Serial analysis of gene expression during early pulmonary infection
To investigate pathogen gene expression during pulmonary cryptococcosis, we generated SAGE libraries from fungal cells harvested by bronchoalveolar lavage from mouse lungs at 8 (21 510 tags) and 24 h (20 129 tags) after infection. The details of the SAGE analysis are presented in Experimental procedures and in Tables S1–S3. A comparison of the two libraries identified 382 tags with differential levels at the two stages of infection suggesting dynamic changes during adaptation to the host environment. The SAGE libraries were also compared with two previously described libraries constructed from cells grown in low-iron medium (LIM) (77 829 tags; Hu et al., 2007) or in YNB broth at 37°C (84 039 tags; Steen et al., 2002). These are referred to as in vitro libraries. In addition, the SAGE data for fungal cells from rabbit CSF (66 217 tags; Steen et al., 2003) were compared with the pulmonary infection libraries (in vivo libraries). Overall, these libraries provided snapshots of RNA abundance in cells from quite different conditions of temperature (i.e. 37°C for both in vitro and in vivo lung libraries, and 39.5°C for rabbit), nutrients (i.e. YNB broth, LIM and host environment) and stress (i.e. presence or absence of host defence responses). The SAGE data were normalized to 20 000 tags per library to facilitate comparisons between all five conditions. For this analysis, we focused mainly on comparisons between in vivo (mouse lung and rabbit CSF) and in vitro (LIM and YNB broth) conditions. Following annotation to match tags to genes, we identified several functional categories containing differentially expressed genes as shown in Tables 1 and 2 and Tables S4 and S5. A notable general observation was that the patterns of expression were most similar for the libraries prepared with cells from LIM and rabbit CSF (Table S2). For the bulk of the analysis described below, we employed the data for the 8 h lung library and the LIM library to define differential expression with the primary goal of identifying genes that showed elevated expression during lung infection.
Table 1. SAGE tags for genes encoding predicted functions in carbon and lipid metabolism, and transport.
|Tag||LIM||Yeast nitrogen base||Lung, 8 h||Lung, 24 h||Rabbit, CSF||TIGR gene number||GenBank accession number||Predicted function and gene name (if known)|
|Glyoxylate cycle and acetate/ethanol/pyruvate metabolism|
| CAGCAGTGTA||3||0||144||46||7||CNF03900||XM_571348||Aldehyde dehydrogenase|
| CGCGAGGGCA||1||9||80||40||0||CNA07740||XM_567122||Acetyl-CoA synthase, ACS1|
| GCCCAGCAGG||0||1||62||25||0||CNH02910||XM_572462||Malate synthase, MLS1|
| CATCACTCTT||13||2||39||48||13||CNJ00950||XM_567475||Pyruvate decarboxylase|
| ACCTTTATAT||0||0||7||3||0||CNC06260||XM_569885||Alcohol dehydrogenase|
|Tricarboxylic acid cycle|
| CATTTTCTCA||0||0||15||6||1||CNG03480||XM_572036||Succinate dehydrogenase|
| GGTTACGCCG||1||55||6||3||1||CNG03490||XM_572038||Malate dehydrogenase|
| AACTTTGTTC||13||0||4||2||15||CNA04610||XM_566837||Isocitrate dehydrogenase|
| CATTTTGATT||8||0||0||0||2||CNF03780||XM_571672||Malic enzyme|
| ACTCAGGTTG||1||65||19||17||3||CNB00300||XM_568771||Fructose 1,6-biphosphate aldolase|
| TGTGAATGTG||1||11||9||6||4||CNB02660||XM_568853||Hexokinase, HXK2|
| GGTCGTTTAT||12||0||0||0||7||CNB04050||XM_569228||Glucose 6-phosphate isomerase|
| TATGCAACAC||6||0||0||0||4||CNF00810||XM_571493||Phosphoglycerate mutase|
| TTTCAGAAGG||0||1||68||27||2||CNI03590||XM_572603||Phosphoenolpyruvate carboxykinase, PCK1|
|Pentose phosphate pathway|
| TAGTGTCCCG||70||6||41||9||32||CNK01050||XM_567793||6-Phosphogluconate dehydrogenase|
|Other functions related to carbon metabolism|
| CATTATGACA||4||0||19||5||5||CNA01100||XM_567089||Glycerol 3-phosphate dehydrogenase|
| ATTCACGCGC||1||0||17||5||3||CND02900||XM_570206||Glutamine:fructose-6-phosphate amidotransferase|
| CCATATGTTT||3||14||15||2||1||CNF03530||XM_571431||Glycogen phosphorylase-like protein|
| GACATTGTTG||2||22||18||8||1||CNC05340||XM_569729||d-arabinitol dehydrogenase|
| CATCCCAAAG||15||0||0||0||20||CNC03700||XM_569599||UDP-glucose pyrophophorylase|
| TATAAGACCG||6||0||0||0||4||CNF03930||XM_571680||Glycogen metabolism-related protein|
| TAACCATATA||19||0||96||17||7||CNJ02970||XM_567576||Butyrylcholinesterase, triacylglycerol lipase, CGL1|
| AACCACACTA||1||34||39||15||14||CNN01770||XM_568678||Similar to Benzodiazepin receptor|
| TCATTTCACT||2||0||33||10||3||CNI01560||XM_572814||Sterol-binding protein|
| GCGAAGTACT||0||0||27||3||0||CNN01550||XM_568632||Acyl-CoA oxidase|
| GCTGCCTTTG||2||13||22||6||1||CNI00240||XM_572730||Enoyl-CoA hydratase/isomerase|
| CAATGGGAAT||2||0||12||3||0||CNI03690||XM_572896||2,4-Dienoyl-CoA reductase (NADPH)|
| TATGTGAATC||3||0||9||3||4||CNM00830||XM_568422||Oxidoreductase, short-chain dehydrogenase/reductase|
| CTACAAATGA||1||0||9||1||1||CNH02950||XM_572465||Oxidoreductase, short-chain dehydrogenase/reductase|
| GATAAGGTGT||4||0||8||6||8||CNI02880||XM_572653||Oxysterol-binding protein|
| TATTATTGTT||0||0||8||1||2||CNA03320||XM_566658||Enoyl-CoA hydratase|
| AAGCAGAGGA||2||9||7||3||5||CNI00710||XM_572753||Long-chain fatty acid CoA ligase|
| TAGTTTATAT||2||0||7||3||2||CNG02680||XM_571931||Hydroxymethylglutaryl-CoA synthase|
| ATTGAATGTA||0||0||7||2||25||CND01120||XM_570351||Fatty acid β-oxidation-related protein|
| TATACGATTT||26||0||612||143||2||CNB02680||XM_568855||Monosaccharide transporter, HXT1|
| CCCATCGTAT||82||0||136||79||24||CNM02430||XM_568258||Plasma membrane iron permease, CFT1|
| CTTGACAGCG||0||5||42||46||0||CNF02960||XM_571371||Acetate transporter, ADY2|
| TATCTGATTT||19||1||34||8||10||CNG04240||XM_572092||Hexose transporter|
| TATTGGTACA||16||0||28||13||0||CNC03960||XM_569629||Phosphate transporter|
| GTCATTTATG||2||0||27||3||2||CNF02890||XM_571700||Neutral amino acid transporter|
| CGGGAATACG||0||0||24||12||1||CNH03640||XM_572526||GPR/FUN34 family protein/acetate transporter, ATO2|
| CTGTTCGGCA||9||3||21||13||11||CND01080||XM_570353||High affinity copper transporter, CTR4|
| CACTGTATGC||1||0||21||18||0||CNE02570||XM_570958||Succinate:fumarate antiporter|
| GCCGGGCTGT||1||15||20||14||2||CNC02540||XM_569547||Organic acid transporter|
| CCGAGAAACG||2||0||19||7||2||CND04380||XM_570596||Monovalent inorganic cation transporter|
| AGCACTTATG||1||0||16||2||0||CNI03490||XM_572882||Maltose permease, trehalose transporter|
| CAGTATCACT||2||0||15||2||2||CNL05010||XM_568040||Gaba permease|
| GTGGTTTTCT||1||0||11||9||2||CNF04760||XM_571440||Neutral amino acid permease|
| CATATATAAC||2||0||7||0||1||CNH00950||XM_572289||Spermine transporter|
| GGAAGAGGAA||1||0||6||3||2||CNE00440||XM_571108||Inner membrane solute transporter MRS4|
| ATCGGTACCC||4||47||0||0||4||CNN01030||XM_568544||Inorganic phosphate transporter|
Table 2. Virulence-associated and stress-response genes.
|Tags||LIM||Yeast nitrogen base||Lungs, 8 h||Lungs, 24 h||Rabbit, CSF||TIGR gene number||GenBank accession number||Predicted function and gene name (if known)|
|TATATGTGTA||35||0||247||60||262||CNG04220||XM_572088||Heat-shock protein 12|
|CACGTCCACG||27||61||95||30||5||CND01490||XM_570285||Cu/Zn superoxide dismutase, SOD1|
|CATTTTATGT||14||0||35||16||24||CNM01520||XM_568451||Heat-shock protein 90|
|TGTTTCTACA||2||1||24||3||3||CNG04220||XM_572088||Heat-shock protein 12|
|CTCTTCATTT||8||4||23||14||10||CNA03160||XM_566757||Heat-shock protein, SKS2|
|GCGGATAAAA||6||5||23||6||8||CNB00910||XM_569098||Stress-response RCI peptide, cation transport-related protein|
|ATGTTTTATT||9||0||20||7||3||CNG02560||XM_572003||UDP-glucuronic acid decarboxylase, UXS1|
|TTGATTTTTT||0||0||16||3||0||CNM01860||XM_568294||Macrolide-binding protein FKBP12|
|GCGACAGCCA||0||1||15||2||1||CNC01490||XM_569482||Peptidyl prolyl cis-trans isomerase, ESS1|
|ACGGAGTGTA||1||0||14||9||2||CND00260||XM_570333||FK506-resistant calcineurin B regulatory subunit|
|TCGATGGCGA||2||0||11||5||14||CNE05040||XM_571033||Glyoxal oxidase precursor|
|CAACGATGAT||5||0||10||3||66||CNF02910||XM_571367||LEA domain protein|
|GCTTAATTAT||0||1||8||2||1||CNN00360||XM_568558||Protein kinase, SCH9|
|GTCTTCATCA||2||0||8||2||1||CND05600||XM_570476||Heat-shock protein 12|
|TTTCTGAAAA||2||0||7||1||1||CND05940||XM_570487||Capsule-associated protein, CAP1|
|TGTTATCGGT||60||4||22||6||54||CNM01520||XM_568451||Heat-shock protein 90|
Adaptation during infection involves changes in central carbon metabolism
Initially, we identified a number of tags for genes involved in carbon metabolism that were expressed at high levels during lung infection. In particular, tags for functions in the glyoxylate pathway, the TCA cycle, gluconeogenesis, lipid catabolism and two-carbon metabolism were elevated relative to the two in vitro SAGE libraries (Table 1). Notably, these results suggested a metabolic emphasis on the generation and utilization of acetyl-CoA upon infection. The position of acetyl-CoA in central carbon metabolism is summarized in Fig. 1. For the glyoxylate cycle, the tag for the gene encoding malate synthase was notably higher in the lung libraries (60 times higher at 8 h and 25 times at 24 h) compared with the in vitro libraries. In contrast, the tag for this gene was not present in the normalized data for the CSF library. Disruption of the MLS1 gene for malate synthase in C. neoformans eliminates the ability of the fungus to grow on acetate but does not affect virulence (Idnurm et al., 2007). Tags for genes encoding the TCA cycle enzymes aconitase and succinate dehydrogenase were also elevated in the lung libraries. However, tags representing three other TCA cycle enzymes were lower in the lung libraries than in one or both of the in vitro libraries (Table 1). The SAGE analysis also revealed that a tag for the gene encoding phosphoenolpyruvate carboxykinase (PCK1), which controls the only irreversible step in gluconeogenesis, was higher in both in vivo libraries (68 and 27 times at 8 and 24 h after infection, respectively, versus the in vitro libraries). This result suggests that glucose may be limiting during early infection resulting in the activation of gluconeogenesis in at least a portion of the cells in the population. Panepinto et al. (2005) found that disruption of PCK1 caused poor growth on lactate and markedly reduced virulence, thus indicating the importance of gluconeogenesis during infection.
Figure 1. The position of acetyl-CoA in central carbon metabolism. Metabolic processes and metabolites are indicated in upper- and lower-case letters respectively. Note that neither the cellular compartments for the various reactions nor the import processes for metabolites such as ethanol and acetate are indicated.
Download figure to PowerPoint
The possibility of glucose limitation is supported in part by observations that the tags for genes encoding glycolytic functions including glucose 6-phosphate isomerase and phosphoglycerate mutase were relatively low in the lung libraries. However, this pattern was not consistent for all glycolytic functions because the tags for fructose 1,6-biphosphate aldolase, hexokinase and phosphofructokinase were present at similar levels between the libraries or elevated in one of the lung libraries. Additionally, the tag for enolase was present at low levels in the lung libraries compared with elevated levels in the libraries from cells grown in LIM or isolated from CSF. Similarly, tags for different genes involved in the pentose phosphate pathway (PPP) showed a mixed pattern of elevated (e.g. 6-phosphogluconate dehydrogenase), similar (e.g. phosphoketolase) or reduced (e.g. transaldolase) expression in the in vivo versus one or both of the in vitro libraries (Table 1).
The SAGE data also indicated that functions for lipid degradation and fatty acid catabolism were elevated during infection. Specifically, a tag for a predicted butyrylcholinesterase (triacylglycerol lipase, CGL1) was one of most abundant in the 8 h lung library, but dropped in abundance at 24 h. The level of this tag was four times higher in the 8 h library versus the LIM library and > 90 times higher than in the YNB broth library; tag abundance was similar in the 24 h library and the LIM library (Table 1). Lavage fluid from mammalian lungs is rich in phospholipids (Rooney et al., 1994; Rose et al., 1994) and fungal lipases such as the putative Cgl1 protein may contribute to carbon acquisition during lung colonization. Interestingly, the expression of this gene was induced during phagocytosis but attempts at disruption were not successful suggesting that the gene is essential (Fan et al., 2005). Fatty acids generated by phospholipases and triacylglycerol lipases would enter the β-oxidation pathway and eventually yield acetyl-CoA. We did identify tags for components in the β-oxidation pathway in the lung libraries [e.g. enoyl-CoA hydratase/isomerase and 2,4-dienoyl-CoA reductase (NADPH)], suggesting a role for β-oxidation upon pulmonary infection (Table 1). We also identified tags for functions in sterol biosynthesis and regulation, including sterol-binding proteins and a hydroxymethylglutaryl-CoA synthase (Parks and Casey, 1995; Olkkonen et al., 2006).
The ability to convert acetate to acetyl-CoA contributes to virulence
The expression of genes encoding enzymes for the production of acetyl-CoA from pyruvate and acetate was elevated during pulmonary infection. In particular, elevated expression was found for the genes for acetyl-CoA synthetase (ACS1), pyruvate decarboxylase and aldehyde dehydrogenase, as well as two acetate transporters (Table 1). Acetate utilization or production is potentially relevant to the pathogenesis of C. neoformans because Himmelreich et al. (2003) showed that it was one of the major metabolites present in infected tissue. Acetyl-CoA is generated by fatty acid catabolism, by decarboxylation of pyruvate via the pyruvate dehydrogenase complex or by direct activation of acetate by the Acs1 enzyme (Fig. 1). We also found tags for functions involved in the conversion of pyruvate, acetaldehyde and ethanol to acetate (e.g. pyruvate decarboxylase and alcohol dehydrogenase) that were elevated in the lung libraries (Table 1). A tag for a related function, succinate:fumarate antiporter, was also elevated and this protein is required for growth on ethanol or acetate in yeast (Table 1; Palmieri et al., 1997). Notably, a tag for aldehyde dehydrogenase was one of the most abundant in both in vivo libraries (144 copies at 8 h and 46 at 24 h). A similar gene in Ustilago maydis is required for growth on ethanol as a sole carbon source (Basse et al., 1996). Taken together, these tags suggest that acetate and acetyl-CoA play important metabolic roles during pulmonary infection.
To examine the role of ACS1 in more detail, we first confirmed the expression of the gene during infection by real-time quantitative PCR (Fig. 2A). In S. cerevisiae, ACS1 is expressed during growth on non-fermentable carbon sources and under aerobic conditions, and Acs1 functions in the acetate utilization pathway (Schüller, 2003). ACS1 expression is also regulated by the Snf1 protein kinase in yeast (Young et al., 2003) and upregulated during the growth of C. albicans in blood (Fradin et al., 2003). We therefore reasoned that loss of Acs1 function would impair the growth of C. neoformans on acetate. Indeed, deletion of ACS1 resulted in a growth defect on acetate and ethanol, and also caused poor growth on glycerol (Fig. 2B). No growth defect was observed on sucrose, arabinose, galactose or lactic acid (data not shown). In contrast, the S. cerevisiae acs1 mutant can grow on ethanol but there are conflicting reports about growth on acetate (De Virgilio et al., 1992; Kratzer and Schüller, 1995). Complementation of the C. neoformans acs1 mutant with ACS1 gene restored growth to wild type (WT) levels (Fig. 2B). The acs1 mutant did not show differences in capsule or melanin formation, or growth at different temperatures, compared with the WT strain H99 (data not shown). However, the mutant did show a delay in disease progression in a mouse inhalation model of cryptococcosis (Fig. 2C). That is, mice infected with the mutant survived 7–10 days longer than those infected with the WT or complemented strains (P < 0.001). These results indicate that the ability to produce acetyl-CoA directly from acetate makes a modest contribution to growth in the host. It is possible that the synthesis of acetyl-CoA from pyruvate, from β-oxidation or by other acetyl-CoA synthetases may limit the impact of loss of Acs1.
Figure 2. Analysis of ACS1 expression during pulmonary infection and the role of the gene for growth on alternative carbon sources and virulence. A. Quantitative real-time PCR to confirm the elevated expression of the ACS1 gene during pulmonary infection relative to growth in vitro. B. Growth of the WT strain H99, the acs1 mutant and the complemented strain on the carbon sources indicated above the panels. C. Virulence of the acs1 mutant compared with the WT strain H99 and the complemented strain. The strains were each inoculated into 10 mice by inhalation and the mice were monitored for signs of illness. The virulence of the acs1 mutant was statistically different from that of the WT (P = 0.0001) or the complemented strain (P = 0.0008) by the Krustal–Wallis test. A representative graph is shown from a total of three experiments, and the acs1 mutant displayed a similar attenuation of virulence in each experiment.
Download figure to PowerPoint
We have considered the possibility that other genes encode acetyl-CoA synthetase in C. neoformans. Our blastp searches with Acs1 and Acs2 from S. cerevisiae identified the C. neoformans gene product that we designated Acs1 (expect value = 0.0, 56% identity). However, two genes encoding proteins of lower similarity were also found and preliminarily named ACS2 and ACS3. Acs2 had an expect value of E-32 (26% identity) and Acs3 had an expect value of E-13 (21% identity). For context, a blastp comparison of Acs1 and Acs2 from S. cerevisiae yielded an expect value of 0.0 with 57% identity. Thus, for C. neoformans, there was one clear orthologue for ACS1/ACS2 and two genes with weaker similarity. Alignments of the yeast and C. neoformans polypeptides are shown in Fig. S1. Preliminary experiments indicated that disruption of the C. neoformans ACS2 gene yielded mutants that lacked notable phenotypes in terms of growth on alternative carbon sources or virulence (data not shown). The construction of double and triple mutants will be needed to determine whether loss of these genes exacerbates the phenotypes of the acs1 mutant.
Transport functions show elevated expression during lung infection
The SAGE data indicated that C. neoformans cells contained elevated transcripts for several putative transporters upon growth in the lung environmental (Table 1). Notably, a tag for a gene encoding a putative glucose transporter (designated HXT1) was the most abundant transcript in both lung libraries, a finding suggestive of glucose limitation (Table S3). This gene shared high similarity to the yeast RGT2 and SNF3 genes, and was also regulated by PKA as determined by SAGE analysis (Hu et al., 2007). We confirmed the elevation of the HXT1 transcript for the lung libraries by quantitative real-time PCR and found that deletion of HXT1 resulted in early melanin production (Fig. S2). The influence on melanin formation is interesting because elevated glucose is known to suppress this process (Zhu and Williamson, 2004). The hxt1 mutant did not show a virulence defect (data not shown).
The other transporters had predicted functions in the movement of molecules such as amino acids, sugars (e.g. trehalose), metals (iron and copper), organic acids (e.g. acetate) and phosphate (Table 1). Trehalose transport is of interest because the trehalose pathway in C. neoformans is involved in survival in the host, the response to high-temperature stress and glycolysis (Petzold et al., 2006). The tag for a putative phosphate transporter (Pho84) was upregulated at both 8 and 24 h during lung infection, and this gene was also induced in C. neoformans during phagocytosis (Fan et al., 2005). In S. cerevisiae, Pho84 is involved in sensing nutrient signals and activates the cAMP-PKA pathway (Giots et al., 2003; Fan et al., 2005). The candidate metal transporters included the high-affinity iron permease (CFT1, Jung et al., 2006; 2008) and a copper transporter (CTR4, Waterman et al., 2007). CFT1 was previously identified as an iron-regulated gene in C. neoformans cells and the gene is also regulated by PKA in C. neoformans (Lian et al., 2005; Hu et al., 2007). CFT1 encodes an iron permease and is required for the use of iron from transferrin and for virulence in C. neoformans (Jung et al., 2008). Overall, the elevated tags for transport functions suggest that specific assimilation activities are required to support fungal proliferation in host tissue.
Elevated expression of virulence-associated and stress-response genes indicates that the lung is a stressful environment for C. neoformans
The SAGE analysis also identified a group of elevated tags representing functions related to virulence and stress (Table 2). For example, two tags for the flavohaemoprotein gene FHB1 (flavohaemoglobin denitrosylase) were abundant at 8 and 24 h after infection. FHB1 was induced in fungal cells during murine macrophage infection and deletion of the gene resulted in hypersensitivity to nitrosative stress and attenuation of virulence in a mouse model (de Jesus-Berrios et al., 2003; Fan et al., 2005). A tag representing the gene encoding Cu,Zn superoxide dismutase (SOD1) was also elevated during lung infection, especially in the 8 h library. Superoxide dismutase has been shown to contribute to the virulence of C. neoformans and C. gattii (Cox et al., 2003; Narasipura et al., 2003; 2005). Interestingly, a tag representing the gene for the Sch9 protein kinase was elevated at 8 h. In C. neoformans, Sch9 modulates capsule formation and thermal tolerance, and contributes to virulence both independently of and in conjunction with the cAMP-PKA pathway (Wang et al., 2004). We also found that a tag for a gene for peptidyl prolyl cis-trans isomerase (Ess1), an enzyme that mediates the folding of target proteins, was elevated at 8 h. Ess1 is dispensable for growth, haploid fruiting and capsule formation, but is required for virulence in C. neoformans (Ren et al., 2005). A tag matching the gene for the FK506-resistant calcineurin B regulatory subunit was also identified in the early infection library. Calcineurin, a serine-threonine-specific calcium-activated phosphatase, is the target of the immunosuppressive drugs cyclosporine A and FK506, and this protein influences the mating, growth at 37°C and virulence in C. neoformans (Fox et al., 2001; Fox and Heitman, 2005). Finally, the in vivo SAGE libraries revealed that the expression of two genes encoding mannitol-1-phosphate dehydrogenase (Mpd1), an enzyme involved in mannitol synthesis, was elevated during infection (Table 2). Mannitol synthesis is thought to be important in cryptococcosis (Chaturvedi et al., 1996), and the MPD1 gene is induced under nitric oxide stress (Chow et al., 2007). Overall, the elevated expression of genes involved in stress response and functions known to contribute to virulence likely reflects C. neoformans adaptation to a hostile environment in the host.
A homologue of SNF1 influences melanin production and virulence
In evaluating the SAGE results for pulmonary infection, we noticed that the differential expression of genes for carbon metabolism, transport and stress showed similarities to the regulatory pattern defined for Snf1 in S. cerevisiae (Young et al., 2003; Hong and Carlson, 2007). Snf1 is a serine/threonine protein kinase that plays a major role in nutrient response and cellular metabolism, especially in gluconeogenesis and growth on alternative carbon sources (Celenza and Carlson, 1986). We therefore identified and characterized a candidate SNF1 homologue in C. neoformans to test the hypothesis that the Snf1 regulatory pathway influences the patterns of expression that we observed during infection (Fig. S3). Initially, we confirmed that a cDNA of the SNF1 gene from C. neoformans was able to restore the growth of a S. cerevisiae snf1 mutant on sucrose and raffinose (data not shown). Subsequently, a SNF1 deletion mutant was constructed in C. neoformans to examine phenotypes related to carbon source utilization and virulence. The snf1 mutant as well as the WT and complemented strains was able to grow on either glucose or sucrose at 30°C and therefore did not show the growth defect on sucrose observed in S. cerevisiae (Fig. 3, Celenza and Carlson, 1984). A similar result was obtained for the plant pathogenic fungus Cochliobolus carbonum in which a SNF1 mutant was able to grow on both glucose and sucrose at 30°C (Tonukari et al., 2000). At 30°C, the C. neoformans snf1 strain also grew as well as the WT and complemented strains on arabinose, fructose, raffinose, galactose, mannose, lactate, acetate, ethanol and glycerol (Fig. 3 and data not shown). Interestingly, different phenotypes were observed at 37°C in that the C. neoformans snf1 mutant displayed a noticeable reduction in growth on sucrose and ethanol, markedly reduced growth on acetate, and similar growth to the WT and complemented strains on other carbon sources (glycerol, lactate, galactose, raffinose and arabinose; Fig. 3 and data not shown).
Figure 3. Growth of the C. neoformans snf1 mutant on alternative carbon sources. Cells of the WT strain (H99), the snf1 mutant and the complemented strain were spotted at decreasing concentrations (from left to right) on the media indicated on the left. YPD and YNB serve as controls for growth on glucose, and YNB with sucrose, sodium acetate or ethanol were used to test alternative carbon sources. The plates were incubated for 2 days at 30°C or 37°C as indicated. Re-introduction of the SNF1 gene into the snf1 mutant restored growth to the level of the WT strain.
Download figure to PowerPoint
Next, we examined the response of the snf1 mutant of C. neoformans to oxidative, osmotic, salt and nitrosative stress. We did not observe significant differences between the mutant and the WT strain when cells were grown on YNB medium supplemented with NaCl, KCl, sorbitol, H2O2 or menadione, suggesting that SNF1 was not required for protection against these osmotic and oxidative stresses (Fig. 4 and data not shown). However, the mutant exhibited increased sensitivity to sodium nitrite at 37°C (Fig. 4). The complemented strain restored the WT level of growth on sodium nitrite at 37°C, indicating that a functional SNF1 gene is required to withstand nitrosative stress. We also tested the snf1 mutant for altered sensitivity to several drugs known to inhibit fungal growth. We found that the mutant displayed increased sensitivity to the antifungal drug amphotericin B that is commonly used to treat cryptococcosis, but a change in sensitivity to fluconazole was not observed (Fig. 4). We also found that the snf1 mutant was more sensitive to rapamycin, perhaps indicating an interaction with the TOR (target of rapamycin) pathway. The mutant showed equal sensitivity to FK506 and cyclosporine A compared with the WT strain (data not shown). We next examined the three main virulence factors of the fungus: the ability to grow at 37°C, capsule formation and melanin production. We did not observe differences in growth or capsule formation between the WT, snf1 mutant and complemented strains at either 30°C or 37°C (data not shown). It was notable, however, that the snf1 mutant produced melanin at the same level as the WT strain at 30°C, but was unable to produce visible melanin in colonies at 37°C (Fig. 4). Complementation of the snf1 mutation with the SNF1 gene again restored the ability of cells to produce melanin at 37°C (Fig. 4).
Figure 4. Growth and melanin production by the C. neoformans snf1 mutant. Cells of the WT strain H99, the snf1 mutant and the complemented strain were spot inoculated in dilutions from left to right on YNB medium to test the response to salt (1.5 M NaCl), nitrosative stress (8 mM NaNO2), amphotericin B (0.5 μg ml−1) and rapamycin (10 μg ml−1). The plates were incubated at 30°C or 37°C for 2 days. Melanin production was tested on L-DOPA medium after 3 days of incubation.
Download figure to PowerPoint
The motivation to examine the role of SNF1 was based in part on the pattern of gene expression observed in the SAGE data and also on evidence that alternative carbon sources are important for growth in the mammalian host (Lorenz and Fink, 2001). We therefore used quantitative real-time PCR to determine whether loss of Snf1 influenced the expression of a set of genes selected from the SAGE analysis. In parallel, we tested the influence of growth on low glucose or acetate on expression of the same genes. As shown in Fig. 5A, loss of Snf1 did not have an appreciable effect on the expression of the metabolic genes (e.g. ACS1, PCK1 and MLS1), or the acetate transporters ADY2 and ATO2, when cells were grown on 2% glucose, 0.2% glucose or 2% acetate. In contrast, growth of the WT strain at the lower glucose level resulted in a substantial elevation in the transcripts for all of the genes. The CFT1 gene showed a different pattern in that growth on the low-glucose medium resulted in elevated expression and Snf1 was required for part of this response. The transcripts for a subset of the genes responded to acetate with increased levels; these included ACS1 (∼2.6-fold), CGL1 (∼2.5-fold), CFT1 (∼3.7-fold), HXT1 (∼3.7-fold) and MLS1 (∼2.0-fold). Of these genes, loss of Snf1 had a positive influence on the level of the HXT1 transcript during growth on acetate. Overall, we concluded that glucose levels play a major role in regulating the genes that we observed by SAGE to have elevated transcripts during lung infection, and that Snf1 plays little role for these genes. These results require cautious interpretation, however, because we have not tested whether Snf1 influences gene expression during infection. We did find that SNF1 transcript levels were increased in the cells grown on low glucose or acetate (Fig. 5B), but they were not substantially increased in cells recovered from infected lungs (Fig. S3).
Figure 5. Influence of glucose, acetate and deletion of SNF1 on the expression of genes identified by SAGE analysis. A. Expression of selected genes as measured by quantitative real-time PCR following growth under the conditions indicated in the legend on the right. B. Expression of the SNF1 gene in cells grown in YNB medium with the carbon sources indicated on the x-axis. C. Expression of the genes encoding laccase or genes involved in the response to stress following growth under the conditions indicated on the right. The transcript of the ACT1 gene was used for normalization.
Download figure to PowerPoint
Given the influence of the snf1 mutation on melanin formation, the response to nitrosative stress and growth on acetate, we also examined whether Snf1 controlled the expression of genes related to these phenotypes. Loss of Snf1 did have an interesting influence on the expression of the LAC1 and LAC2 genes encoding the laccase enzymes for melanin production. The transcript for LAC2 was slightly elevated in WT cells grown on medium with 0.2% glucose or with acetate, and the transcript was more markedly elevated in the snf1 mutant grown on acetate. The LAC1 transcript was ∼8.0-fold higher in WT cells grown on 0.2% glucose compared with cells grown on acetate or 2% glucose, and Snf1 was required for this enhanced transcript level. Interestingly, the LAC1 transcript levels were not influenced by growth on acetate in either strain. Complementation of the snf1 mutation with SNF1 restored the WT pattern of regulation (data not shown). These results suggest that part of the melanin defect in the snf1 mutant results from an influence on LAC1 expression. Snf1 did not appear to appreciably influence levels for stress-related genes (e.g. FHB1, SOD1, etc.) under any of the conditions (Fig. 5C).
Finally, we hypothesized that SNF1 would also be required for virulence because of the mutant phenotypes for growth on alternative carbon sources, melanin production and the response to nitrosative stress at 37°C. To test this idea, we infected A/Jcr mice with the WT strain, the snf1 mutant and the complemented strain by intranasal inoculation. All mice infected with the WT and complemented strains succumbed to infection by days 22–24, while those infected with the snf1 mutant survived to the end of the experiment at day 60 (Fig. 6). Thus the snf1 mutant was avirulent compared with the other strains (P < 0.001). At day 60, three mice infected with snf1 mutant were sacrificed and the lung and brain tissue were analysed for fungal burden. Mutant cells were found at an average burden of 2.95 × 105 colony-forming units (cfu) g−1 (SD = 1.73 × 105) in lung tissue, but were not detected in the brain samples for any of the mice. Histopathology of brain tissue at day 60 also failed to detect cells of the snf1 mutant thus supporting the conclusion that the mutant was unable to disseminate to or persist in the brain (Fig. S4). This observation is interesting, given the melanization defect of the snf1 mutant, because previous work showed that a laccase-defective, non-melanized strain was unable to escape from the lung (Noverr et al., 2004). To specifically test whether the snf1 mutant could disseminate beyond lung tissue, we inoculated mice by inhalation with the mutant and WT strains, and we monitored the numbers of fungal cells in the brains and lungs at day 17. This day was selected because it immediately precedes the time that the mice succumb to infection with the WT strain. This experiment revealed that the snf1 mutant was able to reach the brain, although the fungal burden for the mutant was quite low [average of 48.50 cfu (SD = 14.64)] compared with the burden of WT cells [average of 1.87 × 105 cfu (SD = 1.02 × 104)]. For comparison, the lung tissue at day 17 contained an average of 1.96 × 104 cfu (SD = 3.90 × 103) for the mutant and an average of 1.10 × 108 cfu (SD = 2.48 × 107) for the WT. Combined with our previous analysis, these results indicated that the snf1 mutant reached the brain in low numbers but failed to persist. Histopathology also confirmed the presence of the snf1 mutant in the lung (Fig. S4).
Figure 6. Analysis of virulence in a mouse inhalation model of cryptococcosis. Ten A/Jcr mice were inoculated with the WT strain H99, the snf1 mutant or the complemented strain and monitored for signs of illness. The virulence of the snf1 mutant was significantly different from that of the WT strain (P < 0.0001) and the complemented strain (P < 0.0001) by the Krustal–Wallis test.
Download figure to PowerPoint