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Eukaryot Cell. Feb 2010; 9(2): 251–265.
PMCID: PMC2823005

Comparative Transcript Profiling of Candida albicans and Candida dubliniensis Identifies SFL2, a C. albicans Gene Required for Virulence in a Reconstituted Epithelial Infection Model [down-pointing small open triangle]

Abstract

Candida albicans and Candida dubliniensis are closely related species displaying differences in virulence and genome content, therefore providing potential opportunities to identify novel C. albicans virulence genes. C. albicans gene arrays were used for comparative analysis of global gene expression in the two species in reconstituted human oral epithelium (RHE). C. albicans (SC5314) showed upregulation of hypha-specific and virulence genes within 30 min postinoculation, coinciding with rapid induction of filamentation and increased RHE damage. C. dubliniensis (CD36) showed no detectable upregulation of hypha-specific genes, grew as yeast, and caused limited RHE damage. Several genes absent or highly divergent in C. dubliniensis were upregulated in C. albicans. One such gene, SFL2 (orf19.3969), encoding a putative heat shock factor, was deleted in C. albicans. ΔΔsfl2 cells failed to filament under a range of hypha-inducing conditions and exhibited greatly reduced RHE damage, reversed by reintroduction of SFL2 into the ΔΔsfl2 strain. Moreover, SFL2 overexpression in C. albicans triggered hyphal morphogenesis. Although SFL2 deletion had no apparent effect on host survival in the murine model of systemic infection, ΔΔsfl2 strain-infected kidney tissues contained only yeast cells. These results suggest a role for SFL2 in morphogenesis and an indirect role in C. albicans pathogenesis in epithelial tissues.

Candida dubliniensis is the closest relative of the important opportunistic human pathogen Candida albicans (8, 21, 44). In spite of their close relationship, the two species show significant differences in virulence and epidemiology (42, 43). Although C. dubliniensis has historically been associated with oral infections in HIV-infected patients (33), it is generally less pathogenic than C. albicans, as judged by differences in carriage rates and prevalence in the human host (7, 27, 28, 45) and by differences in virulence in vitro and in murine infection models (8, 12, 23, 41). However, the reasons for these virulence differences are poorly understood and are the focus of investigations to determine their genetic basis.

Among traits important for virulence and variable between C. albicans and C. dubliniensis are adherence to human epithelial tissues and production of hydrolytic enzymes, as well as resistance to antifungal agents, oxidative stress, and phagocytosis by cells of the host immune system (8, 12, 20, 38, 41, 47). A trait critically important for Candida virulence especially in endothelial and epithelial infection models is the ability to undergo the yeast-to-hypha transition (hyphal morphogenesis) (16, 26, 31). Importantly, hyphal morphogenesis in response to many stimuli is consistently slower in C. dubliniensis than in C. albicans (8, 41). Some specific triggers that induce hyphal morphogenesis in C. albicans, such as increased CO2/HCO3 and growth in mammalian tissues (14, 37, 52), do not induce hyphae in C. dubliniensis (14, 23, 41). The transcriptional regulator Nrg1 has been identified as a key regulator suppressing filamentation in C. dubliniensis, and it has been suggested that this suppression of hyphal growth may partially explain why C. dubliniensis is less virulent in the human host than C. albicans (23).

The close phylogenetic relationship between C. albicans and C. dubliniensis may offer opportunities to identify virulence genes in C. albicans in infection models where the two species exhibit differences in virulence. A conceptually similar approach, using two C. albicans strains differing in their ability to invade tissue in an organ model of mammalian infection coupled with microarray-based analysis of gene expression, identified DFG16 as being required for pH sensing during tissue invasion (46). The approach adopted in the present study represents a progression from a study by Moran et al. (22), which analyzed the genome contents of C. albicans and C. dubliniensis by comparative genomic hybridization to C. albicans gene arrays. Although the vast majority of genes are highly conserved between the two species, approximately 200 C. albicans genes (representing ~4.0% of its genome) are absent or highly diverged in C. dubliniensis, including known virulence genes and genes of unknown function (11, 22). Availability of the genome sequences of C. albicans (4, 13) and C. dubliniensis (11) in database-accessible formats (2, 11) has enabled mining of the differences in the genetic repertoire of the two species.

For comparative study of C. albicans and C. dubliniensis gene expression, a suitable human infection model has to fulfill several criteria: chiefly ease of use, reproducibility, and sufficient recovery of fungal material for analysis. Reconstituted human oral epithelium (RHE) represents such a model, having been used extensively to study Candida virulence (reviewed in reference 36), including microarray-based transcriptional profiling (52). The RHE infection model permits quantitative measurements of virulence, and we have recently demonstrated that C. dubliniensis is far less virulent than C. albicans in the RHE model (23, 41). Here we report the results of gene expression profiling in C. albicans and C. dubliniensis coincubated with RHE tissue, representing the first comparative analysis of global transcription in these two species in an infection model. We further describe the identification of one C. albicans gene, which we have named SFL2 (orf19.3969; IPF8627.2), which is critical for filamentation in vitro and virulence in the RHE model.

MATERIALS AND METHODS

Strains and culture conditions.

The C. albicans and C. dubliniensis strains used in this study are listed in Table 1. Growth media were from Oxoid (Basingstoke, Hampshire, United Kingdom), and amino acids were from Sigma-Aldrich Ireland, Ltd. (Tallaght, Dublin, Ireland). All strains were cultured on yeast extract-peptone-dextrose (YPD) agar or in YPD broth at 37°C and 200 rpm, unless indicated otherwise. All liquid media for fungal cell cultures for microarray studies or in transformation experiments were filter sterilized. Hexose solutions (10× concentration) used in agar media were filter sterilized and added to molten agar media shortly before pouring.

Table 1.
Candida strains used in this study

Chemicals and enzymes.

All chemicals used were analytical grade or molecular biology grade and supplied by Sigma-Aldrich, Ambion (Warrington, United Kingdom), or Roche Diagnostics (Mannheim, Germany). Ultrapure Milli-Q water (Millipore Ireland B.V., Cork, Ireland) was used in all experiments. PCRs for cloning of DNA constructs were performed with Expand high-fidelity enzyme (Roche), and diagnostic PCRs were carried out with GoTaq enzyme (Promega, Madison, WI).

RHE inoculation and coincubation with Candida cells and RHE tissue damage measurements.

Inoculation and coincubation of RHE with Candida cells were performed as previously described (41). In brief, Candida cells were grown in semisynchronized cultures (i.e., in 25°C and 37°C serial YPD cultures) (36) and harvested by centrifugation, the cell density was adjusted in phosphate-buffered saline (PBS), and RHE tissues were inoculated as described previously (41). Candida cultures on polycarbonate filter (PCF) membranes (used as RHE support matrix) were initiated in the same way as the RHE cultures. Candida RHE and Candida PCF cultures and RHE-only controls were incubated in MCDB 153 maintenance medium (containing 0.1% glucose; Skinethic Laboratories, Nice, France) in a CO2 incubator at 37°C, with 5% (vol/vol) CO2 and 100% humidity, and sampled for RNA or lactate dehydrogenase (LDH) measurements (see below) at regular time points (30, 90, 360, or 720 min). Activity of human LDH in RHE culture medium was measured with the CytoTox 96 nonradioactive cytotoxicity assay (Promega) (41). The LDH assay product, formazan, was measured by spectrophotometry, and concentrations were determined with its extinction coefficient (15,600 M−1 cm−1). Candida RHE cultures and RHE controls (0.5 cm2) were fixed, sectioned, stained, and examined by light microscopy as previously described (41). Cell morphology in Candida PCF cultures was examined by light microscopy of cell suspensions obtained by rinsing the PCF membranes.

RNA extraction.

For RNA extractions, 2 ml of a solution containing 2 parts (vol/vol) RNAlater solution (Ambion) and 1 part filter-sterilized 10% (wt/vol) saponin (Sigma-Aldrich) in PBS was added to a 4-cm2 Candida RHE or Candida PCF culture. To recover the fungal cells, membranes were rinsed 3 or 4 times with the RNAlater-saponin solution, and the cell suspensions from 2 or 3 Candida RHE or Candida PCF cultures were transferred to a 50-ml centrifuge tube (Sarstedt, Wexford, Ireland) along with the cut filter membranes and immediately frozen and stored at −20°C. For total RNA extraction, cell suspensions containing the filter membranes were thawed at room temperature, vortexed for 5 to 10 s, and centrifuged (3,200 × g for 5 min). The membranes were removed, the tubes were centrifuged as before, and the supernatant was removed by careful aspiration. RNA was extracted with the RNeasy minikit (Qiagen, West Sussex, United Kingdom) with cell disruption performed in a Mikro-Dismembrator S system (Sartorius Stedim Biotech, Göttingen, Germany) for 2 min at 2,000 rpm. Candida cultures (~8 ml) used as the 0-min control (inoculum) in PBS (described above) were harvested by centrifugation as before, taken up in 2 ml RNAlater solution, and stored at −20°C; RNA was extracted from Candida cell pellets as described above. Total RNA was resuspended in nuclease-free water and DNase I treated with the DNAfree Turbo kit (Ambion), measured by 260/280 spectrophotometer readings, and integrity was checked on 1.2% Tris-borate-EDTA (TBE) gels. Absence of DNA in RNA samples after DNase treatment was routinely checked by PCR with primers qP-ACT1F and qP-ACT1R (Table 2).

Table 2.
Primers used in this study

Amplification and Cy labeling of RNA and hybridization to Candida gene arrays.

The Amino Allyl MessageAmp II aRNA amplification kit (Ambion) was used for RNA amplification and labeling, following the manufacturer's protocol. One microgram of total RNA was used for reverse transcription, and amino-allyl-labeled RNA (aaRNA) was linearly amplified by T7-based in vitro transcription (49). aaRNA was Cy labeled with N-hydroxysuccinimide esters of Cy3 and Cy5 dyes (GE Healthcare, Bucks, United Kingdom) and used in hybridizations to C. albicans gene arrays. Candida albicans 70-mer oligoarrays (NRC, Canada) representing 6,320 open reading frames (ORFs) spotted in duplicate on glass slides were used in gene expression profiling of C. albicans or C. dubliniensis RHE cultures 30 min postinoculation (p.i.) versus inoculum cultures (0 min). These experiments were carried out in 3 biological replicates (fresh RHE and Candida cultures set up on separate occasions) with dye swaps performed on biological replicates. C. albicans-spotted cDNA gene arrays (Eurogentec, Seraing, Belgium) representing 6,039 ORFs were used in expression profiling of Candida RHE cultures 90 min p.i., relative to Candida PCF cultures 90 min p.i., performed in two biological replicates with dye swaps carried out within each replicate. Approximately 5 μl of Cy-labeled aaRNA (1 μg of each treatment) was incubated at 70°C for 5 min, chilled on ice for 1 min, added to 55 μl digoxigenin (DIG) EasyHyb solution (Roche Diagnostics), and immediately loaded onto a microarray slide in a hybridization chamber (Corning, NY). Slides were covered with a plastic HybriSlip (Schleicher & Schuell, Keene, NH), incubated stationary in a hybridization oven at 42°C for 16 to 18 h, and washed in 50-ml washing solutions at room temperature. The following washes were performed: 10 min with 1× SSC (0.15 M NaCl plus 0.015 M sodium citrate) plus 0.2% SDS, 10 min with 0.1× SSC plus 0.2% SDS, and 5 min with 0.1× SSC. Slides were briefly dipped in fresh 0.1× SSC and sterile Milli-Q water, dried by centrifugation at 500 × g, and scanned immediately.

Microarray data analysis.

Microarray slides were scanned with a GenePix 4000B scanner (Axon Instruments, Sunnyvale, CA) at a resolution of 10 μm, using the auto PMT setting. Fluorescent intensity data were extracted using GenePix Pro 6.1 software (Axon Instruments). GenePix result (gpr) files were uploaded into ArrayPipe (http://www.pathogenomics.ca/arraypipe/) (10) for further analysis. Log2-transformed ratios of Cy5 to Cy3 intensities were calculated for each detected feature, effects of background subtraction and normalization methods were assessed with MA plots, and variation among technical replicates were assessed by interslide ratio plots. Analysis settings giving normal distribution of intensity ratios and acceptable variation among replicates were background subtraction with the normexp algorithm (35) and loess normalization on each subgrid. To identify consistently expressed genes, only those genes whose expression was detected in at least 2 biological replicates were included in further analysis. Statistical significance of differences in log2 ratios from 0 (no change) within groups was determined with empirical Bayes (eBayes) moderated one-sample t tests (39) and between groups by two-sample Student's t tests.

Sequence retrieval and analyses.

Genomic, exon-only, and predicted protein sequences were retrieved from the Candida Genome database (http://www.candidagenome.org/) or Candida dubliniensis GeneDB (http://www.genedb.org/genedb/cdubliniensis/) for C. albicans (Assembly 21) and C. dubliniensis, respectively, and used in primer design for real-time PCR and PCR-based cloning and for BLAST searches. Real-time PCR primers were designed in SciTools at IDT (http://www.idtdna.com/SciTools/SciTools.aspx?c = US) using the default settings; wherever possible, the length of PCR products was restricted to 80 to 150 bp. Primers were purchased from Sigma-Aldrich.

Extraction of fungal genomic DNA.

Candida genomic DNA for PCR-based cloning, diagnostic PCR, or quantitative PCR (qPCR) was extracted as described previously (40) with modifications. To a cell pellet from a 2-ml overnight culture in a 1.5-ml microcentrifuge tube was added 12 acid-washed glass beads (0.7 to 1 mm; Sigma-Aldrich) and 0.3 ml lysis buffer (40 mM Tris-acetate, 20 mM sodium acetate, 1 mM EDTA, 1% SDS [pH 7.8]); the mixture was vortexed for 1 min and incubated for 30 to 45 min at 65°C, and DNA was extracted as described previously (40).

RT and qPCR for quantitative analysis of gene expression.

Primers used in real-time quantitative PCR (qPCR) are listed with prefix “qP” in Table 2. Reverse transcription (RT) was performed with 250 ng of total RNA, 0.05 μM gene-specific primer, and Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) in 10-μl volumes following the manufacturer's protocol. Reverse transcription real-time qPCR (RT-qPCR) for quantification of gene expression was carried out with Power or Fast Sybr green kits (Applied Biosystems, Foster City, CA), with 25 μl containing 0.4 μM each primer and the ABI 7700 sequence detector or the ABI 7500 Fast real-time PCR system (Applied Biosystems). Each reaction was carried out in duplicate. Cycling conditions were 1 hold at 95°C for 10 min and 40 cycles of 95°C for 30 s, 50°C for 30 s, and 70°C for 1 min. Amplification efficiency of each gene primer set was determined with 7 serial DNA concentration steps (within 0.1 to 100 ng of genomic DNA). All primer sets had amplification efficiencies that were not significantly different from that for the endogenous control gene, ACT1 (P > 0.05; extra-sum-of-squares F test of regression slopes in GraphPad Prism 4.0c; GraphPad Software, Inc.; http://www.graphpad.com/prism/Prism.htm).

As found in previous studies (6), ACT1 expression varied among different growth conditions, but its variability was lower than that of PMA1 and TFB4, also evaluated as normalizing genes (M.J.S., unpublished observation). To account for the variation in ACT1 expression in the normalization of gene expression, the variation in ACT1 expression among nine treatments (inoculum cultures, as well as RHE and PCF cultures at four time points) was quantified with equally loaded RNA. Threshold cycle (CT) values for ACT1 obtained from two independent experiments carried out in three technical replicates were used to calculate an adjustment factor (AF) for each treatment (TR) by the equation AFACT1(TR) = 2CT ACT1(TR) CT ACT1(median), where CT ACT1(TR) is the CT for ACT1 in the treatment and CT ACT1(median) is the median CT of ACT1 of the 9 treatments. AF values were calculated for each C. albicans and C. dubliniensis and ACT1-normalized and AF-adjusted expression (= normalized expression [NE]) of a test gene (G) in each treatment determined from the CT s for G and for ACT1 by the equation NEG(TR)=2−[CT G(TR)CT ACT1(TR)] × AFACT1(TR)−1.

Candida gene knockout constructs.

Gene knockout (ko) constructs for orf19.7304 and SFL2 (orf19.3969) were generated by a PCR-based method (50). The SAT1 flipper cassette (34) was PCR amplified from M13 forward and reverse priming sites in plasmid pSFS2A, with primers having 80 bases at their 5′ ends that were identical to the 5′ upstream or 3′ downstream regions, respectively, of the targeted gene. PCR amplification was with primers M13F/5′ orf19.3969 and M13R/3′ orf19.3969 to replace SFL2 and M13F/5′ orf19.7304 and M13R/3′ orf19.7304 to replace orf19.7304 with the SAT1 flipper. All PCRs were performed in 50-μl reaction mixtures, using a cycling regimen of 1 hold at 95°C for 2 min and then 7 cycles of 95°C for 30 s, 58°C for 30 s, and 70°C for 4 min followed by 28 cycles of 95°C for 30 s and 68°C for 5 min. orf19.4445 was deleted by a split-marker approach. Its 5′- and 3′-flanking regions were PCR amplified from SC5314 genomic DNA with primers orf19.4445F1 and orf19.4445R1/M13F and orf19.4445F2/M13R and orf19.4445R2, respectively. Partial, overlapping SAT1 cassettes were generated by PCR with pSFS2A and primers M13F/orf19.4445R1 and Nourse-split2 and primers Nourse-split1 and M13R/orf19.4445F2. The 5′ and 3′ orf19.4445 flanks were fused to the partial SAT1 cassettes in a thermocycler reaction (1 cycle of 95°C for 2 min and then 10 cycles of 95°C for 30 s and 70°C for 5 min), followed by PCR with primers orf19.4445F1nest and Nourse-split2 (orf19.4445 5′-flanking region with a 3.8-kb SAT1 fragment) or primers orf19.4445R2nest and Nourse-split2 (orf19.4445 3′-flanking region with the 0.9-kb SAT1 fragment). The amplified deletion constructs were purified with the GeneElute PCR clean-up kit (Sigma) and concentrated by ethanol precipitation. DNA pellets (2 to 5 μg) were taken up in 5 μl sterile ultrapure water for transformation.

Candida transformation and confirmation of transformant genotypes.

Candida cells were transformed by electroporation as previously described (23) and selected on YPD plates with 200 μg/ml nourseothricin (Jena Bioscience GmbH, Jena, Germany). Correct integration of SAT1 into the targeted gene locus was checked with primers SAT1check1 or SAT1check2 and primers annealing outside the gene region targeted for knockout.

C. albicans is diploid; therefore, the two alleles of each gene were removed by sequential deletion, utilizing the reusable SAT1 flipper marker (34). The number of alleles in heterozygous and homozygous ko and complemented strains was checked by qPCR with genomic DNA (1 ng per 20-μl reaction) and the applicable “qP” primers (Table 2) annealing within the gene regions targeted for deletion, using the same reagents and instrumentation as described for RT-qPCR. Amplification of ACT1 was used as a normalizing control for loading, and wild-type (WT) SC5314 was used as the calibrator for determination of the number of alleles in each transformant.

Complementation of SFL2-deleted transformants.

To reintroduce SFL2 into ΔΔsfl2 strains, the SFL2 ORF was amplified from SC5314 genomic DNA by PCR (35 cycles of 95°C for 30 s and 68°C for 3 min) with primers orf19.3969complF containing 80 bases with 100% identity to the region immediately upstream of the deleted SFL2 region and orf19.3969complR/M13F, containing 80 bases 100% identical to the SFL2 3′ untranscribed region (UTR) gene region—deleted in ΔΔsfl2 strains—and M13F sequence in reverse complement orientation. The SAT1 cassette was PCR amplified as before with primers M13F/orf19.3969complR and M13R/3′ orf19.3969, containing 80 bases 100% identical to the intergenic region immediately downstream of the deleted SFL2 3′ UTR region. Both fragments were targeted to the SFL2 locus via the 80-nucleotide-long SFL2-flanking sequences; the 98-bp sequence overlapping the SFL2 fragment with the SAT1 cassette was absent in the ΔΔsfl2 strains and permitted in vivo recombination between the two fragments. The two DNA fragments were purified as described above, mixed in equimolar amounts to give 2 to 5 μg DNA in 5 μl, and used to transform Candida cells. Transformants were selected by nourseothricin resistance, and reintroduction of SFL2 was checked by PCR and qPCR as described above.

SFL2 overexpression in C. albicans and expression in C. dubliniensis.

An SFL2 overexpression vector for C. albicans was constructed using Gateway cloning technology (Invitrogen). The SFL2 ORF devoid of its stop codon was amplified from C. albicans genomic DNA using primers orf19.3969GTWF and orf19.3969GTWR and a cycling regimen of 1 hold at 95°C for 3 min and then 7 cycles of 94°C for 15 s, 52°C for 15 s, and 72°C for 2.5 min followed by 26 cycles of 94°C for 15 s, 60°C for 15 s, and 72°C for 2.5 min. The PCR product was cloned in the pDONR207 vector using the Gateway BP clonase (Invitrogen) according to the supplier's instructions. Using Gateway LR clonase (Invitrogen), the SFL2 ORF was then transferred into the CIp-Op2 expression vector, a derivative of CIp10 (25) that harbors a doxycycline-inducible promoter (30) and a Gateway cloning cassette (A. Firon and C. d'Enfert, unpublished results). A control plasmid was constructed using the ORF for green fluorescent protein (GFP). The resulting SFL2 and GFP overexpression plasmids were linearized with StuI to promote targeting at the C. albicans RPS1 locus and transformed into C. albicans strain CEC955, yielding strains CEC1352 and CEC1147, respectively.

To express CaSFL2 in C. dubliniensis strains CD36 and Wü284, the following procedure was used. Plasmid CdpNIM1 was produced by replacing the C. albicans 5′ ADH1 DNA sequence in pNIM1 (30) with the orthologous C. dubliniensis ADH1 sequence, obtained by PCR using the primers CdADH1 F1 and CdADH1 R1 (which contain SacII and XbaI restriction enzyme sites, respectively). The 3′ ADH1 sequence was left unchanged in pNIM1, because it is highly similar in C. dubliniensis. This plasmid was then digested with SalI and BglII to replace the GFP-coding sequence with the SFL2 (orf19.3969) ORF (containing SalI and BglII cutting sites on either end), PCR amplified from C. albicans SC5314 using primers SFL2 F2 and SFL2 R2. The resulting CdpNIM1SFL2 construct and the GFP control, CdpNIM1, were linearized with SacII and KpnI for electroporation into CD36 or Wü284. Transformants were screened by PCR for the presence of SFL2 (CD36/pNIM1SFL2 and Wü284/pNIM1SFL2) or GFP (CD36/pNIM1GFP and Wü284/pNIM1GFP), and maintained on YPD containing 100 μg/ml nourseothricin.

Phenotypic analysis of gene deletion strains in vitro and in vivo.

For germ tube induction tests, Candida cells were grown in YPD overnight at 30°C or 37°C and inoculated into 10% (vol/vol) fetal calf serum (FCS) in water (41). Germ tubes were identified by light microscopy at a 400× magnification as filaments nonconstricted at the septa. To generate microaerophilic conditions, cells were grown in YPD overnight at 30°C with shaking (200 rpm) and collected by centrifugation. Approximately 100 cells in 5 μl PBS were added to 50 μl of yeast extract-2% sucrose (YPSuc) liquid agar (2%; cooled to 50°C), spotted on 10 ml YPSuc agar, overlaid with 15 ml YPSuc agar, and grown for 24 to 48 h at 25°C or 37°C. Spider and Pal's agar media were prepared as described previously (1, 18), and cells were incubated at 30°C or 37°C for 5 days. The effects of a pH shift on morphology were tested by using cells precultured overnight in liquid Lee's medium (15) at pH 4.5 and 30°C, which were diluted 1:500 into fresh Lee's medium at pH 6.5 and 37°C.

C. albicans morphological responses to CO2 were tested by adding cells from an overnight YPD shaking culture (200 rpm) grown at 37°C to give a density of 2 × 106 cells/ml in 25 ml yeast nitrogen base (YNB) medium (Sigma-Aldrich) with 0.1% glucose and an amino acid mixture based on the composition of artificial saliva (51). Cells were then incubated in static cultures at ambient (0.038%) or 5% CO2 at 37°C and examined by light microscopy. To test the sensitivity of gene knockouts and the WT to specific stresses, they were grown in YPD broth at 30°C to mid-exponential phase, and 10-fold serial dilutions of these cells spotted onto YPD plates and YPD plates supplemented with either 1 M NaCl or 5 mM H2O2. Growth was monitored after 24 and 48 h at 30°C (42°C for the heat shock assay).

Virulence of deletion mutants in the mouse systemic model of infection was measured as described previously (23). For each fungal strain, six female BALB/c mice (6 to 8 weeks old) (Harlan, United Kingdom) were inoculated intravenously with 1.2 × 104 to 1.4 × 104 cells/g of body weight and survival was monitored over 28 days. Mice were humanely terminated when they had lost 20% body weight, showed signs of distress, or were no longer able to freely access food and water. Kidneys, spleen, and brain were removed from culled mice, and fungal burdens were determined by homogenizing tissues in sterile saline and plating on YPD agar to determine viable cell counts. In a second experiment, for each strain, five female BALB/c mice were inoculated intravenously with 2.8 × 104 to 3.2 × 104 cells/g of body weight. Mice were culled on day 3 postinfection, and kidneys, liver, lung, spleen, and brain were sampled for fungal organ burdens. In both infection models, half of each kidney was also fixed in formalin, to allow paraffin sections to be produced. Kidney sections (5 μm) were stained by Grocott's methenamine silver stain and poststained with light-green SF yellowish stain, or periodic acid Schiff stained with hematoxylin poststaining.

RESULTS

Candida albicans and Candida dubliniensis show major differences in growth morphology very early during RHE infection.

As shown previously, C. albicans is more virulent in the RHE infection model than C. dubliniensis because C. dubliniensis is unable to form hyphae in this model (23, 41). These earlier studies focused on RHE colonization ≥12 h postinoculation (p.i.) when differences in hyphal formation were most apparent. To identify genes involved in the very early stages of this morphological switch in C. albicans, we conducted a time course study of RHE infection to determine the time points at which the differences in growth morphology between the two species first became evident. To this end, RHE tissues were inoculated with C. albicans SC5314 or C. dubliniensis CD36, incubated in 5% CO2 at 37°C, and sampled at regular time points 1 to 12 h postinoculation for tissue histology and to measure RHE tissue damage (41). C. albicans cells had begun to form germ tubes by 1 h p.i., whereas C. dubliniensis cells grew in the yeast phase (Fig. 1A), in which they remained for the whole duration of the experiment (12 h). These results indicated that major differences in growth morphology between C. albicans and C. dubliniensis occurred within 1 h of RHE colonization. RHE tissue damage (measured as LDH activity release into the RHE medium) by C. albicans started to increase within 4 to 12 h p.i., while damage of RHE tissues inoculated with C. dubliniensis was identical to that in the uninfected control (Fig. 1B).

Fig. 1.
Growth morphology and tissue damage by C. albicans SC5314 and C. dubliniensis CD36 on RHE. Growth on RHE 1 to 2 h p.i. (A) and RHE tissue damage (measured as LDH activity released from RHE Candida cultures) by the two species 1 to 12 h p.i and in uninoculated ...

C. albicans, but not C. dubliniensis, shows early upregulation of virulence genes on RHE.

The above results suggested that significant differences in gene expression underlying the morphological differences between the two species may already be present within <1 h of RHE colonization. Therefore, we chose 30 min p.i., at which time the vast majority of cells grew as yeast (data not shown) for comparative analysis of global gene expression with whole-genome microarrays. Total RNA was extracted from cells of both species on RHE 30 min p.i. and from the 0-min reference controls (Candida cells from the same cell pools used to inoculate the RHE; see Materials and Methods). Cy-labeled amplified RNAs (aaRNAs) from C. albicans or C. dubliniensis RHE cultures 30 min p.i. were cohybridized with aaRNAs from the respective 0-min control cultures to C. albicans oligonucleotide microarrays (NRC, Canada). In three biological replicates, 2,654 and 1,301 consistently expressed genes were identified in C. albicans and C. dubliniensis, respectively.

To identify genes highly expressed in both species in the RHE, the following selection criteria were applied: ≥2-fold upregulation in RHE 30 min p.i. and statistically significant (P < 0.05; eBayes t test) difference from 1 (no change in expression). This yielded 268 genes (10.1% of all expressed genes) in C. albicans and 82 (6.3%) genes in C. dubliniensis, with 47 genes upregulated in both species (see Tables S1 and S2 in the supplemental material). The majority of upregulated genes in the two species fell into the categories of protein synthesis, cellular transport, membrane, amino acid metabolism, or mitochondrial genes (Fig. 2). The array probes contained C. albicans sequences; so given some sequence divergence between C. albicans and C. dubliniensis genes (11, 22), detection of fewer expressed genes in C. dubliniensis was expected. To distinguish between lack of detection due to sequence divergence and that due to absence of expression in C. dubliniensis, we estimated the minimum sequence identity between probe and target sequences for detection of expression in C. dubliniensis by using the array probe sequences for all genes (shown in Tables S1 and S2 in the supplemental material) in BLASTN searches of C. dubliniensis GeneDB (http://www.genedb.org/genedb/cdubliniensis/blast.jsp). The majority of genes (76%) showing consistent expression in C. dubliniensis had probe-target similarities of ≥90% along ≥68 nucleotides (see Table S2 in the supplemental material), indicating that this level of probe-target similarity permitted reliable detection of expression of C. dubliniensis genes with the C. albicans oligoarray.

Fig. 2.
Functional categories of the genes showing significant upregulation in C. albicans and C. dubliniensis in RHE 30 min p.i. relative to 0 min (control). Biological categories of the upregulated genes were assigned with CGD's Gene Ontology Slim Mapper ( ...

Ribosomal protein genes were the largest group showing upregulated expression in RHE 30 min p.i.: 83 (30% of all upregulated genes) and 37 (44%) ribosomal protein genes were ≥2-fold upregulated in C. albicans and C. dubliniensis, respectively (see Tables S1 and S2 in the supplemental material). RT-qPCR confirmed the expression for two ribosome-biogenesis genes in both species: RPS7A, whose dynamics in increase of expression were very similar to those of the hyphally regulated C. albicans gene ECE1 (Fig. 3A) and those of NOP1 (Fig. 3B) in both species. Gene expression profiles were consistent with the respective morphologies of the two species in the RHE. Even at this early stage, C. albicans exhibited upregulation of several hyphally regulated and virulence-associated genes, including ECE1, HWP1, HYR1, ALS3, IHD1, and RBT1 (see Table S1 in the supplemental material), encoding hypha-specific proteins or cell surface adhesins, commonly upregulated in human tissue models (32, 46, 52). With the exception of IHD1, probes for all of these genes had <90% similarity to C. dubliniensis sequences, and no upregulated expression of any hyphally regulated genes was detectable in C. dubliniensis, with the exception of one gene, NIP7, encoding a hyphally induced ribosomal protein (see Table S2 in the supplemental material). The C. albicans gene set was also significantly enriched for genes encoding protein mannosyltransferases: i.e., PMT1, PMT2, PMT4, and PMT6 were >2-fold upregulated in C. albicans, but not in C. dubliniensis (sequence similarity to the PMT gene probes was >90%, except for being 89% for PMT4 [see Table S1 in the supplemental material]). The numbers of genes showing significant (P < 0.05) and ≥2-fold-downregulated expression in the RHE were 176 (6.6% of all expressed genes) for C. albicans and 49 (1.9%) for C. dubliniensis, with 16 genes showing significant downregulation in both species. The majority (>50%) of downregulated genes had unknown functions, and several encoded enzymes for intra- and extracellular metabolite transport (ABC transport proteins as well as peptide and hexose transporters [see Tables S3 and S4 in the supplemental material]).

Fig. 3.
Expression of RPS7A, ECE1, and NOP1 and genes absent from or divergent in C. dubliniensis 0 to 12 h p.i. on RHE or PCF. (A) Expression of RPS7A in C. albicans and C. dubliniensis and ECE1 in C. albicans on RHE; (B) expression of NOP1 in C. albicans and ...

Besides interactions with epithelial cells in the RHE tissue, other factors in the RHE environment, including CO2 levels and microaerophilic conditions, could affect morphology and gene expression in the Candida cells. Therefore, to control for these conditions (29), we also grew C. albicans and C. dubliniensis on the polycarbonate filters (PCF) used as support matrix for the RHE. C. albicans—but not C. dubliniensis—formed hyphal elements when incubated on the polycarbonate filters under the same conditions and in the same growth medium as the RHE cultures (not shown). To profile gene expression, cells of each Candida species were incubated on RHE or PCF for 90 min. aaRNAs from RHE and PCF cultures were cohybridized to Eurogentec's C. albicans cDNA array, and in two biological replicates, consistent expression of 4,613 genes for C. albicans and 1,368 genes for C. dubliniensis was detected. In total, 222 (4.8%) genes in C. albicans (see Table S5 in the supplemental material) and 122 (8.9%) genes in C. dubliniensis (see Table S6 in the supplemental material) showed significantly (P < 0.05) and ≥1.5-fold upregulated expression on RHE 90 min p.i. relative to PCF 90 min p.i.

There were 78 genes that displayed upregulation on RHE relative to PCF in both species (35% and 64% of all upregulated in genes in C. albicans and C. dubliniensis, respectively). Among these were several heat shock protein genes (i.e., HSP12, HSP60, HSP70, HSP78, and HSP104 [see Tables S5 and S6 in the supplemental material]), suggesting similarities in stress response in the two species. Also upregulated in both species were genes encoding enzymes for utilization of C2 compounds (i.e., fatty acid β-oxidation, glyoxylate cycle, and gluconeogenesis), such as POX1-3, ICL1, and PCK1, which was confirmed by RT-qPCR analysis (data not shown).

C. albicans shows early upregulation of several genes absent or divergent in C. dubliniensis.

Following the initial characterization of the C. albicans and C. dubliniensis gene sets, we focused on uncharacterized genes that may be absent from or divergent in C. dubliniensis (22), as increased expression of these genes in C. albicans in the RHE may provide clues to their possible involvement in Candida virulence in this model. In this analysis, genes were preselected based upon ≥1.5-fold upregulation (P < 0.05) in C. albicans and on expression significantly different from expression in C. dubliniensis (P < 0.05; Student's t test). In the RHE experiment 30 min p.i. versus the 0-min experiment, 146 genes fulfilled these criteria (data not shown), 20 of which had unknown functions and 3 of which were predicted to be absent or very divergent in C. dubliniensis (Table 3). One of these genes, HYR1, encoding a hyphal cell wall protein, showed ≥2-fold upregulation in C. albicans in RHE. The other two, orf19.3969 (IPF8627) and orf19.7304 (IPF19812), had only predicted or unknown functions, respectively. Because of sequence relationships with SFL1 described below, we assigned orf19.3969 the name “SFL2.”

Table 3.
C. albicans genes of unknown function or with divergent orthologues in C. dubliniensis showing significanta and ≥1.5-fold upregulation in C. albicans in RHE 30 min p.i.

In the RHE-PCF 90-min p.i. comparison, we identified 24 genes with unknown function that were predicted to be absent from or divergent in C. dubliniensis (10.8% of all upregulated genes in C. albicans) and that had ≥1.5-fold upregulation on the RHE relative to PCF in C. albicans (Table 4). Among these genes was HYR1, also upregulated in RHE 30 min p.i. relative to 0 min (see above), while SFL2 was not among the genes showing differential expression in RHE relative to PCF 90 min p.i. (confirmed by RT-qPCR analysis) (Fig. 3), suggesting similar regulation of this gene under the two conditions.

Table 4.
C. albicans genes of unknown function or with divergent orthologues in C. dubliniensis with significanta and ≥1.5-fold upregulation in C. albicans on RHE relative to expression on PCF 90 min p.i.

RT-qPCR confirmed very early upregulation of SFL2 after inoculation onto RHE. C. albicans SFL2 was >3-fold increased (P < 0.05; t test) 30 min p.i. compared to expression at 0 min and declined 90 to 720 min p.i.: SFL2 expression was also upregulated in cells grown on PCF 30 min p.i., but tended to be lower than expression in RHE 30 min p.i. (Fig. 3). The putative SFL2 orthologue in C. dubliniensis, Cd36_54430, identified based on both sequence homology and synteny (in the Candida Genome Database) (2), showed no appreciable increase in expression under any condition, and its expression levels appeared to be more than 1 order of magnitude lower than expression of C. albicans SFL2. Expression of orf19.7304 in C. albicans increased rapidly within 90 min on both RHE and PCF, with a gradual decline approximately 360 to 720 min p.i. (Fig. 3).

SFL2 is required for C. albicans filamentation in response to multiple environmental signals.

Upregulated expression of SFL2 (orf19.3969) and orf19.7304 in the RHE suggested their possible involvement in C. albicans virulence. BLAST alignments of the predicted protein sequences for SFL2 with its closest C. dubliniensis orthologue, Cd36_54430, and orf19.7304 with its closest orthologue, Cd36_34500, indicated only 50% identity (58% similarity) and 68% identity (78% similarity) between them, respectively. Therefore, we focused on SFL2 and orf19.7304 for functional analysis by targeted gene knockout. Also included in these tests was orf19.4445, as its expression increased in C. albicans upon transfer to the RHE (not shown) and was 3-fold greater than expression on PCF 90 min p.i. (Table 4).

SFL2, orf19.7304, and orf19.4445 were deleted separately in SC5314 by replacement of each gene with the SAT1 flipper cassette (34). Several heterozygous (Δ) and homozygous (ΔΔ) knockout (ko) strains were generated (Table 1), and their phenotypes were tested in a range of filamentation-inducing conditions. Deletion of orf19.7304 and orf19.4445 had no detectable effects on cell morphology in 10% fetal calf serum (FCS) and embedded growth in agar (not shown) and were not investigated any further in this study. However, cells of the Δsfl2 and ΔΔsfl2 ko strains displayed filamentation defects under several conditions. When cells of the ΔΔsfl2 ko strains were precultured at 30°C in YPD and inoculated into 10% FCS and incubated at 37°C, they formed germ tubes at a rate similar to the wild type (WT) (data not shown). However, in the absence of a temperature shift (37°C preculture), germ tube formation in the ΔΔsfl2 strains was reduced by approximately 50% and 7% compared to that in the WT after incubation in 10% serum for 30 min and 45 min, respectively.

SFL2 ko mutants were also impaired in hyphal formation in response to a pH shift. Following a shift from pH 4.5 to pH 6.5 in Lee's liquid medium, after 3 h of incubation, approximately 80% of WT cells had produced germ tubes or true hyphae, whereas Δsfl2 cells exhibited only 10 to 20% germ tube formation, and ΔΔsfl2 cells failed entirely to form hyphae in response to this pH shift (data not shown). Reintroduction of a WT SFL2 allele into the ΔΔsfl2 strains restored filamentation to levels similar to those of the heterozygous Δsfl2 strains (data not shown).

We then tested morphological responses to increased CO2, which induces hyphae in C. albicans (14, 37). To detect CO2-specific morphogenic responses, we used a YNB medium with 0.1% glucose and supplemented with amino acids (YNB-Gluc-AA [see Materials and Methods]) in which the WT formed germ tubes only in 5% CO2 and not in ambient CO2 (Fig. 4). In YNB-Gluc-AA and 5% CO2, the Δsfl2 strain formed fewer and less-elongated germ tubes than the WT, the ΔΔsfl2 strain did not form any germ tubes, and the ΔΔsfl2 strain with reintroduced SFL2 showed germ tube formation similar to that of the Δsfl2 strain.

Fig. 4.
Growth of C. albicans SFL2 knockout and complemented strains under high-CO2 conditions. Shown are cells of wild-type strain SC5314 in ambient (0.038%) CO2 (A) and 5% CO2 (B) and cells of the Δsfl2 strain (CaMS46-2) (C), ΔΔsfl2 ...

We next tested the morphological responses of the mutant strains to microaerophilic conditions that induce filamentation in C. albicans (5). Cells were embedded in YPSuc agar and incubated at 25°C or 37°C; identical results were obtained at both temperatures, and only results for embedded growth at 37°C are shown. The WT strain formed long elongated filaments (Fig. 5A and B). In contrast, only 20% of Δsfl2 colonies produced some filaments, and all ΔΔsfl2 colonies were completely smooth and formed only short chains of yeast cells or pseudohyphae. Reintroduction of the SFL2 wild-type allele into the ΔΔsfl2 strain restored hyphal formation to a level similar to that of the Δsfl2 strain.

Fig. 5.
Growth morphologies of C. albicans SC5314 and SFL2 deletants under agar-embedded (microaerophilic) conditions. (A) Colonies of the WT strain, Δsfl2 strain (CaMS46-2), ΔΔsfl2 strain (CaMS49-1), and ΔΔsfl2 strain ...

When incubated on Pal's agar at 30°C for <72 h, C. albicans grows as smooth colonies; however, when grown at 37°C for >72 h, it forms smooth colonies with a hyphal fringe (D.J.S., unpublished data). Following growth for 96 h at 37°C on Pal's agar, wild-type SC5314 formed colonies with a hyphal fringe, as expected, whereas ΔΔsfl2 colonies lacked a hyphal fringe (Fig. 5C). On Spider agar, the wild-type strain formed wrinkled colonies with a hyphal fringe (data not shown), while the Δsfl2 strain exhibited wrinkled colonies without a fringe and the ΔΔsfl2 strain grew as fringeless, completely smooth colonies. The ΔΔsfl2 strain complemented with a wild-type copy of SFL2 formed wrinkled colonies on Spider agar that were similar to those of the Δsfl2 strain (data not shown).

Deletion of SFL2 had no detectable effect on growth under aerobic conditions in standard media: the doubling time of the ΔΔsfl2 strain during mid-log phase (60 to 330 min) in YPD at 37°C was 60.6 ± 2.1 min (median ± standard error [SE]; n = 2) and essentially identical to that of the parental WT strain (60.8 ± 2.7 min). Growth of ΔΔsfl2 cells in response to heat, salt, or oxidative stress was similar to that of the WT (data not shown).

Overexpression of SFL2 from a doxycycline-inducible promoter in C. albicans resulted in the formation of filaments in liquid YPD at 30°C, while the control (expressing GFP from the same promoter) grew only as yeast (Fig. 6). Moreover, when the SFL2-overexpressing strain CEC1352 was grown on solid YPD or YPD plus 1% FCS at 37°C, colonies appeared wrinkled, whereas the colonies of the GFP-overexpressing strain remained smooth. Expression of CaSFL2 in C. dubliniensis CD36 or Wü284 under the control of the doxycycline-inducible promoter and grown in YPD and doxycycline resulted in elongated cells and pseudohypha-like elements, whereas the GFP-expressing strains grew solely as yeast cells (data not shown).

Fig. 6.
Overexpression of SFL2 in C. albicans. Strains CEC1352 (Table 2; indicated by TETp-SFL2), overexpressing SFL2 from a doxycycline-inducible promoter, and CEC1147 (Table 2; indicated by TETp-GFP), expressing GFP from a doxycycline-inducible promoter, were ...

SFL2 is required for RHE tissue colonization and damage by C. albicans, but not for virulence in the mouse model of systemic infection.

To see if SFL2 deletion affected colonization and tissue damage to the RHE, the SFL2 ko strains were tested in this model. Compared to the WT, which exhibited extensive filamentation in the RHE, the Δsfl2 strain showed only few hyphal elements, and cells of the ΔΔsfl2 strains were completely impaired in hyphal morphogenesis in the RHE; ΔΔsfl2 strains with reintegrated SFL2 showed filamentation levels similar to those of the Δsfl2 strain (Fig. 7). RHE damage mirrored the morphological phenotypes: i.e., sequential deletion of each SFL2 allele resulted in a gradual reduction of tissue damage: LDH release caused by Δsfl2 strains was approximately 40% of that of the WT, and LDH release by ΔΔsfl2 strains was about 50% of LDH release by Δsfl2 strains and significantly lower than that of the WT (P < 0.01; analysis of variance [ANOVA] and Tukey's test), with RHE damage similar to that in the uninfected control. RHE damage by ΔΔsfl2 strains with reintegrated SFL2 was similar to that of the Δsfl2 strains.

Fig. 7.
Growth morphology and tissue damage by C. albicans SFL2 knockout and complemented strains in RHE. (A) Growth morphology in RHE 33 h p.i. Hyphal elements present in SC5314 (WT) and the Δsfl2 and ΔΔsfl2/SFL2 strains are indicated ...

Finally, we tested ΔΔsfl2 strains in in vivo mouse models of systemic infection, using the conventional 28-day model and a 3-day infection model. In these experiments, virulence of the ΔΔsfl2 strain CaMS49-1 was very similar to that in the WT: survival times of mice inoculated with ΔΔsfl2 or WT (SC5314) cells were 16.7 ± 3.9 and 15.3 ± 2.9 days (mean ± SE; n = 6), respectively (P > 0.05; Kaplan-Meier and log rank statistics), with no significant differences (P > 0.05; ANOVA) detected for Candida burdens in the kidney, spleen, or brain (data not shown). To examine whether changes in organ burdens were evident at an earlier time point during infection, mice were infected and then sampled at 3 days postinfection. Again, there was no significant difference in Candida burdens for the kidney, lung, liver, spleen, and brain (data not shown). Interestingly, histology of kidney sections obtained from the 3- and 28-day infection models revealed that the ΔΔsfl2 strain was defective in hypha formation, growing almost exclusively as yeast cells with only a few short filaments (Fig. 8). Consistent with the in vitro phenotypes, Δsfl2 strain-infected renal lesions showed an intermediate phenotype, with some lesions containing long hyphae and others containing short filaments or yeasts (Fig. 8).

Fig. 8.
Morphology of C. albicans SFL2 knockout strains in mouse kidneys. Kidney sections (5 μm; 3 days postinfection) were stained with methenamine silver and poststained with light green. The photographs shown represent two different magnifications. ...

SFL2 encodes a putative DNA-binding heat shock factor protein.

BLASTP searches of the NCBI nonredundant (nr) protein database with the inferred Sfl2p sequence (714 amino acids) gave a significant match with Hsr1p (E = 1e−51, 64% identity; alignment of 129 amino acids at the Sfl2p protein N terminus), described as a heat-shock-related transcription factor (HSF) in Candida tropicalis (CAC12663), and WU-BLAST2 searches of Saccharomyces cerevisiae YeastDB (at http://seq.yeastgenome.org) gave significant (E < 1e−18) matches to several HSF-type proteins, including Sfl1p (19 to 62% identity) and Mga1p (25 to 28% identity). A highly conserved HSF-type DNA-binding domain (pfam00447; E = 6e−16) and an HSF-type DNA-binding domain signature (PS00434; at amino acids 57 to 81) were detected in the N-terminal region of Sfl2p, also present at the same location (amino acids 58 to 82) in its putative C. dubliniensis orthologue, Cd36_54430p. Sfl2p shared about 24% identity and the HSF-type signature with its closest relative in C. albicans, Sfl1p (orf19.454), which is a negative regulator of hyphal morphogenesis (3, 17).

DISCUSSION

The opportunistic pathogens C. albicans and C. dubliniensis have a close phylogenetic relationship and share several morphophysiological traits. Despite this, they display large differences in virulence, with C. albicans being much more pathogenic in the human host and in human infection models than C. dubliniensis. Here, to identify novel virulence-associated genes in C. albicans and begin to characterize commonalities and differences in gene expression between the two species, we compared global gene expression in C. albicans and C. dubliniensis in the RHE model of the oral mucosa. Both species exhibited coordinated upregulation of primary metabolism genes in RHE 30 min postinoculation, indicating conserved responses in general metabolism in the two species. However, whereas C. albicans showed rapid filamentation and caused increased RHE tissue damage, as well as upregulated expression of several known hyphally regulated genes, C. dubliniensis grew only as yeast cells, caused very limited RHE damage, and lacked detectable upregulation of hyphal genes. C. albicans also displayed upregulation of several genes of unknown function that were absent or significantly divergent in C. dubliniensis. One such gene, SFL2 (orf19.3969), showed significant upregulation in C. albicans on the RHE 30 min p.i. and high (~50%) sequence divergence with its likely orthologue in C. dubliniensis (Cd36_54430). When SFL2 was deleted in C. albicans, cells were unable to form hyphae under a variety of conditions, including growth under microaerophilic or high-CO2 conditions, in response to pH shifts, and in mouse kidneys. Overexpression of SFL2 resulted in increased filamentation in C. albicans and the production of pseudohypha-like cells in C. dubliniensis. Deletion of SFL2 in C. albicans had no effect on mouse survival in the systemic infection model; however, SFL2 deletion led to decreased RHE colonization and damage, demonstrating a possible role for SFL2 in virulence in this oral mucosal infection model.

The greater virulence of C. albicans and the limited virulence of C. dubliniensis were consistent with earlier observations (12, 41) and with lower carriage and prevalence of C. dubliniensis in the oral cavities of healthy individuals (42, 43). C. albicans and C. dubliniensis displayed broadly similar patterns of coordinately upregulated expression of ribosomal and other primary metabolism (e.g., protein, amino acid biosynthesis, and mitochondrial) genes (Fig. 2; see Tables S1 and S2 in the supplemental material), indicating high levels of metabolic activity in both species in the RHE. So given their differences in virulence in the RHE, it was surprising that growth initiation—as revealed by the temporal dynamics and magnitude of expression of the primary metabolism genes—appeared to be equally rapid in C. albicans and C. dubliniensis. Moreover, both species exhibited very similar upregulation in the RHE of C2 utilization genes, such as genes for fatty acid β-oxidation, glyoxylate cycle, and gluconeogenesis, as well as heat shock protein genes (see Tables S5 and S6 in the supplemental material). This pattern of gene expression in mammalian tissues has previously been described only for C. albicans (46, 52), and upregulation of C2 utilization and stress response genes also in C. dubliniensis suggests common pathways for physiological adaptation to the RHE environment in the two species. Thus, we hypothesize that only a small set of physiological cues in the RHE, triggering filamentation in C. albicans while failing to do so in C. dubliniensis, may be responsible for the difference in virulence. Filamentation of C. albicans cells growing on the polycarbonate filters used as RHE support matrix indicated that hyphal morphogenesis in C. albicans was induced by the general RHE growth conditions, such as levels of CO2 and composition of the media.

Our results indicated increased expression of many C. albicans virulence and hyphal genes, with lower or undetectable expression of these genes in C. dubliniensis. The use of the C. albicans microarray could have potentially underestimated the number of expressed genes in C. dubliniensis, including expression of several hyphal genes, where probes had lower specificity (<90% similarity) to the corresponding C. dubliniensis sequences (see Table S2 in the supplemental material). While we cannot exclude the possibility that the lack of detectable expression of these genes in C. dubliniensis was due to low probe-target similarity, the almost complete absence of hypha-associated gene expression in this species was consistent with its growth morphology and lower virulence in the RHE model.

Hyphal morphogenesis is a pivotal process for colonization and virulence in mucosal tissues by C. albicans (16, 26, 31) and some C. dubliniensis mutants (23). Accordingly, the reduction in RHE damage by the ΔΔsfl2 strains appeared to be due to their inability to form hyphae in this model (Fig. 7), as no effects on biomass in the RHE and growth rates of the ΔΔsfl2 strains were observed. Key events and genes for epithelial colonization have been identified in C. albicans (29, 52), but less is known about the factors that trigger filamentation in the RHE. CO2 at concentrations present in the oral cavity (48) induce filamentation in C. albicans—but not in C. dubliniensis (23)—both in vitro (37) and in the RHE (14). As shown by Klengel and coworkers (14), CO2/HCO3 directly stimulates activity of adenylyl cyclase for cyclic AMP (cAMP) production and hyphal morphogenesis in C. albicans. Therefore, it is noteworthy that apart from being unable to filament in the RHE, the ΔΔsfl2 strains were also impaired in CO2-induced and microaerophilic (high-CO2 and low oxygen) filamentation (Fig. 4 and and5).5). ΔΔsfl2 strains were still capable of forming germ tubes in 10% serum, although at a slightly reduced rate when no temperature shift was applied. Along with the RHE-PCF comparison, suggesting that SFL2 expression does not specifically respond to contact with the epithelial cells but rather to the environmental culture conditions in the RHE infection model, our results suggest that the inability of the ΔΔsfl2 strains to colonize and damage the RHE was due to their failure to filament in response to the increased CO2 levels or microaerophilic conditions in the RHE. Therefore, it appears that Sfl2 does not have a direct role in epithelial infection, but rather an indirect role in pathogenesis in the RHE model by virtue of its effect on hypha formation in response to CO2 and microaerophilic conditions.

It is widely accepted that the ability to produce hyphae is an important virulence factor in C. albicans, supported by observations indicating that mutants that are defective in hyphal formation are usually less pathogenic in vitro than wild-type strains (16, 26, 31, 52). SFL2 deletion had no detectable effect on survival in the mouse model of systemic infection. However, the finding that in the kidney tissues the ΔΔsfl2 cells grew almost exclusively in the yeast form was unexpected. This suggests that ΔΔsfl2 cells have a capacity to infect mice and affect mouse survival similar to wild-type cells, despite the fact that the ΔΔsfl2 strain does not produce hyphae in the kidney. The possibility that ΔΔsfl2 cells formed hyphae only during the early stages of infection and then reverted to the yeast form during the later stages was not supported by our data, which showed that ΔΔsfl2 strains grew only as yeast cells already at day 3 postinfection (Fig. 8). However, the possibility that ΔΔsfl2 cells might form hyphae prior to the 3-day time point cannot be discounted. Another possible reason for the lack of reduced virulence of ΔΔsfl2 strains in the mouse models might be that formation of hyphae was similar to WT C. albicans in organs other than the kidneys. The possibility that yeast cells alone can cause disease and death in mice is intriguing; however, confirmation of this awaits further testing in infection model experiments in which a range of organs are examined for fungal morphology and burdens as well as inflammatory responses during an infection time course. Although ΔΔsfl2 strains formed only yeasts or short filaments, there was significant immune cell infiltration associated with the renal lesions (Fig. 8) (data not shown). Since Candida virulence is correlated with the extent of immune cell infiltrate in the kidney (19), it is possible that the ΔΔsfl2 strain induces damage in the host similar to the WT and, hence, is similarly virulent. As infection outcome is determined by early renal immune responses in this infection model (19), it will be important in the future to examine the chemokine and cytokine responses to ΔΔsfl2 and WT strains.

Similarities of the inferred Sfl2 protein sequence to heat shock transcription factors and possession of a highly conserved heat-shock factor DNA-binding signature suggest that it may act as a transcriptional regulator. Sfl2p possesses sequence similarity, including the HSF-type DNA-binding domain, to Sfl1p, a nuclear protein involved in regulation of microaerophilic morphogenesis (3, 17). However, unlike SFL2, SFL1 expression is constant across different conditions (17) and Sfl1p suppresses C. albicans hyphal morphogenesis under several conditions, including microaerophilic growth (3). This suggests that these two proteins possess opposing activities, but whether or not they interact has yet to be determined. Given that deletion of SFL2 abrogated hyphal morphogenesis in response to increased CO2 and shift to alkaline pH, Sfl2p may act downstream of the cAMP and pH morphogenesis pathways. Further studies are planned to investigate the roles of Sfl1p and Sfl2p in morphogenesis, including microarray analysis of single and double mutants and localization studies.

The lack of a CO2 response in C. dubliniensis (23) makes it difficult to design experimental approaches to assign a role to the likely SFL2 orthologue, Cd36_55430, in C. dubliniensis. While possession of a conserved HSF-type domain points to some overlap in structure between Sfl2p and Cd36_55430p, the remainder of the sequences showed only ~50% identity, and deletion of Cd36_55430 gave no detectable effects on morphogenesis in C. dubliniensis CD36 in microaerophilic conditions (M.J.S., unpublished results). That expression of SFL2 in C. dubliniensis triggered filamentation indicated that SFL2 acts as a hyphal inducer also in this species. It further suggested that SFL2 and its orthologue, Cd36_55430, either have functionally diverged or that Cd36_55430 expression is repressed in C. dubliniensis. Repression of Cd36_55430 under conditions that induce expression of SFL2 is indeed supported by very low levels of expression in the RHE of Cd36_55430 in C. dubliniensis compared to expression of SFL2 in C. albicans (Fig. 3).

In conclusion, to our knowledge this study is the first that has compared global gene expression in C. albicans and C. dubliniensis in a model of oral infection. Using standard conditions for RHE infection, we have established a baseline comparison of gene expression in the two species in this important model and identified SFL2 as a gene required for virulence in the RHE infection model. SFL2 was significantly upregulated during the very early stages of RHE colonization, and its deletion drastically reduced filamentation and tissue damage of C. albicans in the RHE and also abolished filamentation in 5% CO2, in response to pH, and in microaerophilic conditions. This, along with the relationship of the predicted Sfl2 protein to HSF-type DNA-binding transcription factors, suggests a role for Sfl2 as a regulator of filamentation and opens the way for detailed studies into the roles of Sfl2 in hyphal morphogenesis and Candida virulence.

Supplementary Material

[Supplemental material]

ACKNOWLEDGMENTS

We thank Jan Walker at St. James's Hospital, Dublin, for fixation and staining of the RHE tissue sections; Joachim Morschhäuser, University of Würzburg, for supplying plasmid pSFS2A; and Arnaud Firon for the Gateway expression vector.

This work was supported by a grant from Science Foundation Ireland (Programme Investigator grant no. 04/IN3/B463) to D. Sullivan and grants from the European Commission to C. d'Enfert. (Galar Fungail 2 Marie Curie Research Training Network, MRTN-CT-2003-504148; FINSysB Marie Curie Initial Training Network, PITN-GA-2008-214004).

Footnotes

Supplemental material for this article may be found at http://ec.asm.org/.

[down-pointing small open triangle]Published ahead of print on 18 December 2009.

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