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Genome-wide analysis of Candida albicans gene expression patterns during infection of the mammalian kidney

Genome-wide analysis of Candida albicans gene expression patterns during infection of the... University of Aberdeen Genome-wide analysis of Candida albicans gene expression patterns during infection of the mammalian kidney Walker, Louise Ann; MacCallum, Donna Margaret; Bertram, Gwyneth; Gow, Neil Andrew Robert; Odds, Frank Christopher; Brown, Alistair James Petersen Published in: Fungal Genetics and Biology DOI: 10.1016/j.fgb.2008.10.012 Publication date: Document Version Publisher's PDF, also known as Version of record Link to publication Citation for pulished version (APA): Walker, L. A., MacCallum, D. M., Bertram, G., Gow, N. A. R., Odds, F. C., & Brown, A. J. P. (2009). Genome- wide analysis of Candida albicans gene expression patterns during infection of the mammalian kidney. 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Aug. 2016 Fungal Genetics and Biology 46 (2009) 210–219 Contents lists available at ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi Genome-wide analysis of Candida albicans gene expression patterns during infection of the mammalian kidney Louise A. Walker, Donna M. MacCallum, Gwyneth Bertram, Neil A.R. Gow, Frank C. Odds, Alistair J.P. Brown Aberdeen Fungal Group, School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK article info abstract Article history: Global analysis of the molecular responses of microbial pathogens to their mammalian hosts represents a Received 23 March 2008 major challenge. To date few microarray studies have been performed on Candida albicans cells derived Accepted 22 October 2008 from infected tissues. In this study we examined the C. albicans SC5314 transcriptome from renal infec- Available online 6 November 2008 tions in the rabbit. Genes involved in adhesion, stress adaptation and the assimilation of alternative car- bon sources were up-regulated in these cells compared with control cells grown in RPMI 1640, whereas Keywords: genes involved in morphogenesis, fermentation and translation were down-regulated. When we com- Candida albicans pared the congenic virulent C. albicans strains NGY152 and SC5314, there was minimal overlap between Infection their transcriptomes during kidney infections. This suggests that much of the gene regulation observed Genomics during infections is not essential for virulence. Indeed, we observed a poor correlation between the tran- Microarrays scriptome and phenome for those genes that were regulated during kidney infection and that have been Transcript profiling virulence tested. 2008 Elsevier Inc. Open access under CC BY license. 1. Introduction expression of some adhesins and secreted proteinases is coordi- nated with yeast-hypha morphogenesis (Hube et al., 1994; Staab Candida albicans is a major opportunistic fungal pathogen of hu- et al., 1996; Argimón et al., 2007). High frequency phenotypic mans (Odds, 1988; Calderone, 2002). In many healthy individuals switching of C. albicans cells between distinct epigenetic states that C. albicans exists as a commensal in the oral cavity and the gastro- express different metabolic, morphological and cell surface proper- intestinal and urogenital tracts, generating no obvious pathology. ties is associated with changes in virulence and might help the fun- However, this fungus frequently causes a range of mucosal infec- gus evade host immune responses (Odds, 1997; Soll, 2002). Other tions such as oral thrush and vaginitis (Ruhnke, 2002). In patients properties of C. albicans, which are not virulence factors that inter- with compromised immune defences, C. albicans can establish act directly with the host (Odds et al., 2003), contribute to patho- bloodstream infections that can progress to deep-seated infections genicity. These include the metabolic flexibility to adapt to diverse of major organs such as the kidney, liver and brain, many of which niches in the host (Lorenz and Fink, 2001; Barelle et al., 2006), and are fatal (Filler and Kullberg, 2002; Kullberg and Filler, 2002). robust stress responses that enhance fungal survival following at- Clearly the immune status of the host strongly influences the abil- tack by host immune defences (Wysong et al., 1998; Hwang et al., ity of C. albicans to cause disease (Casadevall and Pirofski, 2003). 2002; Fradin et al., 2005; Enjalbert et al., 2007). Nevertheless, understanding the changes in the fungus that are Over a decade ago it was predicted that the relative contribu- associated with, and contribute to, the development of tissue-dam- tions of specific virulence factors and fitness attributes change aging disease represents a major challenge in the field. temporally and spatially during the establishment and progression Multiple factors are thought to contribute to the virulence of C. of C. albicans infections (Odds, 1994). This idea has been reinforced albicans. Cell surface adhesins promote binding to, and possibly the by data from a number of laboratories on the expression of viru- penetration of, host tissue (Staab et al., 1999; Hoyer et al., 2007; lence-associated genes in a range of infection models. These stud- Phan et al., 2007). Secreted proteinases, lipases and phospholipases ies have generally focused on specific genes that are presumed or are thought to provide nutrients and may promote invasion (Nag- known to be important for the virulence of C. albicans. Members lik et al., 2003; Schaller et al., 2005). Morphological transitions be- of the SAP (secreted aspartyl proteinase), LIP (lipase) and ALS tween yeast and (pseudo)hyphal growth forms have been (agglutinin-like sequence) gene families are regulated in a stage- predicted to promote the dissemination and penetration of C. albi- and niche-specific fashion (reviewed by Brown et al., 2007). More cans cells (Gow et al., 2002, 2003; Sundstrom, 2006), and the recently, the advent of microarray technologies has allowed the generation of unbiased global views of C. albicans gene regulation that make no presumptions about the responses of this pathogen * Corresponding author. Fax: +44 1224 555844. to specific stimuli. Transcript profiling of C. albicans has been E-mail address: [email protected] (A.J.P. Brown). 1087-1845/ 2008 Elsevier Inc. Open access under CC BY license. doi:10.1016/j.fgb.2008.10.012 L.A. Walker et al. / Fungal Genetics and Biology 46 (2009) 210–219 211 performed on a range of in vitro conditions such as serum-stimu- the capsules peeled off and discarded. Slices of cortical tissue, lated morphogenesis, during phenotypic switching and biofilm for- where white microabscesses characteristic of C. albicans infection mation, exposure to various stresses, and carbon and nitrogen were seen, were shaved with a sterile scalpel directly into contain- starvation (Nantel et al., 2002; Lan et al., 2002; Enjalbert et al., ers of liquid N , to snap-freeze the lesion-rich tissue within 2.5 min 2003, 2006; Lorenz et al., 2004; Garcia-Sanchez et al., 2004; Hro- of the animal’s death. Fungal burdens were measured by viable matka et al., 2005). More interestingly from a virulence perspec- counting. Pieces of renal cortex were also fixed in formalin, and tive, expression profiling has been performed on C. albicans cells embedded in paraffin to prepare tissue sections (5 lm). Tissue sec- following exposure to macrophages, neutrophils and blood frac- tions were stained with periodic acid-Schiff’s reagent. tions (Rubin-Bejerano et al., 2003; Lorenz et al., 2004; Fradin Tissue slices from a single rabbit kidney were combined and et al., 2003, 2005), and in ex vivo infection models such as reconsti- fixed in a total of 56 ml RLT buffer (Qiagen, West Sussex, UK) and tuted human epithelium and perfused pig liver (Thewes et al., homogenized for 30 s with an Ultra-Turrax homogeniser with 2007; Zakikhany et al., 2007). These studies have provided new in- 20 mm probe. Homogenate was divided into 7 ml aliquots and sights into C. albicans-host interactions, highlighting the impor- each was layered onto a sucrose gradient comprising 2 ml 20% su- tance of metabolic and stress adaptation in the fungus, as well as crose, 2 ml 40% sucrose and 2 ml 60% sucrose. Gradients were cen- classical virulence attributes. trifuged at 300g for 30 min with the brake switched off. The layer The major challenge has been to extend these analyses into ani- enriched with fungal material, between the 40% and 60% sucrose mal models of systemic infection since these are thought to best shelves, was transferred to microcentrifuge tubes and centrifuged reflect clinical systemic infections. Few studies have been pub- at 13,000g for 10 min. The cell pellets were combined by washing lished because the transcript profiling of C. albicans from infected in 1 ml of RLT lysis buffer, and RNA extracted immediately. Micro- tissues presents significant technical challenges (reviewed by scopic analyses of these pellets indicated that this sucrose gradient Brown et al., 2007). We address two of these technical challenges fractionation protocol is not selective with regard to C. albicans cell in this paper. The first is the need to generate sufficient fungal bio- morphology. Yeast, pseudohyphal and hyphal cells were isolated. mass for a microarray study. Previous expression profiling studies To prepare control cells, C. albicans was grown overnight in NGY of C. albicans cells infecting mouse kidney and liver used various at 30 C, resuspended in RPMI 1640, grown at 37 C for 6 h, and amplification strategies to increase hybridization signals from rel- harvested by centrifugation. These exponentially growing cells atively small amounts of biomass (Andes et al., 2005; Thewes et al., were either immediately frozen in liquid N , or subjected to su- 2007). We have avoided cDNA amplification by generating larger crose density gradient fractionation before they were frozen, as de- amounts of biomass in the rabbit model of systemic candidiasis. scribed above. Other control C. albicans cells were prepared by growth in YPD at 30 Ctoan OD of 0.6, before flash freezing in The second challenge is the ‘‘contamination” of fungal biomass with the mammalian tissue it is intimately associated with. Signif- liquid N , as described above. icant contamination has prevented the analysis of fungal samples (Thewes et al., 2007). We have addressed this by developing meth- 2.3. RNA extraction ods for the enrichment of fungal cells from infected tissues. We compare our expression profiling of C. albicans cells with data from RNA was extracted from C. albicans cells isolated from rabbit other infection models, and discuss the relationship between gene kidneys by procedures modified from those of Hayes et al. regulation and gene essentiality with respect to the virulence of (2002). Briefly, cell pellets were resuspended in 500 ll TRIzol re- this major pathogen. agent (Invitrogen Ltd., Paisley, UK). Glass beads (500 ll) were added and cells were disrupted with a Fastprep cell breakage ma- chine (Thermo Savant, Middlesex, UK) run for three 30 s cycles at 2. Materials and methods 6.5 m/s with chilling on ice for 1 min in between. Samples were centrifuged for 10 min at 12,000g, the supernatants extracted with 2.1. Strains and growth conditions chloroform, and the RNA precipitated with 0.5 volumes of isopro- panol for 20 min at room temperature. Precipitates were harvested The C. albicans clinical isolate SC5314 (Gillum et al., 1984) and by centrifugation, and washed twice with ice-cold 70% ethanol. its congenic derivative NGY152 were used in this study. NGY152 Pellets were resuspended in 200 ll diethylpyrocarbonate-treated is CAI4 (ura3::kimm434/ura3::kimm434: Fonzi and Irwin, 1993) water and RNA re-precipitated with 200 ll LiCl precipitation buffer transformed with CIp10 (URA3: Murad et al., 2000). C. albicans (Ambion, TX, USA) overnight at 20 C. RNA was harvested by cen- was grown in the yeast form at 30 C in YPD (1% yeast extract, trifugation, washed twice with ice-cold ethanol, and resuspended 2% mycological peptone, 2% glucose: Sherman, 1991). To form a in 25 ll DEPC water. RNA was extracted from control samples as mixture of hyphae and pseudohyphae, C. albicans was grown over- described above, except that these cells were sheared mechanically night at 37 C in NGY (0.1% Neopeptone, 0.4% glucose and 0.1% using a microdismembrator (Braun, Melsungen, Germany). The yeast extract), washed and resuspended in RPMI 1640 at 37 C integrity of all RNA samples was confirmed by gel electrophoresis (CLSI, 2002). before use in microarray experiments (Supplementary data). 2.2. Preparation of fungal cells for transcript profiling 2.4. Transcript profiling To prepare C. albicans cells from infected kidneys, strains were grown overnight in NGY at 30 C, washed twice by centrifugation Candida albicans transcript profiling was performed as previ- and resuspension in sterile saline, and injected into the marginal ously described (Copping et al., 2005; Enjalbert et al. 2006). Cy3- ear veins of male NZ white rabbits weighing 2.5 ± 0.5 kg at a dose and Cy5- labeled cDNAs were prepared from total RNA prepara- of 1–4  10 yeast cells/kg body weight. Inoculum sizes were con- tions and hybridized with C. albicans whole genome microarrays firmed by counting viable cells (cfus). The rabbits were given food (Eurogentec, Seraing, Belgium). The microarrays were scanned and water ad libitum. Animal experimentation was done in full with a ScanArray Lite scanner (Perkin–Elmer Life Sciences, Bea- conformity with the laws and requirements of the UK Home Office. consfield, UK) at a resolution of 10 lM. Signals on the slides were Three days after infection the rabbits were terminated by intrave- located with the ScanArray 4000 Microarray Analysis System and nous injection of sodium pentobarbitone and the abdomen rapidly quantified with QuantArray software (version 2.0). Approximately opened. Both kidneys were removed, halved longitudinally, and 85% of C. albicans genes gave expression levels above background 212 L.A. Walker et al. / Fungal Genetics and Biology 46 (2009) 210–219 Table 1 levels in our experiments. The data were normalized with the Low- Impact of fixation and enrichment procedures upon the C. albicans transcriptome. ess algorithm and analysed with Genespring software (Silicon Genes displaying 2-fold regulation or more are listed. Genetics, Redwood City, CA). Genes were viewed as significantly induced or repressed if they were up- or down-regulated by 2-fold or more in three of four array experiments, and if they passed sta- tistical filtering using SAM software using a false discovery rate of <1% (significance analysis of microarrays; Tusher et al., 2001). The complete datasets are available in the Supplementary data and at ArrayExpress (www.ebi.ac.uk/microarray). 2.5. Real-time PCR For qRT-PCR, samples were incubated at room temperature for 15 min using 2 lg RNA, 2 ll DNase I buffer (Invitrogen), 1.5 ll DNase I and 1.5 ll RNase OUT (Invitrogen) in a 20 ll reaction mix to remove any contaminating DNA. cDNA was prepared using Superscript II (Invitrogen) as per the manufacturer’s protocol. Opti- mization of amplification efficiency and real-time RT-PCR SYBR green assays were carried out as described by Avrova et al. (2003). The constitutively expressed gene EFB1 was used as a con- trol for all reactions. The amplification efficiency of the endoge- nous control and the genes of interest were found to be equivalent, thereby allowing the use of the comparative Ct method (DDCt), which allowed comparison of gene expression levels orf19.9556 (0.45-fold change) and orf19.1287 (0.46-fold change). in vivo relative to expression levels in vitro (as per the manufactur- Neither of these genes has a known function. We conclude that this ers instructions; DyNAmo SYBR Green qPCR Kits). Calculations and fixation protocol is rapid and effective, and had a minimal impact statistical analyses were carried out as described in ABI PRISM upon the C. albicans transcriptome. 7700 Sequence Detection System User Bulletin 2 (Applied Biosys- tems, USA). To obtain adequate amounts of fungal biomass from infected tissues sufficient to generate significant microarray signals without 3. Results and discussion RNA amplification steps, we worked with infected rabbits (mean kidney weight 25 g) instead of the more commonly used mouse 3.1. Preparation of fungal biomass from infected tissue model (mean kidney weight 0.17 g). Progression of infection in the rabbit is essentially the same as in the mouse, with primary Our first goal was to extract fungal RNA from infected renal tis- involvement of the kidneys in both species (Hasenclever, 1959; sue in quantities sufficient for transcript profiling. Gene expression Rippon and Anderson, 1978; Morrison et al., 2003). We used a rel- within fungal lesions might change rapidly following the termina- atively high intravenous challenge dose, to induce formation of tion of the animal. Therefore, we only analysed lesions that had profuse visible kidney lesions (microabscesses) within 3 days. The data from our preliminary experiments on fungal fixation, den- been frozen in liquid N within 2.5 min of death, and used proce- dures designed to fix the fungal transcriptome throughout sity gradient enrichment of fungal cells and RNA extraction con- processing. firm the suitability of our approach for the determination of To evaluate the speed of our fixation methods we measured expression profiles of C. albicans cells in vivo. temporal loss of viability following the addition of fixative. C. albi- cans SC5314 cells were added to the fixation buffer and cell viabil- 3.2. In vivo expression profiling of a clinical isolate ity determined at various intervals thereafter by plating onto YPD medium. No viable C. albicans cells were recovered after 15 s of fix- Having established procedures for the fixation and enrichment ation (the most rapid time point that was practical to measure), of C. albicans cells we then applied these methods to the analysis suggesting that our fixation methods were rapid and effective. of expression profiling of C. albicans SC5314 cells harvested and en- To examine the combined effects of fixation and sucrose density riched from rabbit kidney lesions. This fungal RNA was compared gradient fractionation on the C. albicans transcriptome, control C. with control RNA from SC5314 cells growing exponentially in RPMI albicans SC5314 cells were grown in RPMI 1640 and snap-frozen 1640. We used RPMI 1640-grown cells as the control (rather than YPD-grown cells, for example) because this tissue culture medium for transcript profiling. Cells from equivalent cultures were fixed for 15 or 30 min, subjected to density gradient fractionation, and is generally considered to better reflect growth conditions in vivo. Therefore, we reasoned that a comparison with RPMI 1640 is more harvested for transcript profiling. The expression profiles of these processed cells were compared against the control cells in three likely to reveal infection-associated changes in expression, rather independent microarray experiments. The expression of only a than changes associated with transfer from a rich growth medium. small fraction of C. albicans genes in the processed cells differed This view was supported by expression profiling of cells grown in from that of the unprocessed controls. Five genes (0.08% of the gen- YPD and RPMI 1640, which revealed that a different subset of C. ome) were up-regulated, and seven genes (0.11%) were down-reg- albicans genes is up regulated in YPD-grown cells compared with ulated after 15 min of fixation and subsequent centrifugation in vivo-grown cells, when compared with RPMI 1640-grown cells (Table 1). Three genes involved in carbon metabolism (IDF1, PGI1, (Supplementary material). CIT1) and two components of the F F -ATPase complex (ATP1, Relative to the RPMI 1640-grown control cells, 58 C. albicans 1 0 ATP2) and orf19.9556 were included in these gene sets. After genes were reproducibly induced by 2-fold or more in kidney le- 30 min of fixation, zero genes were reproducibly up-regulated, sions compared to the control cells in four independent replicate experiments (Table 2). These included genes involved in the assim- and only two genes were down-regulated (0.03% of the genome) in processed cells compared with unprocessed controls: ilation of fatty acids and other alternative carbon sources (ACO1, L.A. Walker et al. / Fungal Genetics and Biology 46 (2009) 210–219 213 Table 2 Up-regulated genes in C. albicans SC5314 kidney lesions. +, virulence defect; , no virulence defect; n, virulence not tested (according to CGD). ACS1, CIT1, FAA4, MLS1, POX4, SDH12), adhesion (ALS1, ALS2, ALS4), In all cases the qRT-PCR data displayed a high degree of concor- stress adaptation (CTA1, ENA22) and many genes of unknown func- dance with the microarray data (Fig. 1). tion. In total, 50 genes were down-regulated in kidney lesions com- The apparent down-regulation of hypha-specific genes was rel- pared to control cells (Table 3). The down-regulated genes ative to the control RPMI 1640-grown control cells, and does not included functions associated with morphogenesis (ECE1, HYR1, reflect a lack of expression of hypha-specific genes in vivo. Hy- RBT5), fermentation (CDC19, HGT11, HXK2, HXT5, HXT61, HXT62), pha-specific genes display dynamic changes in their expression protein biosynthesis (BEL1, RPL18, RPS13, RPS21) and genes associ- levels during morphogenesis in C. albicans (e.g. HYR1: Bailey ated with the cell surface (ALS10, HYR1, IHD1, PGA54, PGA59, PGA10, et al., 1996). Therefore, the observed regulation of hypha-specific PHR1, RBT5, SUN41). To test the validity of these microarray data- genes might reflect temporal differences in the morphological sets, we examined the expression levels of six genes by qRT-PCR. development of the cells from kidney lesions compared with the 214 L.A. Walker et al. / Fungal Genetics and Biology 46 (2009) 210–219 Table 3 Down-regulated genes in C. albicans SC5314 kidney lesions. +, virulence defect; , no virulence defect; n, virulence not tested (according to CGD). control cells, as well as the heterogeneous morphologies of the fun- gal cells in these lesions. C. albicans SC5314 mainly formed pseudo- hyphae in RPMI 1640, whereas mixed populations of yeast, pseudohyphal and hyphal C. albicans cells were typically observed in sections from infected kidneys. Hyphal morphologies predomi- nate in rabbit and mice kidneys, whereas pseudohyphal and yeast forms tend to predominate in guinea pig renal lesions (Odds et al., 2000). The microarray data also indicated that ALS family members were differentially expressed in C. albicans cells infecting the kid- ney compared with cells growing in RPMI 1640. This is consistent with data from Hoyer’s group on differential ALS gene expression in vitro and in vivo (Hoyer, 2001; Green et al., 2005; Hoyer et al., 2007). Furthermore our data suggest that the C. albicans cells grow- ing in RPMI 1640 and the mouse kidney differ with respect to their Fig. 1. Comparison of qRT-PCR and microarray measurements of fold-regulation for six C. albicans SC5314 genes. carbon metabolism. Most cells infecting the kidney are thought to L.A. Walker et al. / Fungal Genetics and Biology 46 (2009) 210–219 215 assimilate carbon through glycolysis (Barelle et al., 2006). How- heat shock, osmotic stress, oxidative stress and amino acid starva- ever, assuming that these changes in gene regulation reflect bone tion (Enjalbert et al., 2003; Tournu et al, 2005). Minimal regulation fide metabolic changes, our microarray data suggest that the pop- of ADR1 has been reported in transcript profiling studies of in vitro ulation of C. albicans cells in kidney lesions are less glycolytically culture conditions. active than cells growing in RPMI 1640. Rather, alternative path- We also compared our results with data from Hube’s laboratory ways of carbon assimilation such as fatty acid b-oxidation, the gly- on mouse peritoneal infections and human oral infections (Thewes oxylate cycle and the TCA cycle may be more active in cells et al., 2007; Zakikhany et al., 2007). As expected there was greater infecting the kidney. These pathways are known to be activated overlap between the data from the kidney and peritoneal infec- during phagocytosis by macrophages and neutrophils, and in a tions than between the datasets for either of the systemic infec- subset of cells infecting kidney tissue (Prigneau et al., 2003; Lorenz tions (kidney or peritoneal) and the mucosal infections (Fig. 2B). et al., 2004; Barelle et al., 2006). However they are not essential for Six C. albicans genes were up-regulated in the rabbit, mouse and virulence in the mouse model of systemic candidiasis (Barelle et al., human infections. Two of these encode functions involved in the 2006; Piekarska et al., 2006; Ramirez and Lorenz, 2007; Zhou and utilization of alternative carbon sources (ACO1, CIT1) and one en- Lorenz, 2008). codes a stress-related function (ENA22), once again reinforcing We compared our microarray data on rabbit renal infections the view that stress and metabolic adaptation contribute to the fit- with those from two other laboratories that have examined the ness of this pathogen in its host. Twenty-six genes were up-regu- in vivo transcriptome of C. albicans. Andes and co-workers (2005) lated in both the rabbit and mouse infections (Fig. 2B). These examined the C. albicans transcriptome during mouse kidney infec- included three genes involved in iron assimilation (FRE30), oxida- tions, using YPD-grown cells as their comparator. They reported tive stress response (CTA1) and central carbon metabolism (ACS1, that 19% of all genes displayed >2-fold regulation in renal tissue MLS1), reinforcing the view that these properties are important compared with YPD-grown controls. They also observed up-regu- for virulence. lation of glyoxylate cycle, lipid metabolism and stress genes, and the down regulation of genes involved in translation. However, 3.3. In vivo expression profiling of a congenic virulent strain there is limited overlap between their data and ours with respect to the C. albicans genes that were up- or down-regulated during re- The above data suggest that C. albicans genes associated with nal infection (Fig. 2A). This is probably due in part to the different some virulence factors, fitness attributes and other functions are control conditions used in these studies: exponential RPMI 1640- regulated during infection. We tested this further by examining a grown cells in our case versus YPD-grown cells in the mouse renal second C. albicans strain in the rabbit renal model. We chose the study (Andes et al., 2005). Also, different microarray formats were strain NGY152 because this strain is a virulent, prototrophic, con- used: Eurogentec microarrays were used in our case, whereas ar- genic derivative of SC5314 (MacCallum and Odds, 2005). We con- rays from the Biotechnology Research Institute, National Research firmed the comparable virulence levels of these strains in the Council, Montreal were used by Andes and co-workers. Finally of rabbit model by measuring fungal burdens in both kidneys of in- course, different mammalian models were used: rabbits versus fected animals after 72 h of infection. For SC5314, the kidney bur- 6 6 mice. These parameters might explain why only two C. albicans dens from one rabbit were 4.0  10 and 4.6  10 cfu/g, and for a 6 6 genes were up-regulated in both datasets: ADR1 and ZRT2, both second rabbit were 1.2  10 and 1.5  10 cfu/g. For NGY152, the 6 6 of which are putative zinc finger transcription factors. ZRT2 is also kidney burdens in the first rabbit were 4.4  10 and 3.7  10 cfu/ 6 6 transcriptionally induced during interactions with macrophages g, and in the second were 2.3  10 and 3.8  10 cfu/g. Animals (Lorenz et al., 2004), but down-regulated in vitro in response to infected with both strains displayed signs of clinical deterioration after three days. Furthermore histological analyses confirmed that kidney lesions generated by SC5314 and NGY152 were of similar size, and that SC5314 and NGY152 cells infecting the kidney dis- played similar morphologies (Fig. 3). Therefore, the gross patholog- ical effects of both strains were similar. Fig. 4 illustrates the consistency of the replicate in vivo expres- sion profiles for C. albicans SC5314 and NGY152 and reveals signif- icant differences between the transcriptomes of these closely related strains. Only a small number of C. albicans NGY152 genes were regulated reproducibly when cells from kidney lesions were compared to control cells grown in RPMI 1640 (Table 4). These dif- ferences were not caused by technical issues. NGY152 RNA isolated from cells infecting the kidney was of good quality (Supplementary data) and equivalent proportions of C. albicans genes gave signifi- cant signals on the SC5314 and NGY152 microarrays (Section 2.4). To confirm the dramatic differences in the expression profiles of these closely related strains in vivo we performed qRT-PCR on the same set of transcripts that were used to validate the initial SC5314 microarray experiments: DIP51, orf19.6079, FRP3, CTA1, FAA4 and PHR1. No significant regulation was observed for any of these transcripts in NGY152, in contrast to their strong regulation in SC5314 (Supplementary data). Therefore our qRT-PCR data val- idated our microarray experiments. Of the four genes that were Fig. 2. Comparison of the renal C. albicans SC5314 transcriptome with other in vivo up-regulated in NGY152 (DDR48, GPM1, HSP12, PDC11), none were microarray studies. The numbers of genes displaying >2-fold regulation in each in common with those genes that were up-regulated during study are illustrated in the Venn diagrams. (A) This rabbit renal study compared SC5314 infections. However of the five that were down-regulated with the mouse kidney study of Andes and co-workers (2005). (B) This rabbit renal in NGY152 (ADH1, ECE1, SOD5; IPF8762, PCK1), the first three were study compared with the mouse intraperitoneal study of Thewes et al. (2007) and the human oral candidiasis study of Zakikhany et al. (2007). also down-regulated during SC5314 infections. The functions of 216 L.A. Walker et al. / Fungal Genetics and Biology 46 (2009) 210–219 Fig. 3. Histological analyses indicate that C. albicans SC5314 and NGY152 generate equivalent sizes of lesions and display similar cell morphologies in rabbit renal infections. Scale bars = 50 lm. (A) Low magnification. (B) Higher magnification. Fig. 4. Comparison of the replicate microarray experiments for the C. albicans SC5314 and NGY152 renal infections. Each line represents a single gene, and each line is colour- coded on the basis of whether the corresponding gene was up- (red) or down-regulated (green) in the first SC5314 experiment: R1–R4, rabbits 1–4; K1–K2, kidneys 1–2. (For interpretation of colour mentioned in this figure the reader is referred to the web version of the article.) these genes that were up- or down-regulated in NGY152 further PHO84 and PGA29 mRNAs are up-regulated 5-fold following OCH1 reinforce the view that morphogenesis, stress and metabolic adap- disruption in this strain background. Therefore a lack of respon- tation contribute to disease progression. However, these data are siveness in the NGY152 transcriptome does not account for our also consistent with the idea that, while C. albicans gene regulation observations in this study. might occur during renal infections, much of this regulation is not essential for the infection process. 3.4. Comparison of C. albicans expression profiles from different Candida albicans strain NGY152 is transcriptionally responsive kidneys to other conditions. For example, over 600 genes are regulated in response to OCH1 inactivation (Carol Munro, personal communica- We compared the microarray data from individual kidneys in- tion). (OCH1 encodes a mannosyltransferase involved in the glyco- fected with C. albicans NGY152. This was done by calculating sylation of cell wall mannoproteins: Bates et al., 2006). The CRH11 pair-wise correlation coefficients for the global expression patterns and SAP9 transcripts are down-regulated more than 5-fold, and the for each kidney against all of the other kidneys. The mean correla- L.A. Walker et al. / Fungal Genetics and Biology 46 (2009) 210–219 217 Table 4 Regulated genes in C. albicans NGY152 kidney lesions. +, virulence defect; n, virulence not tested (according to CGD). No genes in common with subset of up-regulated in C. albicans SC5314 cells. Also down-regulated in C. albicans SC5314 cells. Fig. 5. Comparison of genome-wide expression patterns for C. albicans NGY152 from different kidneys from the same animal versus kidneys from different animals. Mean correlation coefficients (±SD) for the pairwise comparisons of kidney ** infections using the whole microarray dataset for each kidney: , significant at p < 0.01. Fig. 6. Comparison of the in vivo transcriptome (i.e. the subset of C. albicans SC5314 genes that were regulated during renal infections) with the in vivo phenome (i.e. the subset of C. albicans genes that affect the virulence of C. albicans, as defined by the tion coefficient for the left and right kidneys from the same rabbit Candida Genome Database @ August 2007) (Supplementary data). was significantly higher than for the mean correlation coefficient for kidneys from different rabbits (Fig. 5; Supplementary data). This indicates that the C. albicans expression profiles for cells infecting ulence were up-regulated in the rabbit kidney lesions. These were different kidneys in the same animal were more similar than the ALS1 and ALS2 (both GPI-anchored cell surface adhesins: Hoyer expression profiles from different animals (i.e. there is more biolog- et al., 1995, 2001), CTA1 (which encodes catalase that contributes ical variation between animals than between kidneys in the same to oxidative stress protection: Wysong et al., 1998), and a gene animal). This is consistent with the idea that the behaviour of C. albi- of unknown function (orf19.1239). However, only a relatively small cans is affected by the properties of the host and that variation be- proportion of C. albicans genes have been virulence tested and the tween individual hosts can affect the expression profile of the ‘‘phenome” of C. albicans is still very much incomplete. Indeed pathogen. Our observation is also consistent with experimental var- according to the Candida Genome database, only five of the C. albi- iation in survival time that is generally observed for individual ani- cans genes that were up-regulated in the rabbit kidney lesions have mals infected with equivalent inocula in mammalian models of been virulence tested to date (Table 2). Four of these five genes are disseminated candidiasis (MacCallum and Odds, 2005). required for virulence. We examined the relationship between the transcriptome and 3.5. Comparison of in vivo phenome (virulence) with in vivo expression phenome further by looking at the genes that were down-regulated (profiling) in the rabbit kidney (Fig. 6). We reasoned that, if there was a correla- tion between gene regulation and essentiality for infection, down- Our data suggest that changes in expression occur during infec- regulated genes would not display a virulence defect. However this tion, but that many of these changes may not be essential for infec- was not the case. Seven of these down-regulated genes have been tion (Section 3.3). To test this we examined the overlap between subjected to virulence testing (Table 3). Of these, six are required the subset of C. albicans genes whose expression was induced for virulence: ALS3 (another GPI-anchored cell surface adhesin), in vivo (Table 2) and the subset of C. albicans genes that are essen- CDC19 (pyruvate kinase), ERG3 (ergosterol biosynthesis), SOD5 (a tial for virulence (i.e. those genes that have been annotated as hav- superoxide dismutase), SUN41 (a cell wall glycosidase involved in ing an impact upon virulence by the Candida Genome Database: biofilm formation) and PHR1 (a pH-regulated cell surface glycosi- www.candidagenome.org)(Fig. 6; Supplementary data). Four of dase). Therefore in our experiments, there was a poor correlation be- the 148 C. albicans genes that have been shown to contribute to vir- tween in vivo expression and virulence phenotype. 218 L.A. Walker et al. / Fungal Genetics and Biology 46 (2009) 210–219 This poor correlation between the transcriptome and phenome Appendix A. Supplementary data is not surprising when Saccharomyces cerevisiae genomic datasets are considered. Genome-wide comparisons between the regulation Supplementary data associated with this article can be found, in of genes and their contribution to fitness under equivalent growth the online version, at doi:10.1016/j.fgb.2008.10.012. conditions revealed a poor correlation between the transcriptome and the ‘‘phenome” (Giaever et al., 2002). Several factors probably References account for this. For example, the inactivation of individual genes Andes, D., Lepak, A., Pitula, A., Marchillo, K., Clark, J., 2005. 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Genome-wide analysis of Candida albicans gene expression patterns during infection of the mammalian kidney

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University of Aberdeen Genome-wide analysis of Candida albicans gene expression patterns during infection of the mammalian kidney Walker, Louise Ann; MacCallum, Donna Margaret; Bertram, Gwyneth; Gow, Neil Andrew Robert; Odds, Frank Christopher; Brown, Alistair James Petersen Published in: Fungal Genetics and Biology DOI: 10.1016/j.fgb.2008.10.012 Publication date: Document Version Publisher's PDF, also known as Version of record Link to publication Citation for pulished version (APA): Walker, L. A., MacCallum, D. M., Bertram, G., Gow, N. A. R., Odds, F. C., & Brown, A. J. P. (2009). Genome- wide analysis of Candida albicans gene expression patterns during infection of the mammalian kidney. 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Aug. 2016 Fungal Genetics and Biology 46 (2009) 210–219 Contents lists available at ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi Genome-wide analysis of Candida albicans gene expression patterns during infection of the mammalian kidney Louise A. Walker, Donna M. MacCallum, Gwyneth Bertram, Neil A.R. Gow, Frank C. Odds, Alistair J.P. Brown Aberdeen Fungal Group, School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK article info abstract Article history: Global analysis of the molecular responses of microbial pathogens to their mammalian hosts represents a Received 23 March 2008 major challenge. To date few microarray studies have been performed on Candida albicans cells derived Accepted 22 October 2008 from infected tissues. In this study we examined the C. albicans SC5314 transcriptome from renal infec- Available online 6 November 2008 tions in the rabbit. Genes involved in adhesion, stress adaptation and the assimilation of alternative car- bon sources were up-regulated in these cells compared with control cells grown in RPMI 1640, whereas Keywords: genes involved in morphogenesis, fermentation and translation were down-regulated. When we com- Candida albicans pared the congenic virulent C. albicans strains NGY152 and SC5314, there was minimal overlap between Infection their transcriptomes during kidney infections. This suggests that much of the gene regulation observed Genomics during infections is not essential for virulence. Indeed, we observed a poor correlation between the tran- Microarrays scriptome and phenome for those genes that were regulated during kidney infection and that have been Transcript profiling virulence tested. 2008 Elsevier Inc. Open access under CC BY license. 1. Introduction expression of some adhesins and secreted proteinases is coordi- nated with yeast-hypha morphogenesis (Hube et al., 1994; Staab Candida albicans is a major opportunistic fungal pathogen of hu- et al., 1996; Argimón et al., 2007). High frequency phenotypic mans (Odds, 1988; Calderone, 2002). In many healthy individuals switching of C. albicans cells between distinct epigenetic states that C. albicans exists as a commensal in the oral cavity and the gastro- express different metabolic, morphological and cell surface proper- intestinal and urogenital tracts, generating no obvious pathology. ties is associated with changes in virulence and might help the fun- However, this fungus frequently causes a range of mucosal infec- gus evade host immune responses (Odds, 1997; Soll, 2002). Other tions such as oral thrush and vaginitis (Ruhnke, 2002). In patients properties of C. albicans, which are not virulence factors that inter- with compromised immune defences, C. albicans can establish act directly with the host (Odds et al., 2003), contribute to patho- bloodstream infections that can progress to deep-seated infections genicity. These include the metabolic flexibility to adapt to diverse of major organs such as the kidney, liver and brain, many of which niches in the host (Lorenz and Fink, 2001; Barelle et al., 2006), and are fatal (Filler and Kullberg, 2002; Kullberg and Filler, 2002). robust stress responses that enhance fungal survival following at- Clearly the immune status of the host strongly influences the abil- tack by host immune defences (Wysong et al., 1998; Hwang et al., ity of C. albicans to cause disease (Casadevall and Pirofski, 2003). 2002; Fradin et al., 2005; Enjalbert et al., 2007). Nevertheless, understanding the changes in the fungus that are Over a decade ago it was predicted that the relative contribu- associated with, and contribute to, the development of tissue-dam- tions of specific virulence factors and fitness attributes change aging disease represents a major challenge in the field. temporally and spatially during the establishment and progression Multiple factors are thought to contribute to the virulence of C. of C. albicans infections (Odds, 1994). This idea has been reinforced albicans. Cell surface adhesins promote binding to, and possibly the by data from a number of laboratories on the expression of viru- penetration of, host tissue (Staab et al., 1999; Hoyer et al., 2007; lence-associated genes in a range of infection models. These stud- Phan et al., 2007). Secreted proteinases, lipases and phospholipases ies have generally focused on specific genes that are presumed or are thought to provide nutrients and may promote invasion (Nag- known to be important for the virulence of C. albicans. Members lik et al., 2003; Schaller et al., 2005). Morphological transitions be- of the SAP (secreted aspartyl proteinase), LIP (lipase) and ALS tween yeast and (pseudo)hyphal growth forms have been (agglutinin-like sequence) gene families are regulated in a stage- predicted to promote the dissemination and penetration of C. albi- and niche-specific fashion (reviewed by Brown et al., 2007). More cans cells (Gow et al., 2002, 2003; Sundstrom, 2006), and the recently, the advent of microarray technologies has allowed the generation of unbiased global views of C. albicans gene regulation that make no presumptions about the responses of this pathogen * Corresponding author. Fax: +44 1224 555844. to specific stimuli. Transcript profiling of C. albicans has been E-mail address: [email protected] (A.J.P. Brown). 1087-1845/ 2008 Elsevier Inc. Open access under CC BY license. doi:10.1016/j.fgb.2008.10.012 L.A. Walker et al. / Fungal Genetics and Biology 46 (2009) 210–219 211 performed on a range of in vitro conditions such as serum-stimu- the capsules peeled off and discarded. Slices of cortical tissue, lated morphogenesis, during phenotypic switching and biofilm for- where white microabscesses characteristic of C. albicans infection mation, exposure to various stresses, and carbon and nitrogen were seen, were shaved with a sterile scalpel directly into contain- starvation (Nantel et al., 2002; Lan et al., 2002; Enjalbert et al., ers of liquid N , to snap-freeze the lesion-rich tissue within 2.5 min 2003, 2006; Lorenz et al., 2004; Garcia-Sanchez et al., 2004; Hro- of the animal’s death. Fungal burdens were measured by viable matka et al., 2005). More interestingly from a virulence perspec- counting. Pieces of renal cortex were also fixed in formalin, and tive, expression profiling has been performed on C. albicans cells embedded in paraffin to prepare tissue sections (5 lm). Tissue sec- following exposure to macrophages, neutrophils and blood frac- tions were stained with periodic acid-Schiff’s reagent. tions (Rubin-Bejerano et al., 2003; Lorenz et al., 2004; Fradin Tissue slices from a single rabbit kidney were combined and et al., 2003, 2005), and in ex vivo infection models such as reconsti- fixed in a total of 56 ml RLT buffer (Qiagen, West Sussex, UK) and tuted human epithelium and perfused pig liver (Thewes et al., homogenized for 30 s with an Ultra-Turrax homogeniser with 2007; Zakikhany et al., 2007). These studies have provided new in- 20 mm probe. Homogenate was divided into 7 ml aliquots and sights into C. albicans-host interactions, highlighting the impor- each was layered onto a sucrose gradient comprising 2 ml 20% su- tance of metabolic and stress adaptation in the fungus, as well as crose, 2 ml 40% sucrose and 2 ml 60% sucrose. Gradients were cen- classical virulence attributes. trifuged at 300g for 30 min with the brake switched off. The layer The major challenge has been to extend these analyses into ani- enriched with fungal material, between the 40% and 60% sucrose mal models of systemic infection since these are thought to best shelves, was transferred to microcentrifuge tubes and centrifuged reflect clinical systemic infections. Few studies have been pub- at 13,000g for 10 min. The cell pellets were combined by washing lished because the transcript profiling of C. albicans from infected in 1 ml of RLT lysis buffer, and RNA extracted immediately. Micro- tissues presents significant technical challenges (reviewed by scopic analyses of these pellets indicated that this sucrose gradient Brown et al., 2007). We address two of these technical challenges fractionation protocol is not selective with regard to C. albicans cell in this paper. The first is the need to generate sufficient fungal bio- morphology. Yeast, pseudohyphal and hyphal cells were isolated. mass for a microarray study. Previous expression profiling studies To prepare control cells, C. albicans was grown overnight in NGY of C. albicans cells infecting mouse kidney and liver used various at 30 C, resuspended in RPMI 1640, grown at 37 C for 6 h, and amplification strategies to increase hybridization signals from rel- harvested by centrifugation. These exponentially growing cells atively small amounts of biomass (Andes et al., 2005; Thewes et al., were either immediately frozen in liquid N , or subjected to su- 2007). We have avoided cDNA amplification by generating larger crose density gradient fractionation before they were frozen, as de- amounts of biomass in the rabbit model of systemic candidiasis. scribed above. Other control C. albicans cells were prepared by growth in YPD at 30 Ctoan OD of 0.6, before flash freezing in The second challenge is the ‘‘contamination” of fungal biomass with the mammalian tissue it is intimately associated with. Signif- liquid N , as described above. icant contamination has prevented the analysis of fungal samples (Thewes et al., 2007). We have addressed this by developing meth- 2.3. RNA extraction ods for the enrichment of fungal cells from infected tissues. We compare our expression profiling of C. albicans cells with data from RNA was extracted from C. albicans cells isolated from rabbit other infection models, and discuss the relationship between gene kidneys by procedures modified from those of Hayes et al. regulation and gene essentiality with respect to the virulence of (2002). Briefly, cell pellets were resuspended in 500 ll TRIzol re- this major pathogen. agent (Invitrogen Ltd., Paisley, UK). Glass beads (500 ll) were added and cells were disrupted with a Fastprep cell breakage ma- chine (Thermo Savant, Middlesex, UK) run for three 30 s cycles at 2. Materials and methods 6.5 m/s with chilling on ice for 1 min in between. Samples were centrifuged for 10 min at 12,000g, the supernatants extracted with 2.1. Strains and growth conditions chloroform, and the RNA precipitated with 0.5 volumes of isopro- panol for 20 min at room temperature. Precipitates were harvested The C. albicans clinical isolate SC5314 (Gillum et al., 1984) and by centrifugation, and washed twice with ice-cold 70% ethanol. its congenic derivative NGY152 were used in this study. NGY152 Pellets were resuspended in 200 ll diethylpyrocarbonate-treated is CAI4 (ura3::kimm434/ura3::kimm434: Fonzi and Irwin, 1993) water and RNA re-precipitated with 200 ll LiCl precipitation buffer transformed with CIp10 (URA3: Murad et al., 2000). C. albicans (Ambion, TX, USA) overnight at 20 C. RNA was harvested by cen- was grown in the yeast form at 30 C in YPD (1% yeast extract, trifugation, washed twice with ice-cold ethanol, and resuspended 2% mycological peptone, 2% glucose: Sherman, 1991). To form a in 25 ll DEPC water. RNA was extracted from control samples as mixture of hyphae and pseudohyphae, C. albicans was grown over- described above, except that these cells were sheared mechanically night at 37 C in NGY (0.1% Neopeptone, 0.4% glucose and 0.1% using a microdismembrator (Braun, Melsungen, Germany). The yeast extract), washed and resuspended in RPMI 1640 at 37 C integrity of all RNA samples was confirmed by gel electrophoresis (CLSI, 2002). before use in microarray experiments (Supplementary data). 2.2. Preparation of fungal cells for transcript profiling 2.4. Transcript profiling To prepare C. albicans cells from infected kidneys, strains were grown overnight in NGY at 30 C, washed twice by centrifugation Candida albicans transcript profiling was performed as previ- and resuspension in sterile saline, and injected into the marginal ously described (Copping et al., 2005; Enjalbert et al. 2006). Cy3- ear veins of male NZ white rabbits weighing 2.5 ± 0.5 kg at a dose and Cy5- labeled cDNAs were prepared from total RNA prepara- of 1–4  10 yeast cells/kg body weight. Inoculum sizes were con- tions and hybridized with C. albicans whole genome microarrays firmed by counting viable cells (cfus). The rabbits were given food (Eurogentec, Seraing, Belgium). The microarrays were scanned and water ad libitum. Animal experimentation was done in full with a ScanArray Lite scanner (Perkin–Elmer Life Sciences, Bea- conformity with the laws and requirements of the UK Home Office. consfield, UK) at a resolution of 10 lM. Signals on the slides were Three days after infection the rabbits were terminated by intrave- located with the ScanArray 4000 Microarray Analysis System and nous injection of sodium pentobarbitone and the abdomen rapidly quantified with QuantArray software (version 2.0). Approximately opened. Both kidneys were removed, halved longitudinally, and 85% of C. albicans genes gave expression levels above background 212 L.A. Walker et al. / Fungal Genetics and Biology 46 (2009) 210–219 Table 1 levels in our experiments. The data were normalized with the Low- Impact of fixation and enrichment procedures upon the C. albicans transcriptome. ess algorithm and analysed with Genespring software (Silicon Genes displaying 2-fold regulation or more are listed. Genetics, Redwood City, CA). Genes were viewed as significantly induced or repressed if they were up- or down-regulated by 2-fold or more in three of four array experiments, and if they passed sta- tistical filtering using SAM software using a false discovery rate of <1% (significance analysis of microarrays; Tusher et al., 2001). The complete datasets are available in the Supplementary data and at ArrayExpress (www.ebi.ac.uk/microarray). 2.5. Real-time PCR For qRT-PCR, samples were incubated at room temperature for 15 min using 2 lg RNA, 2 ll DNase I buffer (Invitrogen), 1.5 ll DNase I and 1.5 ll RNase OUT (Invitrogen) in a 20 ll reaction mix to remove any contaminating DNA. cDNA was prepared using Superscript II (Invitrogen) as per the manufacturer’s protocol. Opti- mization of amplification efficiency and real-time RT-PCR SYBR green assays were carried out as described by Avrova et al. (2003). The constitutively expressed gene EFB1 was used as a con- trol for all reactions. The amplification efficiency of the endoge- nous control and the genes of interest were found to be equivalent, thereby allowing the use of the comparative Ct method (DDCt), which allowed comparison of gene expression levels orf19.9556 (0.45-fold change) and orf19.1287 (0.46-fold change). in vivo relative to expression levels in vitro (as per the manufactur- Neither of these genes has a known function. We conclude that this ers instructions; DyNAmo SYBR Green qPCR Kits). Calculations and fixation protocol is rapid and effective, and had a minimal impact statistical analyses were carried out as described in ABI PRISM upon the C. albicans transcriptome. 7700 Sequence Detection System User Bulletin 2 (Applied Biosys- tems, USA). To obtain adequate amounts of fungal biomass from infected tissues sufficient to generate significant microarray signals without 3. Results and discussion RNA amplification steps, we worked with infected rabbits (mean kidney weight 25 g) instead of the more commonly used mouse 3.1. Preparation of fungal biomass from infected tissue model (mean kidney weight 0.17 g). Progression of infection in the rabbit is essentially the same as in the mouse, with primary Our first goal was to extract fungal RNA from infected renal tis- involvement of the kidneys in both species (Hasenclever, 1959; sue in quantities sufficient for transcript profiling. Gene expression Rippon and Anderson, 1978; Morrison et al., 2003). We used a rel- within fungal lesions might change rapidly following the termina- atively high intravenous challenge dose, to induce formation of tion of the animal. Therefore, we only analysed lesions that had profuse visible kidney lesions (microabscesses) within 3 days. The data from our preliminary experiments on fungal fixation, den- been frozen in liquid N within 2.5 min of death, and used proce- dures designed to fix the fungal transcriptome throughout sity gradient enrichment of fungal cells and RNA extraction con- processing. firm the suitability of our approach for the determination of To evaluate the speed of our fixation methods we measured expression profiles of C. albicans cells in vivo. temporal loss of viability following the addition of fixative. C. albi- cans SC5314 cells were added to the fixation buffer and cell viabil- 3.2. In vivo expression profiling of a clinical isolate ity determined at various intervals thereafter by plating onto YPD medium. No viable C. albicans cells were recovered after 15 s of fix- Having established procedures for the fixation and enrichment ation (the most rapid time point that was practical to measure), of C. albicans cells we then applied these methods to the analysis suggesting that our fixation methods were rapid and effective. of expression profiling of C. albicans SC5314 cells harvested and en- To examine the combined effects of fixation and sucrose density riched from rabbit kidney lesions. This fungal RNA was compared gradient fractionation on the C. albicans transcriptome, control C. with control RNA from SC5314 cells growing exponentially in RPMI albicans SC5314 cells were grown in RPMI 1640 and snap-frozen 1640. We used RPMI 1640-grown cells as the control (rather than YPD-grown cells, for example) because this tissue culture medium for transcript profiling. Cells from equivalent cultures were fixed for 15 or 30 min, subjected to density gradient fractionation, and is generally considered to better reflect growth conditions in vivo. Therefore, we reasoned that a comparison with RPMI 1640 is more harvested for transcript profiling. The expression profiles of these processed cells were compared against the control cells in three likely to reveal infection-associated changes in expression, rather independent microarray experiments. The expression of only a than changes associated with transfer from a rich growth medium. small fraction of C. albicans genes in the processed cells differed This view was supported by expression profiling of cells grown in from that of the unprocessed controls. Five genes (0.08% of the gen- YPD and RPMI 1640, which revealed that a different subset of C. ome) were up-regulated, and seven genes (0.11%) were down-reg- albicans genes is up regulated in YPD-grown cells compared with ulated after 15 min of fixation and subsequent centrifugation in vivo-grown cells, when compared with RPMI 1640-grown cells (Table 1). Three genes involved in carbon metabolism (IDF1, PGI1, (Supplementary material). CIT1) and two components of the F F -ATPase complex (ATP1, Relative to the RPMI 1640-grown control cells, 58 C. albicans 1 0 ATP2) and orf19.9556 were included in these gene sets. After genes were reproducibly induced by 2-fold or more in kidney le- 30 min of fixation, zero genes were reproducibly up-regulated, sions compared to the control cells in four independent replicate experiments (Table 2). These included genes involved in the assim- and only two genes were down-regulated (0.03% of the genome) in processed cells compared with unprocessed controls: ilation of fatty acids and other alternative carbon sources (ACO1, L.A. Walker et al. / Fungal Genetics and Biology 46 (2009) 210–219 213 Table 2 Up-regulated genes in C. albicans SC5314 kidney lesions. +, virulence defect; , no virulence defect; n, virulence not tested (according to CGD). ACS1, CIT1, FAA4, MLS1, POX4, SDH12), adhesion (ALS1, ALS2, ALS4), In all cases the qRT-PCR data displayed a high degree of concor- stress adaptation (CTA1, ENA22) and many genes of unknown func- dance with the microarray data (Fig. 1). tion. In total, 50 genes were down-regulated in kidney lesions com- The apparent down-regulation of hypha-specific genes was rel- pared to control cells (Table 3). The down-regulated genes ative to the control RPMI 1640-grown control cells, and does not included functions associated with morphogenesis (ECE1, HYR1, reflect a lack of expression of hypha-specific genes in vivo. Hy- RBT5), fermentation (CDC19, HGT11, HXK2, HXT5, HXT61, HXT62), pha-specific genes display dynamic changes in their expression protein biosynthesis (BEL1, RPL18, RPS13, RPS21) and genes associ- levels during morphogenesis in C. albicans (e.g. HYR1: Bailey ated with the cell surface (ALS10, HYR1, IHD1, PGA54, PGA59, PGA10, et al., 1996). Therefore, the observed regulation of hypha-specific PHR1, RBT5, SUN41). To test the validity of these microarray data- genes might reflect temporal differences in the morphological sets, we examined the expression levels of six genes by qRT-PCR. development of the cells from kidney lesions compared with the 214 L.A. Walker et al. / Fungal Genetics and Biology 46 (2009) 210–219 Table 3 Down-regulated genes in C. albicans SC5314 kidney lesions. +, virulence defect; , no virulence defect; n, virulence not tested (according to CGD). control cells, as well as the heterogeneous morphologies of the fun- gal cells in these lesions. C. albicans SC5314 mainly formed pseudo- hyphae in RPMI 1640, whereas mixed populations of yeast, pseudohyphal and hyphal C. albicans cells were typically observed in sections from infected kidneys. Hyphal morphologies predomi- nate in rabbit and mice kidneys, whereas pseudohyphal and yeast forms tend to predominate in guinea pig renal lesions (Odds et al., 2000). The microarray data also indicated that ALS family members were differentially expressed in C. albicans cells infecting the kid- ney compared with cells growing in RPMI 1640. This is consistent with data from Hoyer’s group on differential ALS gene expression in vitro and in vivo (Hoyer, 2001; Green et al., 2005; Hoyer et al., 2007). Furthermore our data suggest that the C. albicans cells grow- ing in RPMI 1640 and the mouse kidney differ with respect to their Fig. 1. Comparison of qRT-PCR and microarray measurements of fold-regulation for six C. albicans SC5314 genes. carbon metabolism. Most cells infecting the kidney are thought to L.A. Walker et al. / Fungal Genetics and Biology 46 (2009) 210–219 215 assimilate carbon through glycolysis (Barelle et al., 2006). How- heat shock, osmotic stress, oxidative stress and amino acid starva- ever, assuming that these changes in gene regulation reflect bone tion (Enjalbert et al., 2003; Tournu et al, 2005). Minimal regulation fide metabolic changes, our microarray data suggest that the pop- of ADR1 has been reported in transcript profiling studies of in vitro ulation of C. albicans cells in kidney lesions are less glycolytically culture conditions. active than cells growing in RPMI 1640. Rather, alternative path- We also compared our results with data from Hube’s laboratory ways of carbon assimilation such as fatty acid b-oxidation, the gly- on mouse peritoneal infections and human oral infections (Thewes oxylate cycle and the TCA cycle may be more active in cells et al., 2007; Zakikhany et al., 2007). As expected there was greater infecting the kidney. These pathways are known to be activated overlap between the data from the kidney and peritoneal infec- during phagocytosis by macrophages and neutrophils, and in a tions than between the datasets for either of the systemic infec- subset of cells infecting kidney tissue (Prigneau et al., 2003; Lorenz tions (kidney or peritoneal) and the mucosal infections (Fig. 2B). et al., 2004; Barelle et al., 2006). However they are not essential for Six C. albicans genes were up-regulated in the rabbit, mouse and virulence in the mouse model of systemic candidiasis (Barelle et al., human infections. Two of these encode functions involved in the 2006; Piekarska et al., 2006; Ramirez and Lorenz, 2007; Zhou and utilization of alternative carbon sources (ACO1, CIT1) and one en- Lorenz, 2008). codes a stress-related function (ENA22), once again reinforcing We compared our microarray data on rabbit renal infections the view that stress and metabolic adaptation contribute to the fit- with those from two other laboratories that have examined the ness of this pathogen in its host. Twenty-six genes were up-regu- in vivo transcriptome of C. albicans. Andes and co-workers (2005) lated in both the rabbit and mouse infections (Fig. 2B). These examined the C. albicans transcriptome during mouse kidney infec- included three genes involved in iron assimilation (FRE30), oxida- tions, using YPD-grown cells as their comparator. They reported tive stress response (CTA1) and central carbon metabolism (ACS1, that 19% of all genes displayed >2-fold regulation in renal tissue MLS1), reinforcing the view that these properties are important compared with YPD-grown controls. They also observed up-regu- for virulence. lation of glyoxylate cycle, lipid metabolism and stress genes, and the down regulation of genes involved in translation. However, 3.3. In vivo expression profiling of a congenic virulent strain there is limited overlap between their data and ours with respect to the C. albicans genes that were up- or down-regulated during re- The above data suggest that C. albicans genes associated with nal infection (Fig. 2A). This is probably due in part to the different some virulence factors, fitness attributes and other functions are control conditions used in these studies: exponential RPMI 1640- regulated during infection. We tested this further by examining a grown cells in our case versus YPD-grown cells in the mouse renal second C. albicans strain in the rabbit renal model. We chose the study (Andes et al., 2005). Also, different microarray formats were strain NGY152 because this strain is a virulent, prototrophic, con- used: Eurogentec microarrays were used in our case, whereas ar- genic derivative of SC5314 (MacCallum and Odds, 2005). We con- rays from the Biotechnology Research Institute, National Research firmed the comparable virulence levels of these strains in the Council, Montreal were used by Andes and co-workers. Finally of rabbit model by measuring fungal burdens in both kidneys of in- course, different mammalian models were used: rabbits versus fected animals after 72 h of infection. For SC5314, the kidney bur- 6 6 mice. These parameters might explain why only two C. albicans dens from one rabbit were 4.0  10 and 4.6  10 cfu/g, and for a 6 6 genes were up-regulated in both datasets: ADR1 and ZRT2, both second rabbit were 1.2  10 and 1.5  10 cfu/g. For NGY152, the 6 6 of which are putative zinc finger transcription factors. ZRT2 is also kidney burdens in the first rabbit were 4.4  10 and 3.7  10 cfu/ 6 6 transcriptionally induced during interactions with macrophages g, and in the second were 2.3  10 and 3.8  10 cfu/g. Animals (Lorenz et al., 2004), but down-regulated in vitro in response to infected with both strains displayed signs of clinical deterioration after three days. Furthermore histological analyses confirmed that kidney lesions generated by SC5314 and NGY152 were of similar size, and that SC5314 and NGY152 cells infecting the kidney dis- played similar morphologies (Fig. 3). Therefore, the gross patholog- ical effects of both strains were similar. Fig. 4 illustrates the consistency of the replicate in vivo expres- sion profiles for C. albicans SC5314 and NGY152 and reveals signif- icant differences between the transcriptomes of these closely related strains. Only a small number of C. albicans NGY152 genes were regulated reproducibly when cells from kidney lesions were compared to control cells grown in RPMI 1640 (Table 4). These dif- ferences were not caused by technical issues. NGY152 RNA isolated from cells infecting the kidney was of good quality (Supplementary data) and equivalent proportions of C. albicans genes gave signifi- cant signals on the SC5314 and NGY152 microarrays (Section 2.4). To confirm the dramatic differences in the expression profiles of these closely related strains in vivo we performed qRT-PCR on the same set of transcripts that were used to validate the initial SC5314 microarray experiments: DIP51, orf19.6079, FRP3, CTA1, FAA4 and PHR1. No significant regulation was observed for any of these transcripts in NGY152, in contrast to their strong regulation in SC5314 (Supplementary data). Therefore our qRT-PCR data val- idated our microarray experiments. Of the four genes that were Fig. 2. Comparison of the renal C. albicans SC5314 transcriptome with other in vivo up-regulated in NGY152 (DDR48, GPM1, HSP12, PDC11), none were microarray studies. The numbers of genes displaying >2-fold regulation in each in common with those genes that were up-regulated during study are illustrated in the Venn diagrams. (A) This rabbit renal study compared SC5314 infections. However of the five that were down-regulated with the mouse kidney study of Andes and co-workers (2005). (B) This rabbit renal in NGY152 (ADH1, ECE1, SOD5; IPF8762, PCK1), the first three were study compared with the mouse intraperitoneal study of Thewes et al. (2007) and the human oral candidiasis study of Zakikhany et al. (2007). also down-regulated during SC5314 infections. The functions of 216 L.A. Walker et al. / Fungal Genetics and Biology 46 (2009) 210–219 Fig. 3. Histological analyses indicate that C. albicans SC5314 and NGY152 generate equivalent sizes of lesions and display similar cell morphologies in rabbit renal infections. Scale bars = 50 lm. (A) Low magnification. (B) Higher magnification. Fig. 4. Comparison of the replicate microarray experiments for the C. albicans SC5314 and NGY152 renal infections. Each line represents a single gene, and each line is colour- coded on the basis of whether the corresponding gene was up- (red) or down-regulated (green) in the first SC5314 experiment: R1–R4, rabbits 1–4; K1–K2, kidneys 1–2. (For interpretation of colour mentioned in this figure the reader is referred to the web version of the article.) these genes that were up- or down-regulated in NGY152 further PHO84 and PGA29 mRNAs are up-regulated 5-fold following OCH1 reinforce the view that morphogenesis, stress and metabolic adap- disruption in this strain background. Therefore a lack of respon- tation contribute to disease progression. However, these data are siveness in the NGY152 transcriptome does not account for our also consistent with the idea that, while C. albicans gene regulation observations in this study. might occur during renal infections, much of this regulation is not essential for the infection process. 3.4. Comparison of C. albicans expression profiles from different Candida albicans strain NGY152 is transcriptionally responsive kidneys to other conditions. For example, over 600 genes are regulated in response to OCH1 inactivation (Carol Munro, personal communica- We compared the microarray data from individual kidneys in- tion). (OCH1 encodes a mannosyltransferase involved in the glyco- fected with C. albicans NGY152. This was done by calculating sylation of cell wall mannoproteins: Bates et al., 2006). The CRH11 pair-wise correlation coefficients for the global expression patterns and SAP9 transcripts are down-regulated more than 5-fold, and the for each kidney against all of the other kidneys. The mean correla- L.A. Walker et al. / Fungal Genetics and Biology 46 (2009) 210–219 217 Table 4 Regulated genes in C. albicans NGY152 kidney lesions. +, virulence defect; n, virulence not tested (according to CGD). No genes in common with subset of up-regulated in C. albicans SC5314 cells. Also down-regulated in C. albicans SC5314 cells. Fig. 5. Comparison of genome-wide expression patterns for C. albicans NGY152 from different kidneys from the same animal versus kidneys from different animals. Mean correlation coefficients (±SD) for the pairwise comparisons of kidney ** infections using the whole microarray dataset for each kidney: , significant at p < 0.01. Fig. 6. Comparison of the in vivo transcriptome (i.e. the subset of C. albicans SC5314 genes that were regulated during renal infections) with the in vivo phenome (i.e. the subset of C. albicans genes that affect the virulence of C. albicans, as defined by the tion coefficient for the left and right kidneys from the same rabbit Candida Genome Database @ August 2007) (Supplementary data). was significantly higher than for the mean correlation coefficient for kidneys from different rabbits (Fig. 5; Supplementary data). This indicates that the C. albicans expression profiles for cells infecting ulence were up-regulated in the rabbit kidney lesions. These were different kidneys in the same animal were more similar than the ALS1 and ALS2 (both GPI-anchored cell surface adhesins: Hoyer expression profiles from different animals (i.e. there is more biolog- et al., 1995, 2001), CTA1 (which encodes catalase that contributes ical variation between animals than between kidneys in the same to oxidative stress protection: Wysong et al., 1998), and a gene animal). This is consistent with the idea that the behaviour of C. albi- of unknown function (orf19.1239). However, only a relatively small cans is affected by the properties of the host and that variation be- proportion of C. albicans genes have been virulence tested and the tween individual hosts can affect the expression profile of the ‘‘phenome” of C. albicans is still very much incomplete. Indeed pathogen. Our observation is also consistent with experimental var- according to the Candida Genome database, only five of the C. albi- iation in survival time that is generally observed for individual ani- cans genes that were up-regulated in the rabbit kidney lesions have mals infected with equivalent inocula in mammalian models of been virulence tested to date (Table 2). Four of these five genes are disseminated candidiasis (MacCallum and Odds, 2005). required for virulence. We examined the relationship between the transcriptome and 3.5. Comparison of in vivo phenome (virulence) with in vivo expression phenome further by looking at the genes that were down-regulated (profiling) in the rabbit kidney (Fig. 6). We reasoned that, if there was a correla- tion between gene regulation and essentiality for infection, down- Our data suggest that changes in expression occur during infec- regulated genes would not display a virulence defect. However this tion, but that many of these changes may not be essential for infec- was not the case. Seven of these down-regulated genes have been tion (Section 3.3). To test this we examined the overlap between subjected to virulence testing (Table 3). Of these, six are required the subset of C. albicans genes whose expression was induced for virulence: ALS3 (another GPI-anchored cell surface adhesin), in vivo (Table 2) and the subset of C. albicans genes that are essen- CDC19 (pyruvate kinase), ERG3 (ergosterol biosynthesis), SOD5 (a tial for virulence (i.e. those genes that have been annotated as hav- superoxide dismutase), SUN41 (a cell wall glycosidase involved in ing an impact upon virulence by the Candida Genome Database: biofilm formation) and PHR1 (a pH-regulated cell surface glycosi- www.candidagenome.org)(Fig. 6; Supplementary data). Four of dase). Therefore in our experiments, there was a poor correlation be- the 148 C. albicans genes that have been shown to contribute to vir- tween in vivo expression and virulence phenotype. 218 L.A. Walker et al. / Fungal Genetics and Biology 46 (2009) 210–219 This poor correlation between the transcriptome and phenome Appendix A. Supplementary data is not surprising when Saccharomyces cerevisiae genomic datasets are considered. Genome-wide comparisons between the regulation Supplementary data associated with this article can be found, in of genes and their contribution to fitness under equivalent growth the online version, at doi:10.1016/j.fgb.2008.10.012. conditions revealed a poor correlation between the transcriptome and the ‘‘phenome” (Giaever et al., 2002). Several factors probably References account for this. For example, the inactivation of individual genes Andes, D., Lepak, A., Pitula, A., Marchillo, K., Clark, J., 2005. 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