is that the azole 1.6. It is possible that overexpression in the resistant isolate is resistance phenotype observed in this series is due in part to Ϫ gradually lost over time or during storage at 80°C. It is also transcriptional overexpression of efflux pump mRNAs. While possible that growth in culture modifies the expression of both the term “overexpression” has been previously applied to clin- the susceptible and resistant isolates. There is some indication ical isolates, the nuclear run-on data presented above are the that the MIC for the susceptible isolate has increased over first to actually monitor transcription rather than increased time, which might reflect increased expression of the pumps in mRNA levels that can be the result of several cellular pro- this isolate. The MIC for the resistant isolate continues to cesses. remain above the upper limit of the assay. The CDR probe used for this analysis cross-hybridized with In vivo, C. albicans is likely to grow under a variety of many members of the CDR gene family, and it is not known conditions, which do not always include a rich medium con- specifically which CDR mRNA have increased transcription. It taining glucose. Therefore, it was important to examine gene should be noted that these experiments do not rule out other expression after the diauxic shift and during stationary phase. mechanisms of elevating mRNA levels, such as increasing Most of the genes studied (ACT1, ERG11, MDR1, CDR2, and mRNA half-lives. In addition, increased levels of mRNA do Downloaded from CDR4) were repressed or down-regulated by 3 days of growth not always lead to an increased expression of protein or in- (post-diauxic shift) and were not detectable at 8 days of growth creased enzymatic activity. Further analysis is necessary to (stationary phase) in both susceptible and resistant cells (Fig. clarify these issues. 2). The surprising finding was that mRNA was detected for the As shown above, mRNA levels of ERG11, MDR1, CDR1, CDR1 gene at both 3 and 8 days in both the susceptible and and CDR2, which have all been correlated with azole resistance resistant isolates. This may be the result of persistent transcrip-

C. albicans aac.asm.org tion or selective protection from degradation of the CDR1 in , rely on the exact growth phase of the cells. message. This suggests that CDR1 pump expression is impor- Nuclear run-on analysis has demonstrated that at least one tant for the continued survival of both susceptible and resistant reason for the observed increases of mRNA is an increase in cells under these conditions, perhaps by removing toxins, mRNA transcription. However, cell growth and expression of these genes was conducted in vitro. Little is known about the which would accumulate during long-term growth, from the by on June 25, 2007 cell. The overexpression of CDR1 in the resistant isolate con- growth environments and growth stages of C. albicans in vivo. tinues at these late time points, suggesting that the resistant The growth stages may vary greatly in vivo in oral (both phenotype persists throughout the growth of the cells. At- pseudomembranous and erythematous), vaginal, and systemic tempts to monitor the exact phase of cell growth at these time infections. Growth may also vary in biofilms compared to points, using genes expected to be expressed in these phases growth in a flask (1). Depending upon the type of infection, the such as SUR1 (C. albicans information Web page), were un- yeast may exist in several different states of growth (i.e., hyphal successful (data not shown). or pseudohyphal), and the cells may be growing exponentially Since the diauxic shift represents a shift in growth from or in a phase resembling the stationary phase. These distinct glucose to a nonfermentable carbon source, gene expression phases of growth are likely to influence the expression of re- was monitored on glucose, glycerol, and acetate. As seen in sistance genes. This is likely to have major implications for Fig. 3, there was a modest reduction in gene expression in both azole drug resistance and drug therapy. the susceptible and resistant isolates for CDR1, CDR2, MDR1, The lack of gene amplification and the increases in transcrip- and ERG11 when the cells were grown on the alternate carbon tion observed for the efflux pumps direct attention to the pro- sources. Despite these changes in pump and target enzyme moter and associated trans-acting factors as important mech- expression, no change in MIC was observed when cells were anisms of azole resistance, at least in this series of isolates. grown in these carbon sources (data not shown), suggesting Clearly, a thorough characterization of the molecular mecha- that these minor reductions in gene expression do not signifi- nisms of gene transcription will be important for a greater cantly impact drug susceptibility. understanding of in vivo azole resistance in C. albicans. Southern analysis indicated that gene amplification was not a cause of the elevated mRNA levels of CDR1, CDR2,or MDR1 in this series (Fig. 4). Previous data have shown the ACKNOWLEDGMENTS same for ERG11 in this series (35) and for CDR1 in a second series of isolates (21). To date, there is only one example of We thank Spencer Redding (University of Texas Health Science gene amplification associated with azole resistance: a chromo- Center at San Antonio) for the use of his isolates. We thank Simone some duplication in Candida glabrata (20). Sanchez for assistance with the Southern blot analysis. We thank Nuclear run-on analysis demonstrated that CDR and MDR1 Kieren Marr for helpful comments and discussions and the other members of our laboratory for their support and comments on the mRNAs were transcribed at higher rates, 2.6-fold and 9.3-fold, manuscript. respectively, in the resistant isolate than in the susceptible This research was supported by NIH NIDR grant RO1 DE-11367. isolate (Fig. 5). No increase in transcription for ERG11 was T.C.W. is supported by a New Investigator Award from the M. J. observed. Previous Northern blot analysis data of RNA pre- Murdock Charitable Trust and is the recipient of a New Investigator pared at an OD600 of 1.0 showed increased mRNA levels of Award in Molecular Pathogenic Mycology from the Burroughs Well- CDR and MDR1 to be 5-fold and 25-fold, respectively (35). come Fund. VOL. 44, 2000 ANALYSES OF ANTIFUNGAL DRUG RESISTANCE IN C. ALBICANS 2303

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