Response of gene expression in Saccharomyces cerevisiae to amphotericin B and nystatin measured by microarrays

Liang Zhang1,2, Yan Zhang1,3, Yiming Zhou1,3, Shuang An3, Yuxiang Zhou1,2,3 and Jing Cheng1,2,3,*

1Department of Biological Science and Biotechnology, and 2State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing 100084; 3Beijing National Biochip Research & Engineering Center, Jia 2 Qinghua West Road, Beijing 100084, People’s Republic of China

Received 7 September 2001; returned 18 November 2001; revised 14 December 2001; accepted 4 January 2002.


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The response of the yeast Saccharomyces cerevisiae to two polyene antibiotics, amphotericin B and nystatin, was studied by genomic expression profiling. The two agents produced highly similar expression pattern changes, which was consistent with their known identical mechanisms of action on cell membranes. Detailed analysis was focused on the amphotericin B-treated sample in this study. Our data showed that genes involved in mitochondrial ribosomal protein synthesis were more severely repressed than those in the cytoplasm, which might contribute to the cytotoxicity of amphotericin B. To counteract the leakage of intracellular nutrients and ions from the pores in the cell membrane caused by amphotericin B, c. 17 genes involved in transport facilitation were induced, presumably to allow more efficient uptake of nutrients and ions. The expression level of five genes involved in ergosterol synthesis dropped and three genes related to cell wall biogenesis were induced, indicating that the cell membrane and cell wall were also affected by the presence of polyene antibiotics. It was observed that the pleiotropic drug resistance network in yeast cells was activated after exposure to amphotericin B, possibly contributing to the acquisition of amphotericin B resistance. Part of the gene expression alteration measured by microarray was confirmed by quantitative RT–PCR.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The antifungal polyenes include three main compounds: natamycin, nystatin and amphotericin B. Despite its toxicity, amphotericin B remains the drug of choice for the treatment of systemic fungal infections, including those due to newly emerging and azole-resistant pathogens.1 Amphotericin B, a heptene, acts by increasing the permeability of the fungal cell. It can bind to ergosterol components in the membrane and cause the formation of cell membrane pores, leading to leak-age of cell components such as proteins and potassium.2 Amphotericin B also interacts with cholesterol in human cell membranes, which probably accounts for its toxic effects and consequently limits its usage.3 Despite more than 40 years of amphotericin B use, resistance among Candida spp. is still very rare; however, it is slowly increasing.4 Nystatin, a tetra-ene, has a similar mechanism of action to amphotericin B.5

The yeast Saccharomyces cerevisiae is an ideal eukaryotic organism to study gene expression caused by environmental insults. This organism has a relatively small sequenced genome of c. 6000 genes,6 allowing the construction of a genome-scale DNA microarray. Moreover, the function of c. 68.2% of the S. cerevisiae 6000 open reading frames (ORFs) is known, by genetics, biochemistry or homology,7 which will no doubt aid our understanding of the mechanisms of action of stimuli by chemical agents. The yeast genome-scale DNA microarrays have been employed to monitor global responses to environmental stresses and a variety of chemical agents.8,9 In addition, yeast microarrays are used to study the responses of yeast cells to antifungal agents at the molecular level10 and to predict the mode of action of novel agents with antifungal activity.11

In this study, we monitored the global expression profile changes of 5935 yeast genes caused by treatment with amphotericin B and nystatin. We anticipated a panoramic view of the responses of yeast cells to the two polyene antifungal antibiotics at the molecular level. Efforts have also been undertaken to validate the microarray data in classifying the action of chemical agents by gene expression profiling, since amphotericin B and nystatin have similar physiological and biochemical antifungal mechanisms.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Primer design and PCR amplification

The S. cerevisiae genomic DNA sequence was downloaded from ftp//ftp.ebi.ac.uk/pub/databases/yeast/orf_seqs/dnaseq. pir in November 1999. Altogether there are 6294 ORFs. The primer selection program PRIMER 0.5 (Whitehead Institute, Boston, MA, USA) was used to design the ORF primers adjacent to the 3' terminus. The main design standard was that all the primers should have very similar annealing temperatures, and the length of PCR products should be between 200 and 1000 base pairs.

ORF amplification and construction of DNA microarrays

Genomic DNA of S. cerevisiae strain S288C was isolated according to Hoffman12 and used as a template for amplification reactions in 96-well PCR plates. The success rate for PCR amplifications with the primer pairs was c. 95% based on the results of agarose gel electrophoresis. PCR products were pre-cipitated with isopropanol and redissolved in 50% dimethyl sulphoxide (DMSO). A commercial arrayer PixSys5500 (Car-tesian Technologies, Irvine, CA, USA) was employed to spot the DNA in DMSO on to poly-L-lysine-treated glass slides. Altogether, 5935 yeast ORFs were arrayed. Additionally, con-trol samples including three different Arabidopsis thaliana DNA fragments (GenBank accession numbers AC004146, AC007661 and U09332), 12 spots of salmon sperm DNA and 12 spots of DMSO were also arrayed. The centre-to-centre distance between two adjacent spots was 225 µm. To verify reproducibility, two identical subgrids (38 x 20 mm for each) of DNA were arrayed on the same glass slide. The detailed procedures for preparing and blocking poly-L-lysine glass slides and denaturing the DNA to be arrayed can be found on the website: http://cmgm.stanford.edu/pbrown/protocols.

Yeast strain and drug exposure

S. cerevisiae strain L1190 (MATa/{alpha}) was exposed to drug treatment. Amphotericin B and nystatin were obtained from Sigma Chemical Company (St Louis, MO, USA) and dis-solved in DMSO at a concentration of 1 mg/mL. The MICs of the two polyene antibiotics for L1190 were measured according to a previous study.11 A 100 mL culture of YPD (1% yeast extract, 2% peptone and 2% D-glucose) medium was grown to OD600 = 0.8 at 30°C and divided into three flasks, each con-taining 30 mL of culture. Amphotericin B and nystatin were added to cultures to 0.5 x MIC (final concentrations were 2.5 mg/L of both antibiotics). To the control sample was added the same volume of DMSO. After exposure for one doubling time (90 min), yeast cells were collected by centrifugation at 5000 rpm for 5 min at room temperature, flash frozen in liquid nitrogen then transferred to a –85°C refrigerator. Cells were kept at –85°C until they were processed for RNA.

RNA preparation, generation of Cy-labelled cDNA and hybridization

Total RNA was isolated with a hot acidic phenol method.13 Reverse transcription (RT) was performed in a total volume of 40 µL containing 40 µg of total RNA, 1.5 µg of oligo(dT)15 (Promega, Madison, WI, USA), 500 µM each of dATP, dTTP and dGTP, 50 µM dCTP, 25 µM Cy5/Cy3-dCTP (Amersham-Pharmacia, Piscataway, NJ, USA), 40 U RNase inhibitor (Promega) and 400 units M-MLV transcriptase (Gibco, Gaithersburg, MD, USA) in 1x first strand buffer. Three poly(A)+ RNAs, synthesized in vitro from the three arrayed A. thaliana genes, were added to the yeast RNA as internal controls at 0.1, 1 and 10 ng, corresponding to c. 3–300 transcripts/cell (assuming the mass of total RNA in a yeast cell is 0.5 pg).14 Briefly, RNA sample and oligo(dT)15 were heated to 70°C for 10 min, quick chilled on ice, and then dNTPs, reaction buffer and enzyme were added and the reaction carried out at 37°C for 2 h. After completion of the reaction, RNA was removed by alkaline hydrolysis. cDNA was precipitated by ethanol and dissolved in 7.5 µL of H2O. Afterwards, 1.5 µL of 20x SSC, 1 µL salmon sperm DNA (10 mg/mL) and 0.2 µL 10% SDS were added to this cDNA solution, boiled for 2 min and cooled to room temperature. To obtain cDNA at a higher concentration, two independent reactions were performed for each RNA sample under identical conditions and the cDNA precipitants redissolved in a total of 20 µL hybridization buffer. After the Cy3- and Cy5-labelled cDNAs were pooled, 40 µL of the mixed cDNA solution was allowed to hybridize to the array at 62°C for 10–14 h. When the hybridization was complete, the arrays were washed for 5 min in 200 mL of washing buffer A (0.1% SDS/0.6x SSC) at 50°C, then for 5 min in 200 mL of washing buffer B (0.03x SSC) at room temperature. The glass slides were centrifuged to dryness in 50 mL conical tubes at 1000 rpm for 1 min.

Scanning and data analysis

Arrays were scanned with ScanArray 4000 (Gsi Lumonics, Kanata, OT, USA) and array images were analysed with ImaGene software version 3.0 (BioDiscovery, Los Angeles, CA, USA). To normalize fluorophore-specific variation, spots containing three exogenous DNA fragments of A. thaliana were applied to each quadrant during the arraying process. These elements were used to autobalance the photomultiplier sensitivity setting between the Cy5 and Cy3 channels such that the ratios between the fluorescent intensity of the A. thali-ana DNA spots were close to a value of 1.0. After subtracting the signal intensity on the negative control spots (DMSO spots), the signal was regarded as a real hybridization signal if the spot fluorescent intensity was two-fold higher than the local background. Each experiment was conducted by dye swap.15 If expression ratios were >1.5 or <0.67 with both dye-swap arrays, the corresponding genes were then considered to be expressed differentially, and the average ratio from the change in expression was calculated and treated as the true ratio between the two arrays.

Quantitative RT–PCR

Total RNA was extracted as described above and treated with DNase I to eliminate traces of genomic DNA. A fraction of 40 µg total RNA was used to synthesize the first strand of cDNA in 40 µL RT volume. Afterwards, 2 µL of RT reaction products were used as template for PCR amplification in the LightCycler PCR System (Roche Molecular Biochemicals, Mannheim, Germany). The relative RNA transcript abundance was obtained by calculating the ratio of the fluorescent intensity (cross point value) of the treated and that of the control sample by employing the SYBR Green I monitoring method.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Global responses of yeast cells to amphotericin B and nystatin

It was found that the stronger the hybridization signals on the arrayed DNA spots, the more reliable were the measured expression alterations. Therefore, we used the same two RT reactions to generate a higher concentration of Cy-labelled cDNA. Of the 5935 yeast genes on the array, 1455 (24.5%) were responsive to amphotericin B treatment, of which 1250 were reduced >1.5-fold, and 205 were increased >1.5-fold. After nystatin treatment, 684 genes were reduced >1.5-fold and 334 genes were increased >1.5-fold. Comparison of the genes responsive to amphoteri-cin B and nystatin showed that of the 1018 (17.2%) nystatin-responsive genes, 771 (75.7%) were also detected in the amphotericin B-treated sample with the same tendency to expression variation despite quantitative differences in the level of change. These should probably not be ascribed to differences in effect of the two antibiotics on yeast cells, but to variation in the hybridization kinetics under different coverslips. It is often observed that even if samples of the same cDNA solution are hybridized to different arrays containing the same DNA spots, there can be a slightly different hybridization signal intensity, which can result in some genes, especially those with low transcript levels, being detected differently on each array. Even so, in our large data pool, there were no examples of a gene that was induced by amphotericin B being reduced by nystatin or vice versa. These results indicate that amphotericin B and nystatin induced highly similar expression profiles in yeast cells, which was consistent with their known similar pharmacological function. All data are available on our website (http://www.capitalbiochip.com/eexp.htm).

Analysis of the amphotericin B-responsive genes

Because the expression profile changes in each gene were in the same directions in cells exposed to amphotericin B and nystatin, we focused our attention on the amphotericin B-responsive genes. These genes are listed in Tables 1 and 2.


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Table 2. Genes induced by more than two-fold after treatmeant with 2.5 mg/L amphotericin B for 90 min (n = 77a)
 
The grouped distribution of genes that were either up- or down-regulated after amphotericin B exposure (Figures 1 and 2) provided information pertinent to the antifungal mechanisms of amphotericin B and to the response of yeast cells to counteract deleterious effects of amphotericin B at the molecular level.



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Figure 1. Distribution of amphotericin B responsive genes in the most representative classes. (a) Repressed; (b) induced. Genes were grouped into these categories according to the S. cerevisiae Genome Database and the Munich Information Center for Protein Sequence (http://www.mips.biochem.mpg.de/proj/yeast).

 


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Figure 2. Comparison of expression measurements by quantitative RT–PCR and microarray. By exposing yeast cells to 2.5 mg/L amphotericin B, the expression changes in the selected nine genes show a good agreement between the two methods.

 
Under environmental stresses, it is usually observed that the expression levels of some ribosomal protein genes de-crease.9,16,17 The modest reduction in ribosomal protein genes allows energy to be reshuffled for increased expression of genes involved in protective responses, while maintaining a basal protein synthesis.17 In the repressed ribosomal protein genes, our results indicated that the mitochondrial ribosomal protein (MRP) genes were repressed more severely (more than three-fold) than the nuclear ribosomal protein genes (between 1.5- and 2-fold). A previous report demonstrated that mitochondrial ribosomal protein transcripts showed a slight increase after yeast cells were exposed to a DNA damaging agent.17 It is known that, except for a VAR1 gene, the other ribosomal proteins are encoded by nuclear genes, synthesized in the cytoplasm and imported into the mitochondria.18,19 Inactivation or absence of the MRP can result in a block in mitochondrial translation and finally lead to a complete loss of mitochondrial DNA.20 It is possible that MRP genes are in a common regulation mode and are more sensitive to amphotericin B. The inhibitory action of amphotericin B on MRP genes should be taken into account for its cytotoxicity to both fungal and mammalian cells. Among the genes repressed by amphotericin B treatment, the ADE gene group was notably represented (Table 1). In yeast, ADE genes encode enzymes involved in AMP synthesis. It is proposed that these purine biosynthesis genes are down-regulated under oxidative stress, and the down-regulation mechanism could be an important way for cells to resist oxidative stress by increasing NADPH regeneration.21 However, our results do not support the hypothesis that amphotericin B causes an intracellular oxidative stress, since the expression alterations of other genes were not consistent with this conclusion. For example, CYC1-encoding isoform-1 of cytochrome c is an aerobic gene, whereas CYC7-encoding isoform-2 of cytochrome c is a hypoxic gene.22,23 It was unexpected that CYC1 and CYC7 genes were both down-regulated to the same extent when yeast cells were exposed to amphotericin B. In addition, CUP1A, CUP1B and SOD2 genes, which are reported to be activated by oxidative stress,24 were all repressed by amphotericin B treatment. Therefore, it was difficult to link change tendencies of intracellular reactive oxygen with amphotericin B exposure.


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Table 1.  Genes repressed by more than three-fold after treatment with 2.5 mg/L amphotericin B for 90 min (n = 77a)
 
A striking induction was observed with genes involved in transport facilitation. It is known that the cell membrane is the primary target of amphotericin B action. The leakage of ions and nutrients induced by formation of pores in the membrane is partly compensated by the increased gene expression of transmembrane transporters. For example, the ZRT1 gene, which encodes the high-affinity zinc transporter protein, was induced 53.5-fold, allowing an efficient uptake of zinc from the extracellular environment to counteract the ion leakage caused by amphotericin B. It is noteworthy that the ZRC1 gene, which was previously reported to be induced in response to a decrease in intracellular zinc levels,25 was repressed 3.1-fold after treatment with amphotericin B. It is possible that there is an uncharacterized regulation mechan-ism between ZRT1 induction and ZRC1 repression to retain the zinc homeostasis after ion leakage caused by amphotericin B. Another adaptation mechanism to nutrient leakage caused by amphotericin B was the activation of the PHO pathway. The induced genes, PHO5, PHO8, PH011, PHO12, PHO84 and SPL2, are known to be regulated by PHO regulatory systems.26 All enzymes encoded by these genes contribute to increased levels of free intracellular phosphate, which is an essential nutrient in the biosynthesis of diverse cellular components. Other than the transmembrane transporter genes, the TIS11 gene, which normally supplies energy for nutrient and ion transport, was also induced by a factor of 8.7. The induction of genes involved in transport facilitation was also observed in our laboratory when yeast cells were exposed to ethanol at 0.5 x MIC (data not shown). Ethanol is also known to have primary deleterious effects on the cell membrane. It is possible that the increase in expression level of transmembrane transporter genes is a common adaptation mechanism to perturbations of cell membranes.

In our experiments, several genes involved in the ergosterol biosynthesis pathway were expressed at a decreased level [ERG4 (1.8-fold), ERG6 (1.8-fold), ERG9 (2.5-fold), ERG11 (2.1-fold) and ERG24 (2.1-fold)]. Concerning genes involved in lipid metabolism, it is noteworthy that some were down-regulated, such as OLE1 and its modulator FAA4, whereas others were up-regulated. INO1, which encodes inositol-1-phosphate synthase, catalysing the formation of inositol-1-phosphate from glucose-6-phosphate, was induced 6.4-fold. It is known that the INO1 gene is the most highly regulated gene in phospholipid synthesis.27 In addition, GIT1, which encodes a putative inositol transporter, and CIT2, which is considered to possess a function similar to GIT1,28 were both up-regulated. These changes in lipid metabolism might suggest a reconstruction of the cell membrane in response to the membrane perturbation caused by amphotericin B. Besides the cell membrane change in response to amphotericin B treatment, three genes involved in cell wall biogenesis, MET5, SPS100 and YMR251W, were induced, revealing that the cell wall may also interfere with the effect of polyene antibiotics, in agreement with previous observations.29,30

In the yeast S. cerevisiae, two networks of genes involved in multidrug resistance (MDR) have been identified. The first one is regulated by Yap1p31 and Cad1p.32 Additionally, an ATP-binding cassette (ABC) transporter, Ycf1p, is required for cadmium resistance mediated by Yap1p.3336 Our results revealed that the expression levels of CAD1 and YCF1 did not change, and YAP1 was repressed by a factor of 2.0. The second network, called pleiotropic drug resistance (PDR),37 is regulated by the transcription factors Pdr1p and Pdr3p. We could not detect the signals of PDR1 and PDR3, perhaps because their transcriptional levels were too low. However, three ABC transporter genes, SNQ2, PDR5 and YOR1, together with PDR15, a member of the PDR network family, were found to have raised their expression levels by factors of 2.1, 2.5, 2.9 and 4.1, respectively. It has been reported that the double deletion of both PDR1 and PDR3 genes results in drug hypersensitivity and strongly reduced expression of SNQ2, PDR5 and YOR1.33,34 Taking all the above information together, it was postulated that amphotericin B possibly triggers the PDR network in yeast cells. In addition to three ABC transporter genes, SNQ2, PDR5 and YOR1, two other MDR transporter-encoding genes, ATR1 and SGE1, which are energerized by proton motive force,38 also increased their expression levels.

We note that two genes, RGT1 and YHR115C, were completely repressed by amphotericin B. The function of YHR115C is as yet unknown; RGT1 functions primarily in regulation of glucose transport. In S. cerevisiae, four HXT genes (HXT1–4) encode hexose transporters, and Rgt1p is proposed to be a repressor of HXT gene expression.39 How-ever, it was not anticipated that HXT4 would also be repressed by a factor of 3.3, accompanied by complete repression of RGT1, and there were no alterations in the expression levels of HXT1, HXT2 and HXT3. Possibly there are more complicated regulation mechanisms between the RGT1 and HXT genes.

Quantitative RT–PCR

To compensate for the sensitivity limitation of northern blots, an alternative method employing a quantitative RT–PCR assay based on LightCycler technology was developed.4042 The LightCycler is able to quantify particular dsDNA PCR products by means of SYBR Green I, which fluoresces only when bound to dsDNA. The relative amount of transcript in each cDNA sample is quantified by direct comparison of the cross point value between test and control samples employing the LightCycler algorithmic software. We calculated the fold-alteration (FA) by the formula FA = 2(CP1 – CP2), where CP1 represents the crossing point of an RNA sample from treated cells, and CP2 represents the crossing point of an RNA sample from untreated cells. Nine genes were selected to compare their changes at the transcription level in the amphotericin B-treated sample and the control by using microarray and LightCycler quantitative RT–PCR methods, respectively. The results shown in Figure 2 indicated a good agreement between measures of changed transcripts obtained by the two methods.

Conclusion

In conclusion, the yeast genome-scale microarray is a highly efficient technology platform to study the mechanisms of action of variants of compounds with antifungal activities. In our next study, we will transfer our focus from these known compounds to those from Chinese traditional herbal drugs, which have been used widely in folk medicine to inhibit or kill fungi but whose mechanisms of action have not been characterized.


    Acknowledgements
 
We thank Dr Guoqiang Chen for the gift of S. cerevisiae strain L1190. This work was supported by the National Natural Science Foundation of China (grants 39889001 and 39825108) and the National Key Basic Research Program of China (grant G19990116).


    Footnotes
 
* Corresponding author. Tel: +86-10-62773059; Fax: +86-10-62566806; E-mail: jcheng{at}tsinghua.edu.cn Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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