Genome-wide expression profiling of the response to short-term exposure to fluconazole in Cryptococcus neoformans serotype A
© Florio et al; licensee BioMed Central Ltd. 2011
Received: 22 December 2010
Accepted: 11 May 2011
Published: 11 May 2011
Fluconazole (FLC), a triazole antifungal drug, is widely used for the maintenance therapy of cryptococcal meningoencephalitis, the most common opportunistic infection in AIDS patients. In this study, we examined changes in the gene expression profile of the C. neoformans reference strain H99 (serotype A) following FLC treatment in order to investigate the adaptive cellular responses to drug stress.
Simultaneous analysis of over 6823 transcripts revealed that 476 genes were responsive to FLC. As expected up-regulation of genes involved in ergosterol biosynthesis was observed, including the azole target gene ERG11 and ERG13, ERG1, ERG7, ERG25, ERG2, ERG3 and ERG5. In addition, SRE1 which is a gene encoding a well-known regulator of sterol homeostasis in C. neoformans was up-regulated. Several other genes such as those involved in a variety of important cellular processes (i.e. lipid and fatty acid metabolism, cell wall maintenance, stress and virulence) were found to be up-regulated in response to FLC treatment. Conversely, expression of AFR1, the major transporter of azoles in C. neoformans, was not regulated by FLC.
Short-term exposure of C. neoformans to FLC resulted in a complex altered gene expression profile. Some of the observed changes could represent specific adaptive responses to the antifungal agent in this pathogenic yeast.
Cryptococcus neoformans is a basidiomycetous fungal pathogen that causes meningoencephalitis in predominantly immunocompromised hosts [1, 2], that is the most devastating manifestation of cryptococcal disease and is fatal unless treated . Cryptococcosis appears to be a significant opportunistic infection in solid-organ transplant recipients, with a prevalence rate ranging from 0.26% to 5% and overall mortality of 42% . Notably, cryptococcal meningitis was reported to occur in 46% of patients from an Indian HIV-positive cohort . Although the introduction of highly active antiretroviral therapy has led to a decrease in the number of cryptococcal infections in AIDS patients in most developed countries, this is not the case in developing countries where the incidence of HIV/AIDS and cryptococcal meningitis continue to rise . As fluconazole (FLC) became increasingly used due to the need for life-long maintenance therapy in HIV/AIDS patients, FLC resistance was hence detected at relatively high frequency in C. neoformans clinical isolates from India, Africa and Cambodia [7–9].
Increased FLC resistance in vitro was shown to be predictive of treatment failures and infection relapses . Recently, the mechanism underlying the heteroresistance to FLC was elucidated , that is an adaptive mode of azole resistance previously associated with FLC therapy failure cases . This mechanism is based on duplications of multiple chromosomes in response to drug pressure . Interestingly, Sionov et al.  observed that the number of disomic chromosomes positively correlated with the duration of exposure to FLC, whereas the duplication of chromosome 1 was closely associated with two genes, ERG11, the target of FLC , and AFR1, the major transporter of azoles in C. neoformans [11, 15]. Such genomic plasticity enables cells to cope with drug stress and was observed in C. neoformans strains of both serotypes, A (C. neoformans var. grubii) and D (C. neoformans var. neoformans) .
The recent sequencing of the C. neoformans genome  has stimulated the development of C. neoformans-specific microarrays that made possible to address hypotheses about global responses to overcome stresses during growth in the human host [17, 18]. Regardless of the source (i.e. host-derived or antifungal drugs), toxic compounds exert constant selective pressure on the fungus that responds by developing mechanisms necessary for survival .
With the aim to identify genes required for adaptive growth in the presence of sub-inhibitory concentrations of FLC, we investigated here the transient response of C. neoformans to FLC by analyzing differences in gene expression prior and after FLC exposure of strain H99, a reference strain of serotype A. Thus, genome-wide transcriptional profiling of over 6823 C. neoformans genes identified 476 genes with significant expression changes. Apart from genes involved in ergosterol biosynthesis (e.g. ERG11), genes involved in other important cellular functions, such as those encoding the sterol homeostasis regulator Sre1  or phospholipase B1 (Plb1) , were shown to be induced by FLC treatment. In addition, AFR1 was not found FLC-responsive, suggesting indirectly that this gene is responsible for long-term FLC adaptation in C. neoformans.
Strain, growth conditions and RNA isolation
C. neoformans var. grubii serotype A strain (H99) was obtained from David S. Perlin , kept as 20% glycerol stock at -80°C and sub-cultured, as required, on YEPD (1% yeast extract, 2% peptone, 2% glucose) agar plates at 30°C. For RNA isolation independent overnight cultures were diluted 1:100 in liquid YEPD and grown at 30°C or 37°C with agitation for 3 h to reach a density of 3 × 107 CFU/ml. At this point cultures were equally divided into two aliquots to which either FLC at a concentration of 10 mg/l or distilled water was added, followed by incubation at 30°C or 37°C for 90 min. After this treatment, cultures were centrifuged at 4°C and 5500 × g and total RNA was extracted as previously described .
Microarray design and preparation
C. neoformans H99 microarrays were designed following the Agilent Array Design guidelines (Earray platform) by first creating two separate sets of 60-base nucleotide probes for each of 6967 open reading frame (ORF) sequences as downloaded from the Broad Institute website http://www.broadinstitute.org/annotation/genome/cryptococcusneoformans/MultiHome.html. The probe selection was performed using the GE Probe Design Tool; probes were filtered following their base composition and distribution, cross-hybridization potential, and melting temperature, to yield final duplicate probes representing 6823 ORFs to cover 97.9% of the whole C. neoformans H99 genome. C. neoformans custom arrays were manufactured in the 8 × 15k format by Agilent Technologies (Santa Clara, CA, USA). For quality control and normalization purposes, 157 probes were selected randomly and spotted 10 times throughout each array. Standard controls (Agilent Technologies) were also included.
cRNA synthesis, labeling and hybridization
RNA sample preparation was performed on three biological triplicates of H99 cells grown at 30°C, as described above. Prior to the labeling/amplification step, purity and integrity of the RNA samples were determined using Agilent RNA 6000 Nano LabChip kit on the Agilent 2100 bioanalyzer (Agilent Technologies). Agilent's One-Color Quick Amp Labeling kit (Agilent Technologies) was used to generate fluorescently labeled cRNA probes according to the manufacturer's instructions. The method uses T7 RNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3-labeled CTP. The labeled cRNAs were purified with the RNeasy Mini kit (Qiagen, Hilden, Germany) and quantified using NanoDrop ND-1000 UV-VIS spectrophotometer. Aliquots (600 ng) of Cy3-labeled cRNAs were fragmented and hybridized for 17 h at 65°C to each array using the Gene Expression Hybridization kit (Agilent Technologies) and according to the manufacturer's instructions.
Microarray imaging and data analysis
Changes in the gene expression of C. neoformans H99 cells exposed to FLC
BROAD ID (CNAG_*****)
C. n. gene name
S. c. gene name
Sterol regulatory element-binding protein 1
C-4 methyl sterol oxidase
C-8 sterol isomerase
C-22 sterol desaturase
Lanosterol 14 alpha-demethylase
C-5 sterol desaturase
Amino acid transporter
Efflux protein EncT
ABC transporter PMR5
Efflux protein EncT
Low-affinity zinc ion transporter
Small oligopeptide transporter
Vacuolar membrane protein
Aflatoxin efflux pump AFLT
Mitochondrial inner membrane protein
Hexose transport-related protein
Endoplasmic reticulum protein
Siderochrome-iron (Ferrioxamine) uptake transporter
Phosphatidylinositol transfer protein SFH5
Gamma-aminobutyric acid transporter
Cell wall maintenance
Chitin synthase 7
Glucan 1,3 beta-glucosidase protein
Chitin synthase 2, CHS2
Putative O-acetyl transferase
Lipid and fatty acid metabolism
Lincomycin-condensing protein lmbA
Heat shock protein 70
Thiol-specific antioxidant protein 3
Low temperature-responsive protein
Response to drug-related protein
Amino acid metabolism
Branched-chain alpha-keto acid dehydrogenase E1-alpha subunit
Glutamate synthase (NADH)
Aromatic amino acid aminotransferase I
Saccharopine dehydrogenase (NADP+, L-glutamate-forming)
Succinate-semialdehyde dehydrogenase (NAD(P)+)
Aldehyde dehydrogenase (ALDDH)
Glycogen (Starch) synthase
Protein biosynthesis, modification, transport, and degradation
AGC-group protein kinase
Mitogen-activated protein kinase CPK1
Endoplasmic oxidoreductin 1
Peptidyl-prolyl cis-trans isomerase D
Proteolysis and peptidolysis-related protein
Serine/threonine-protein kinase ATG1
Protein N-terminal asparagine amidohydrolase
2-oxoglutarate metabolism-related protein
Isocitrate dehydrogenase (NADP+)
Oxoglutarate dehydrogenase (Succinyl-transferring)
Succinate-CoA ligase (ADP-forming)
Isocitrate dehydrogenase (NAD+), putative
Type I phosphodiesterase/nucleotide pyrophosphatase family protein
Glucose-methanol-choline (GMC) oxidoreductase
Carbonic anhydrase 2
Cell cycle control
G1/s-specific cyclin pcl1 (Cyclin hcs26)
Putative uncharacterized protein
Meiotic recombination-related protein
Chromatin and chromosome structures
Nonhistone protein 6
Putative uncharacterized protein
DNA-binding protein hexbp
Putative uncharacterized protein
Rho GDP-dissociation inhibitor 1
G-protein beta subunit GPB1
Sulfite reductase (NADPH)
Fumarate reductase (NADH)
Temperature associated repressor
Antiphagocytic protein 1
AFG1 family mitochondrial ATPase
Integral to membrane protein
Integral membrane protein
Quantitative RT-PCR (qRT-PCR) validation of gene expression
Expression of selected differentially regulated genes as identified by the microarray analysis was quantitatively assessed with qRT-PCR in an i-Cycler iQ system (Bio-Rad Laboratories, Hercules, CA, USA). All primers and probes (see Additional file 1) were designed with Beacon Designer 2 (version 2.06) software (Premier Biosoft International, Palo Alto, CA, USA) and synthesized by MWG Biotech (Florence, Italy). qRT-PCRs were carried out as previously described . The annealing temperature used for all primers was 65°C. Each reaction was run in triplicate on three separate occasions. For relative quantification of target gene expression, ACT1 was used as a normalizer gene . Changes (n-fold) in gene expression relative to that of the control were determined from mean ACT1-normalized expression levels.
Oxidative stress and cell wall inhibitor assays
Susceptibilities to hydrogen peroxide (H2O2) and cell wall inhibitors were measured with exponentially growing cells in liquid YEPD at 30°C or 37°C pre-treated or not with FLC (10 mg/l) for 90 min as described elsewhere with modifications [26, 27]. The cells were next washed with sterile PBS and diluted to an OD650 of 1.0 in PBS. For the oxidative stress assays, aliquots of the cell suspensions were transferred to Eppendorf tubes where H2O2 (Sigma, Milan, Italy) was added to 20 mM and incubated at 30°C or 37°C for 2 h. Viability was determined after appropriate dilution of the samples with PBS by plating 100 μl in triplicate on solid YEPD. The CFU were counted after incubation for 72 h at 30°C or 37°C. For the cell wall inhibitor assays, dilutions of the cell suspensions were made in PBS and 5 μl of these were grown on YEPD plates containing 0.5% Congo red (Sigma, C-6767), 0.5, 1.0 and 1.5 mg ml-1 calcofluor white (Sigma, F-3543), 0.01%, 0.03% and 0.06% SDS (Sigma) and 0.2, 0.5 and 1.0 mg ml-1 caffeine (Sigma, C-0750). Plates were incubated for 48 h at 30°C or 37°C and photographed.
Results and Discussion
Experimental design and global gene expression results
The transcript profiles of C. neoformans H99 cells exposed to 10 mg/l of FLC (1/2 × MIC) for one doubling time (90 min) at 30°C were compared with profiles of untreated cells. A total of 476 genes were found responsive to FLC treatment under the test conditions, consisting of a single concentration and a single time point as described elsewhere [28–30]. The threshold value used in the present analysis was at least a twofold difference of gene expression between the experimental conditions, which is a value generally accepted in fungal genome-wide expression profiling . Given that approximately 95% of the genes (6434/6823) spotted on the microarrays gave validated data, the above mentioned number indicate that 7.4% of the total number of genes in the C. neoformans H99 genome exhibited transcriptional changes, with 231 genes being upregulated and 245 downregulated upon FLC treatment.
Gene Ontology (GO) term analysis for the C. neoformans FLC response
Small molecule metabolic process
Alcohol metabolic process
Sterol metabolic process
Steroid metabolic process
Phytosteroid metabolic process
Steroid biosynthetic process
Ergosterol biosynthetic process
Small molecule metabolic process
Alcohol metabolic process
Cellular ketone metabolic process
Cellular amino acid and derivative metabolic process
Organic acid metabolic process
Amine metabolic process
Gamma-aminobutyric acid metabolic process
Effect of FLC on genes involved in ergosterol biosynthesis and related pathways
Earlier efforts to profile the response of yeast cells (S. cerevisiae or C. albicans) to the short-term exposure to azole drugs implicated genes in the ergosterol biosynthetic pathway as major players [28, 29], thus indicating that this pathway is the target of azoles and is responsive to modulations in ergosterol levels. As shown in Table 1, we found that eight ERG genes (ERG1, ERG2, ERG3, ERG5, ERG7, ERG11, ERG13 and ERG25) exhibited increases in expression (2.09- to 3.95-fold) upon FLC treatment. This was a predictable result from the inhibition of Erg11 function by FLC, which is the rate-limiting step of the ergosterol biosynthetic pathway. Indeed, the idea of a compensatory response to re-establish the plasma membrane ergosterol levels  may account for the observed upregulation of either early (ERG13, ERG7 and ERG1) or late (ERG25, ERG2, ERG3 and ERG5) genes of the ergosterol pathway, in addition to upregulation of ERG11 itself (Table 1, ergosterol biosynthesis).
ERG13 encodes the enzyme hydroxymethylglutaryl-CoA synthase that catalyzes the production of hydroxymethylglutaryl-CoA from acetyl-CoA and acetoacetyl-CoA, and acts in the mevalonate biosynthesis, a precursor required for the biosynthesis of ergosterol. Acetyl-CoA is converted to carbon dioxide and water by enzymes (e.g. isocitrate dehydrogenase) that function in the TCA cycle, a central metabolic process in the mitochondria leading to produce, after oxidative phosphorylation, chemical energy in the form of ATP and NADH. Presumably, as a result of feedback control, we observed that several TCA cycle enzymes were downregulated in response to FLC (Table 1, TCA cycle), suggesting that C. neoformans may direct the cellular acetyl-CoA content to lipid (sterol) biosynthesis and metabolism to counterbalance ergosterol alteration.
Our particular interest was the up-regulation (4.04-fold) of SRE1, that belongs to a group of sterol regulatory element-binding proteins (SREBPs), first characterized in mammalian cells as regulator of lipid homeostasis . While C. neoformans Sre1 regulates genes encoding ergosterol biosynthetic enzymes, SRE1 was shown to be required for growth and survival in the presence of azoles and also for virulence in a mouse model of cryptococcosis [18, 20, 35]. In addition, C. neoformans Sre1 stimulates ergosterol production in response to sterol depletion when the oxygen-dependent ergosterol synthesis is limited by hypoxia . Consistently, C. neoformans mutants in the SREBP pathway showed reduction in ergosterol levels, increased sensitivity not only to low oxygen but also to several chemical agents, including azole antifungals, CoCl2 and reactive oxygen species (ROS)-generating compounds. Most importantly, these mutants showed reduced virulence in mice .
Effect of FLC on genes involved in cell structure and maintenance
Consequent to depletion of ergosterol and the concomitant accumulation of 14-methylated sterols, several plausible hypotheses on the mode of action of azoles were suggested by Vanden Bossche  two decades ago including alterations in membrane functions, synthesis and activity of membrane-bound enzymes, mitochondrial activities and uncoordinated activation of chitin synthesis. Transcript levels of several genes involving lipid and fatty acid metabolism decreased in the current study (Table 1), possibly in agreement with a remodelling of the cell membrane in response to reduced ergosterol levels. Conversely, expression of PLB1, that encodes Plb1, a known virulence factor in C. neoformans, was increased 2.18-fold. Phospholipases cleave fatty acid moieties from larger lipid molecules, releasing arachidonic acid for the production of eicosanoids that are utilized by the pathogenic yeasts C. neoformans and C. albicans to produce immunomodulatory prostaglandins . In addition, cell wall-linked cryptococcal Plb1 contributes to cell wall integrity and is a source of secreted enzyme .
It was also expected that exposure to FLC would affect genes responsible for cell wall integrity. Two chitin synthase genes were found to be significantly up-regulated (2.20-fold for CHS2 and 3.62-fold for CHS7), concomitantly with down-regulated expression (4.35-fold) of the chitin deacetylase CDA3 (homolog to S. cerevisiae CDA2) (Table 1, cell wall maintenance). In C. albicans, activation of chitin synthesis, which is mediated by the PKC-, Ca2+/calcineurin-, and HOG- cell wall signalling pathways, appears to be an adaptive response to caspofungin treatment. Hence, subculturing caspofungin-resistant cells in the absence of caspofungin resulted in wild-type levels of chitin content . While this form of drug tolerance is rationally accepted for a drug damaging the cell wall integrity (caspofungin is known to reduce β-glucan synthesis), it is also possible that exposure to azoles induces a salvage mechanism involving the up-regulation of chitin synthesis. Although known as a relatively minor cell wall component, chitin is thought to contribute significantly to cryptococcal wall strength and integrity . Chitosan, the enzymatically deacetytaled form of chitin, helps to maintain cell integrity and is necessary for maintaining normal capsule width and retention of cell wall melanin . Consistently, up-regulation was observed for BGL2 (2.61-fold) that encodes the glucantransferase (also termed glucosyltransferase) Bgl2, a major cell wall constituent described in a wide range of yeast species.
Effect of FLC on genes involved in cell stress and virulence
We found that FLC induced the expression of several genes involved in oxidative-stress response (Table 1, cell stress). One of these genes, GRE2, was induced 3.54-fold, consistent with the previous observation that transcripts from GRE2 and other stress-induced genes (YDR453C and SOD2) were increased in S. cerevisiae exposed to azoles . Interestingly, loss of Gre2 is impairing tolerance to ergosterol biosynthesis disrupting agents (i.e. clotrimazole and ketoconazole), further supporting an association between GRE2 and ergosterol metabolism . YHB1 that encodes a flavo-haemoglobin able to detoxify nitric oxide in C. albicans and C. neoformans was down-regulated 2.32-fold in our study, which is opposed to its established relevance in vivo . A strong reduction in the expression of FHB1 (the C. neoformans ortholog of YHB1) was also observed during growth of C. neoformans at 37°C compared to 25°C, indicating that regulation of this gene or its product at the posttranslational level may occur in response to environmental changes . In contrast, CTA1 encoding catalase in S. cerevisiae was induced (2.81-fold) by FLC exposure. Together with TSA3 (2.09-fold) encoding thiol-specific antioxidant protein 3 (Table 1, cell stress) and other responsive genes with oxidoreductase activity (Table 1, oxidoreduction), these genes may function in response to oxidative stress. Accordingly, the stress-related gene encoding Ssa1 was also up-regulated (2.48-fold). This C. neoformans protein (Hsp70 family member) acts in vivo as transcriptional co-activator of laccase  and is important for the production of melanin, which is a free-radical scavenger playing a protective role in stress resistance .
The C. neoformans polysaccharide capsule is a complex structure that is required for virulence [46, 47]. Interestingly, the capsule-associated gene CAS3  was found to be up-regulated (12.16-fold) upon exposure to the drug (Table 1, capsule synthesis). This gene encodes a protein belonging to a seven-member protein family that includes Cap64. Treatment with FLC did not significantly change expression of the essential capsule-producing genes, CAP10, CAP59, CAP60 and CAP64. Since the cryptococcal cell wall is needed for the localization or attachment of known or putative virulence factors other than capsule (i.e. melanin, Plb1 and Bgl2), it could be hypothesized that FLC induces alterations in the cell wall which in turns affects the expression of these factors. An alternative hypothesis would be that FLC acts as a stress-generating molecule and triggers enhanced expression of virulence determinant(s) that enable to survive in hostile environments.
Effect of FLC on genes involved in cellular transport
Several genes involved in small molecule transport and vesicular transport were either up- or down-regulated in response to FLC (Table 1, transport). These include DUR3 (plasma membrane transporter for urea, up-regulated by 4.78-fold), MEP2/AMT2 (ammonium permease, up-regulated by 3.78-fold) and AQY1 (aquaporin water channel, up-regulated by 2.73-fold), which all belong to the group of C. neoformans genes regulated by osmotic stress . It is possible that defects in the plasma membrane resulting from inhibition of ergosterol biosynthesis by FLC affects transport of small molecules through the membrane. Analysis of the H99 genome sequence  predicted 54 ATP-Binding Cassette (ABC) transporters and 159 major facilitator superfamily (MFS) transporters, suggesting wide transport capabilities of this environmental yeast . However, we found only two S. cerevisiae transporter homologues with significant increased expression. One is PDR15 that is a member of the ABC transporter subfamily exporting antifungals and other xenobiotics in fungi . The other gene is ATR1 that encodes a multidrug resistance transport protein belonging to the MFS class of transporters. ATR1 expression was recently shown to be upregulated by boron and several stress conditions . To date, Afr1 (encoded by AFR1; also termed CneAfr1) and CneMdr1 are the only two efflux pumps associated with antifungal drug resistance in C. neoformans . Since Afr1 is the major efflux pump mediating azole resistance in C. neoformans [11, 15], the absence of altered AFR1 expression could be expected. Not surprisingly, we noticed downregulated expression (2.35-fold) of FLR1 (for fluconazole resistance) encoding a known MFS multidrug transporter in yeast, that is able to confer resistance to a wide range of dissimilar drugs and other chemicals . This may suggest that both AFR1 and FLR1 do not participate to the short-term stress induced by FLC in C. neoformans.
Effect of FLC on the susceptibility to cell wall inhibitors
Effect of FLC on the susceptibility to H2O2
Although exposure to azoles has been already investigated in several other fungal species and the transcriptional profile of differentially expressed genes was obtained using a single FLC concentration and time point, our study reveals several interesting findings. First, we demonstrated that short-term exposure of C. neoformans to FLC resulted in a complex altered gene expression profile. These genes included not only genes commonly responding to diverse environmental stresses, such as oxidative and drug stresses, but also genes encoding virulence factors (i.e. Plb1, Sre1 and capsule). Second, we corroborated the potential of genome-wide transcriptional analyses to envisage alternative therapeutic strategies for cryptococcosis. Apart from ergosterol and its biosynthesis, there are yet few other targets to be exploited in anticryptococcal therapy. Therefore, elucidation of molecular processes underlying the physiological responses of cryptococcal cells to FLC could serve not only to identify novel treatment approaches but also to potentiate the inhibitory effects of existing azole drugs. Our findings show that the phenomena described can apply to the in vivo situation, i.e. during azole maintenance therapy in the host, but transcriptional analyses using different growth conditions of H99 cells, mimicking stress conditions encountered during a human meningeal infection, may reveal new fields to pursue for anticryptococcal therapy.
This work was supported by grants from the Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Lazzaro Spallanzani (Strategic Research Program 2006) to GF, from the Università Cattolica del S. Cuore (Fondi Ateneo Linea D1-2009) to MS, and from the Swiss Research National Foundation 31003A_127378 to DS.
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