SEARCH

SEARCH BY CITATION

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The pathogenic fungus Cryptococcus neoformans generally initiates infection in mammalian lung tissue and subsequently disseminates to the brain. We performed serial analysis of gene expression (SAGE) on C. neoformans cells recovered from the lungs of mice and found elevated expression of genes for central carbon metabolism including functions for acetyl-CoA production and utilization. Deletion of the highly expressed ACS1 gene encoding acetyl-CoA synthetase revealed a requirement for growth on acetate and for full virulence. Transcripts for transporters (e.g. for monosaccharides, iron, copper and acetate) and for stress-response proteins were also elevated thus indicating a nutrient-limited and hostile host environment. The pattern of regulation was reminiscent of the control of alternative carbon source utilization and stress response by the Snf1 protein kinase in Saccharomyces cerevisiae. A snf1 mutant of C. neoformans showed defects in alternative carbon source utilization, the response to nitrosative stress, melanin production and virulence. However, loss of Snf1 did not influence the expression of a set of genes for carbon metabolism that were elevated upon lung infection. Taken together, the results reveal specific metabolic adaptations of C. neoformans during pulmonary infection and indicate a role for ACS1 and SNF1 in virulence.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Cryptococcus neoformans causes life-threatening meningoencephalitis in patients with immune deficiency (Casadevall and Perfect, 1998). The fungus is found in the environment as desiccated cells and/or basidiospores produced by sexual reproduction or monokaryotic fruiting; inhalation of these cells initiates a pulmonary infection in mammals (Lin and Heitman, 2006). Human exposure to C. neoformans is thought to be common based on the presence of antibodies in the majority of normal individuals, and many cases of cryptococcosis in immunocompromised individuals may result from reactivation of latent asymptomatic infections (Goldman et al., 2001). C. neoformans is a facultative intracellular pathogen during early stages of murine pulmonary infection (Feldmesser et al., 2000; 2001). In fact, Feldmesser et al. (2000) found that the percentage of fungal cells internalized in alveolar macrophages reached a peak during the first 24 h of infection. Most of the yeast cells were extracellular by 24 h, a time associated with macrophage cytotoxicity, and both intracellular and extracellular cryptococci were observed at 48 h and 7 days. Fungal cells eventually disseminate from the lung via the bloodstream and reach the brain to cause meningoencephalitis.

Although the disease process is well characterized, relatively few studies have examined C. neoformans gene expression during growth in vivo (Rude et al., 2002; Steen et al., 2003; Fan et al., 2005). In an initial study, a transcriptional profile was generated by differential display reverse transcription polymerase chain reaction (RT-PCR) for C. neoformans cells during meningitis in an immunosuppressed rabbit model of meningitis (Rude et al., 2002). This analysis revealed elevated expression for the gene ICL1 encoding isocitrate lyase, a key enzyme in the glyoxylate cycle, suggesting that this cycle might be important for fungal growth in the host. However, disruption of ICL1 did not influence virulence in two animal models nor cause a growth defect in macrophages (Rude et al., 2002). Similarly, deletion of the MLS1 gene encoding malate synthase (another glyoxylate cycle enzyme) did not influence virulence (Idnurm et al., 2007). Subsequent transcriptional profiling of C. neoformans from the cerebrospinal fluid (CSF) of infected rabbits by serial analysis of gene expression (SAGE) revealed relatively high expression of genes involved in energy production, stress response and small molecule transport, as well as carbohydrate, amino acid and lipid metabolism (Steen et al., 2003). The transcriptional response of C. neoformans cells upon phagocytosis by murine macrophages has also been examined (Fan et al., 2005). The fungus responds to phagocytosis with elevated expression of genes at the MAT locus and in the cAMP/protein kinase A (PKA) pathway. Additionally, elevated expression was seen for genes involved in autophagy, peroxisome function, membrane transport, lipid metabolism and the response to oxidative stress (Fan et al., 2005). These studies provide the first insights into C. neoformans gene expression during infection or upon phagocytosis in vitro. Further studies are needed, however, because gene expression profiles could vary substantially for chronic versus acute infections or in different host tissues, as demonstrated for other pathogens such as Candida albicans (Lorenz and Fink, 2001; Fradin et al., 2003; Barelle et al., 2006).

Studies in other pathogenic fungi examined gene expression during infection and explored the roles of glycolytic functions, the glyoxylate cycle, gluconeogenesis and β-oxidation in virulence (Lorenz and Fink, 2001; Idnurm and Howlett, 2002; Wang et al., 2003; Lorenz et al., 2004; Solomon et al., 2004; Barelle et al., 2006; Piekarska et al., 2006; Ramirez and Lorenz, 2007; Schöbel et al., 2007; Thewes et al., 2007; Olivas et al., 2008). For example, the transcriptional response of C. albicans upon phagocytosis by macrophages includes the upregulation of functions for the glyoxylate cycle, gluconeogenesis and fatty acid degradation (Prigneau et al., 2003; Lorenz et al., 2004). In addition, phagocytosis by neutrophils induced an amino acid deprivation response in both C. albicans and Saccharomyces cerevisiae (Rubin-Bejerano et al., 2003). Mutants defective in genes encoding glyoxylate pathway functions (e.g. isocitrate lyase), gluconeogenesis functions (phosphoenolpyruvate carboxykinase) and glycolytic functions (phosphofructokinase and pyruvate kinase) are attenuated for virulence (Lorenz and Fink, 2001; Barelle et al., 2006). For β-oxidation, deletion of the FOX2 gene encoding the second enzyme in the pathway also resulted in attenuated virulence (Piekarska et al., 2006; Ramirez and Lorenz, 2007). However, this phenotype may result from an influence on the glyoxylate cycle because deletion of the PEX5 gene for peroxisomal biogenesis did not cause a virulence defect (Piekarska et al., 2006). In human blood, differentially expressed genes in C. albicans encoded functions for a general stress response, an antioxidative response, the glyoxylate cycle and virulence factors (Fradin et al., 2003). Barelle et al. (2006) used GFP fusions to examine the expression of genes in the glyoxylate cycle, gluconeogenesis and glycolysis in more detail in C. albicans. They found that the genes for the glyoxylate pathway and gluconeogenesis were repressed by the concentration of glucose found in blood and that the genes were induced during phagocytosis (as found by other investigators). Interestingly, these genes were not expressed in fungal cells in infected kidneys. In contrast, glycolytic genes were not induced upon phagocytosis but were expressed in cells in infected kidneys. Barelle et al. (2006) concluded that the glyoxylate cycle and gluconeogenesis may be important early in infection and that glycolysis is important during systemic disease. In light of these results, the finding that components of the glyoxylate cycle were not required for virulence in C. neoformans suggests that this fungus may have different nutritional requirements during infection (Idnurm et al., 2007). Similarly, deletion of the glyoxylate cycle genes for isocitrate lyase and malate synthase did not result in virulence defects in Aspergillus fumigatus (Schöbel et al., 2007; Olivas et al., 2008).

In this study, we examined the early transcriptome changes that occur upon C. neoformans deposition in the murine lung. We performed SAGE analysis on C. neoformans cells recovered from lungs at 8 and 24 h after infection and compared the data with previously described SAGE libraries from cells grown in culture, and from cells harvested from the central nervous system of infected rabbits. The SAGE data suggested that murine pulmonary infection represents a nutrient-limiting environment for invading C. neoformans cells; specifically, genes in several functional categories showed elevated transcripts including alternative carbon source utilization, central carbon and lipid metabolism, stress response and virulence. To examine carbon source utilization in more detail, we characterized the ACS1 gene encoding acetyl-CoA synthetase and the SNF1 gene encoding a predicted serine/threonine protein kinase in C. neoformans. Loss of ACS1 resulted in poor growth on acetate and a mild attenuation of virulence. The SNF1 gene was examined because the pattern of gene expression resembled the regulatory influence of the well-characterized Snf1 protein kinase in S. cerevisiae; this protein mediates glucose sensing, alternative carbon source utilization and the response to stress. Deletion of the SNF1 gene in C. neoformans gene resulted in poor growth on acetate and ethanol at 37°C, reduced melanin production and a complete loss of virulence in a murine model of cryptococcosis.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

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.
TagLIMYeast nitrogen baseLung, 8 hLung, 24 hRabbit, CSFTIGR gene numberGenBank accession numberPredicted function and gene name (if known)
  1. Tags are listed by abundance based on the 8 h library; these tags have higher levels in this library relative to the low-iron medium (LIM) library. The remaining tags in the shaded rows are listed by their abundance in the LIM library. The tag counts were normalized to 20 000 tags for each library.

  2. CSF, cerebrospinal fluid.

Glyoxylate cycle and acetate/ethanol/pyruvate metabolism
 CAGCAGTGTA30144467CNF03900XM_571348Aldehyde dehydrogenase
 CGCGAGGGCA1980400CNA07740XM_567122Acetyl-CoA synthase, ACS1
 GCCCAGCAGG0162250CNH02910XM_572462Malate synthase, MLS1
 CATCACTCTT132394813CNJ00950XM_567475Pyruvate decarboxylase
 ACCTTTATAT00730CNC06260XM_569885Alcohol dehydrogenase
Tricarboxylic acid cycle
 CCAATGATTA101968CND02620XM_570245Aconitase
 CATTTTCTCA001561CNG03480XM_572036Succinate dehydrogenase
 GGTTACGCCG155631CNG03490XM_572038Malate dehydrogenase
 AACTTTGTTC1304215CNA04610XM_566837Isocitrate dehydrogenase
 CATTTTGATT80002CNF03780XM_571672Malic enzyme
 TTGGCTCCCA51002CNC01700XM_569517Fumarase
Glycolysis
 ACTCAGGTTG16519173CNB00300XM_568771Fructose 1,6-biphosphate aldolase
 TGGTTTTTGT001110CNJ01080XM_567487Phosphofructokinase
 TGTGAATGTG111964CNB02660XM_568853Hexokinase, HXK2
 GTCGTAGAGT8139357CNC00160XM_569379Enolase
 GGTCGTTTAT120007CNB04050XM_569228Glucose 6-phosphate isomerase
 TATGCAACAC60004CNF00810XM_571493Phosphoglycerate mutase
Gluconeogenesis
 TTTCAGAAGG0168272CNI03590XM_572603Phosphoenolpyruvate carboxykinase, PCK1
Pentose phosphate pathway
 GTATTGACC07256137CNK00070XM_567776Phosphoketolase
 CATTACTGCA5017725CND02280XM_570275Oxidoreductase
 TAGTGTCCCG70641932CNK01050XM_5677936-Phosphogluconate dehydrogenase
 AAGTGTGGTA1220018CNK03170XM_567910Transaldolase
Other functions related to carbon metabolism
 CATTATGACA401955CNA01100XM_567089Glycerol 3-phosphate dehydrogenase
 ATTCACGCGC101753CND02900XM_570206Glutamine:fructose-6-phosphate amidotransferase
 CCATATGTTT3141521CNF03530XM_571431Glycogen phosphorylase-like protein
 GACATTGTTG2221881CNC05340XM_569729d-arabinitol dehydrogenase
 CAACGGGGGG3507111CNH00170XM_572212Phosphomannomutase
 CTTCCTGTTC2319620CNN00430XM_568570Phosphoglucomutase
 CATCCCAAAG1500020CNC03700XM_569599UDP-glucose pyrophophorylase
 TATAAGACCG60004CNF03930XM_571680Glycogen metabolism-related protein
Lipid metabolism
 TAACCATATA19096177CNJ02970XM_567576Butyrylcholinesterase, triacylglycerol lipase, CGL1
 AACCACACTA134391514CNN01770XM_568678Similar to Benzodiazepin receptor
 TCATTTCACT2033103CNI01560XM_572814Sterol-binding protein
 GCGAAGTACT002730CNN01550XM_568632Acyl-CoA oxidase
 GCTGCCTTTG2132261CNI00240XM_572730Enoyl-CoA hydratase/isomerase
 CAATGGGAAT201230CNI03690XM_5728962,4-Dienoyl-CoA reductase (NADPH)
 TATGTGAATC30934CNM00830XM_568422Oxidoreductase, short-chain dehydrogenase/reductase
 CTACAAATGA10911CNH02950XM_572465Oxidoreductase, short-chain dehydrogenase/reductase
 GATAAGGTGT40868CNI02880XM_572653Oxysterol-binding protein
 TATTATTGTT00812CNA03320XM_566658Enoyl-CoA hydratase
 AAGCAGAGGA29735CNI00710XM_572753Long-chain fatty acid CoA ligase
 TAGTTTATAT20732CNG02680XM_571931Hydroxymethylglutaryl-CoA synthase
 ATTGAATGTA007225CND01120XM_570351Fatty acid β-oxidation-related protein
Transporters
 TATACGATTT2606121432CNB02680XM_568855Monosaccharide transporter, HXT1
 CCCATCGTAT8201367924CNM02430XM_568258Plasma membrane iron permease, CFT1
 CTTGACAGCG0542460CNF02960XM_571371Acetate transporter, ADY2
 TATCTGATTT19134810CNG04240XM_572092Hexose transporter
 TATTGGTACA16028130CNC03960XM_569629Phosphate transporter
 GTCATTTATG202732CNF02890XM_571700Neutral amino acid transporter
 CGGGAATACG0024121CNH03640XM_572526GPR/FUN34 family protein/acetate transporter, ATO2
 CTGTTCGGCA93211311CND01080XM_570353High affinity copper transporter, CTR4
 CACTGTATGC1021180CNE02570XM_570958Succinate:fumarate antiporter
 GCCGGGCTGT11520142CNC02540XM_569547Organic acid transporter
 CCGAGAAACG201972CND04380XM_570596Monovalent inorganic cation transporter
 AGCACTTATG101620CNI03490XM_572882Maltose permease, trehalose transporter
 CAGTATCACT201522CNL05010XM_568040Gaba permease
 GTGGTTTTCT101192CNF04760XM_571440Neutral amino acid permease
 CATATATAAC20701CNH00950XM_572289Spermine transporter
 GGAAGAGGAA10632CNE00440XM_571108Inner membrane solute transporter MRS4
 ATCGGTACCC447004CNN01030XM_568544Inorganic phosphate transporter
Table 2.  Virulence-associated and stress-response genes.
TagsLIMYeast nitrogen baseLungs, 8 hLungs, 24 hRabbit, CSFTIGR gene numberGenBank accession numberPredicted function and gene name (if known)
  1. Tags are listed by abundance based on the 8 h library and show higher levels in this library relative to the low-iron medium (LIM) library. The remaining tags in the shaded rows are listed by their abundance in the LIM library. The tag counts were normalized to 20 000 tags for each library.

  2. CSF, cerebrospinal fluid.

TATATGTGTA35024760262CNG04220XM_572088Heat-shock protein 12
CACGTCCACG276195305CND01490XM_570285Cu/Zn superoxide dismutase, SOD1
ATAAGCTTTC120741425CNG00600XM_571772Mannitol-1-phosphate dehydrogenase
CATTTTATGT140351624CNM01520XM_568451Heat-shock protein 90
TAACATAATG16030317CNA01260XM_567249Glutathione S-transferase
TGTTTCTACA212433CNG04220XM_572088Heat-shock protein 12
CTCTTCATTT84231410CNA03160XM_566757Heat-shock protein, SKS2
GCGGATAAAA652368CNB00910XM_569098Stress-response RCI peptide, cation transport-related protein
GCTGCCTCTG0421321CNA01500XM_566622Alternative oxidase
ATGTTTTATT902073CNG02560XM_572003UDP-glucuronic acid decarboxylase, UXS1
AGATGGACGA4191984CNG01060XM_571865Cyclophilin-like protein
AATTGATGAG0018102CNC07160XM_569844Flavohaemoprotein, FHB1
TTGATTTTTT001630CNM01860XM_568294Macrolide-binding protein FKBP12
GCGACAGCCA011521CNC01490XM_569482Peptidyl prolyl cis-trans isomerase, ESS1
ACGGAGTGTA101492CND00260XM_570333FK506-resistant calcineurin B regulatory subunit
TCGATGGCGA2011514CNE05040XM_571033Glyoxal oxidase precursor
CAACGATGAT5010366CNF02910XM_571367LEA domain protein
GCTTAATTAT01821CNN00360XM_568558Protein kinase, SCH9
GTCTTCATCA20821CND05600XM_570476Heat-shock protein 12
TTTCTGAAAA20711CND05940XM_570487Capsule-associated protein, CAP1
TGTGTAAAAG00610CNE00710XM_570793Mannitol-1-phosphate dehydrogenase
TCAGAAGTTG78811151CNC04200XM_569667Thioredoxin
TGTTATCGGT60422654CNM01520XM_568451Heat-shock protein 90
CATAATTGGC35113114CNM02070XM_568283Heat-shock protein

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.

image

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 (< 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.

image

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 (= 0.0001) or the complemented strain (= 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).

image

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).

image

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).

image

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 (< 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).

image

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 (< 0.0001) and the complemented strain (< 0.0001) by the Krustal–Wallis test.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Gene expression during pulmonary cryptococcosis

In this study, we employed SAGE to compare the C. neoformans transcriptomes at 8 and 24 h of murine pulmonary infection with expression patterns from in vitro culture or from CSF in a rabbit model of experimental meningitis. These comparisons provided two main insights into the pathogenesis of C. neoformans. First, lung infection resulted in elevated expression of fungal genes encoding functions for the production and utilization of acetyl-CoA. These included enzymes in the glyoxylate pathway, gluconeogenesis, β-oxidation and the conversion of pyruvate, ethanol and acetate to acetyl-CoA. Two candidate acetate transporters also showed elevated expression. This pattern was set against a background of elevated or reduced expression of specific components of the glycolytic pathway and the TCA cycle. As proposed for C. albicans (Fradin et al., 2003), this pattern may reflect variations in gene expression profiles in subpopulations of cells exposed to different host environments (e.g. extracellular and phagocytized cells). More interestingly, it could also reflect adjustments to the expression levels of pathway components to enhance the production of specific metabolic intermediates necessary for growth in host tissue. Given the early times of infection that we analysed, it is also possible that some of the changes in gene expression for metabolic functions reflect the mobilization of glycogen and/or triglyceride stores to generate glucose and acetyl-CoA. A role for acetyl-CoA as a key metabolic intermediate was tested by deleting the ACS1 gene encoding a predicted acetyl-CoA synthase; the resulting mutant was unable to grow on acetate and showed a delayed ability to cause disease. It is likely that production of acetyl-CoA by other reactions (e.g. β-oxidation, other acetyl-CoA synthetases) partially compensated for loss of ACS1 (Fig. 1).

The second insight from this study was that the transcript profiles observed during lung infection suggested a role for the C. neoformans homologue of the yeast Snf1 kinase, a well-characterized regulator of alternative carbon source utilization and the stress response in S. cerevisiae. Deletion of SNF1 in C. neoformans revealed an involvement in carbon source utilization, virulence and the regulation of melanin production. Interestingly, the snf1 mutant was able to disseminate in quite low numbers to the brain following infection but was unable to persist, perhaps due to problems with carbon source utilization and/or the response to host defence mechanisms. SNF1 was specifically required for the elevated transcript levels of the LAC1 gene during growth on low glucose thus revealing a new regulatory function for melanin formation in C. neoformans. However, loss of SNF1 did not influence the expression of the genes for carbon source utilization that we identified in the SAGE analysis of lung infection. Instead, we found that the transcript levels of these genes were elevated in cells grown under low-glucose conditions, a result consistent with the hypothesis that glucose is limiting during pulmonary infection.

Glucose limitation and alternative carbon sources during infection

The availability of glucose and local microenvironments in the mammalian host can have a marked influence on the expression of functions to exploit carbon sources other than glucose (e.g. acetate, lactic acid, fatty acids). For example, Barelle et al. (2006) examined gene expression in C. albicans during infection in light of the observation that glucose is present at sufficiently high levels in blood (0.06–0.1%) to limit expression of functions for alternative carbon source utilization. These studies indicated that C. albicans differentially regulates carbon assimilation pathways depending on the stage of infection (e.g. during interactions with phagocytic cells versus systemic infection) and the host tissue. Similarly, our comparison of the SAGE data for infections of the lung versus the CSF revealed that the patterns of gene expression were quite different, in support of the idea that there may be tissue-specific patterns of gene expression and adaptation for C. neoformans. The patterns of gene expression found by Thewes et al. (2007) for C. albicans during intraperitoneal infection and ex vivo liver infection also indicated that at least some of the cells experience a low-glucose environment. These investigators found upregulation of the PCK1 gene encoding phosphoenolpyruvate carboxykinase for gluconeogenesis as well as upregulation of genes for the synthesis of acetyl-CoA from pyruvate. However, specific glycolytic enzymes and TCA cycle enzymes were also upregulated indicating that some of the cells were utilizing six-carbon sugars and respiration for energy production. We found similar sets of genes to be upregulated during cryptococcal pulmonary infection, and many of these were also regulated by glucose in vitro. The secretions from respiratory airways generally have a very low concentration of glucose in healthy individuals (< 0.05 mM), and acute illness, inflammation or diabetes can elevate glucose levels (Philips et al., 2003). For example, bronchial aspirates from intubated patients were found to have a mean glucose concentration of 3.5 mM and, interestingly, glucose levels correlated with risk of infection for some bacterial pathogens (Philips et al., 2005). We hypothesize that many of the C. neoformans cells entering the lung experience glucose starvation. This idea is supported by the expression patterns that we observed and, in particular, by the identification of a candidate hexose transporter (HXT1) that was the most highly expressed gene (612 tags) in the 8 h lung library. This tag was present at much lower levels in the LIM (26 tags) and the CSF (2 tags) libraries. Our in vitro analysis indicated that the expression of the gene was dramatically higher at 0.2% glucose compared with 2% glucose. Thus the expression of this gene may be a useful sensor/readout of glucose levels (although other unknown factors could also influence the expression of the gene). The hxt1 mutant also showed precocious melanin formation and this phenotype is consistent with a role for Hxt1 in glucose sensing.

During cryptococcal meningoencephalitis, a low-glucose environment is thought to occur in brain tissue and a three- to fivefold drop in glucose concentrations was found in the CSF of infected rabbits (Perfect et al., 1980; Kwon-Chung et al., 2000; Rude et al., 2002). However, our analysis of the library from experimental meningitis in the rabbit model (Steen et al., 2003) did not reveal elevated expression of the glucose responsive genes that were observed in the pulmonary infection libraries. These results indicate that a more focused comparison of C. neoformans gene expression in different host tissues is needed.

The role of gluconeogenesis in the virulence of C. neoformans has been explored as a result of the identification of the PCK1 gene for phosphoenolpyruvate carboxykinase as a downstream target of Vad1, a DEAD-box RNA helicase (Panepinto et al., 2005). The tag for PCK1 was elevated in our SAGE analysis of pulmonary infection, but not in the macrophage internalized cryptococcal cells (Fan et al., 2005), suggesting a difference between the pulmonary infection and the intracellular environment. Interestingly, a pck1 mutant of C. neoformans was unable to grow on lactate or to cause disease (Panepinto et al., 2005). This observation led to the proposal that three-carbon sources, rather than two-carbon molecules such as acetate, are preferred during infection. Our finding that a acs1 mutant did not grow on acetate or ethanol and had reduced virulence is consistent with this idea. Similarly, icl1 and mls1 mutants also cannot use acetate as a sole carbon source but retain virulence (Rude et al., 2002; Idnurm et al., 2007). Further characterization of the icl1 mutant revealed that the mutant also does not grow on ethanol and shows poor growth on lactate and glycerol (J. Perfect, unpubl. results).

Roles for acetyl-CoA and acetate during adaptation to the lung environment

Acetyl-CoA is a central metabolite in the balance between carbohydrate metabolism and fatty acid catabolism (Fig. 1). Regulation of the balance may be a specific adaptation to the host environment by C. neoformans, as suggested by the abundance of transcripts for enzymes mediating the production and utilization of acetyl-CoA during pulmonary infection. It is possible that the expression pattern that we observed reflects the utilization of ethanol and acetate as carbon sources during infection. However, as mentioned above, mutants that cannot utilize acetate in culture retain virulence in C. neoformans. One can speculate that the production of acetyl-CoA may be specifically important during infection by C. neoformans because acetyl-CoA is an important precursor for the synthesis of chitin in the cell wall and for O-acetylation of the capsule via acetyltransferase activity (in addition to its key metabolic role). β-1,4 N-acetyl glucosamine has recently been shown to play a role in attachment of the capsule to the cell wall of C. neoformans and treatment with chitinase can release capsule polysaccharide (Rodrigues et al., 2008). Acetylation of capsule polysaccharide plays a role in the ability of capsule polysaccharide to inhibit neutrophil migration and a mutant lacking capsular acetyl groups is hypervirulent (Janbon et al., 2001; Ellerbroek et al., 2004). Thus, enhanced acetyl-CoA production may meet the demands of capsule-related biosynthetic functions during infection.

An additional intriguing aspect of pathogen metabolism during infection is the production of exported metabolites that may condition the host environment and contribute to virulence. The SAGE data for pulmonary infection are particularly interesting in light of the characterization of the metabolites produced by C. neoformans in culture and in cryptococcomas from rat lung and brain (Bubb et al., 1999; Wright et al., 2002; Himmelreich et al., 2003). These studies revealed that trehalose, mannitol, glycerol, acetate, ethanol and glycerophosphorylcholine were particularly abundant among the > 30 metabolites that were detected. Wright et al. (2002) discussed the implications of these metabolites for virulence including the contribution of mannitol to defence against oxidative killing and the possibility that acetate acidifies the extracellular environment. In fact, direct measurement of cerebral cryptococcomas revealed a relatively low pH of 5.4–5.6. Wright et al. (2002) went on to demonstrate that C. neoformans supernatants at pH 5.5 induced necrosis in neutrophils, reduced superoxide production and influenced chemotaxis by these cells. Our observed elevated expression of transcripts for enzymes involved in the conversion of pyruvate to metabolites such as acetaldehyde, acetate and ethanol in C. neoformans during pulmonary infection is consistent with the appearance of these metabolites in cryptococcomas. In particular, we found tags for two candidate acetate transporters that were elevated during infection and we hypothesize that one or both of these proteins may contribute to acetate export. In addition to an influence on the host immune response, acidification of the local environment could also contribute to the availability of iron by triggering its release from transferrin. Indeed, Friedman et al. (2006) have shown that Staphylococcus aureus remodels its metabolism in response to iron limitation (a key feature of the host environment) to produce acidic products including lactic acid. For fungal pathogens, Thewes et al. (2007) also found a pattern of expression for pH-responsive genes in C. albicans during infection, as well as upregulated expression of transporters for iron, zinc and phosphate. These authors also noted the possible connection between pH modulation of the host environment and iron acquisition. As mentioned earlier, we found that the transcript for the iron permease Cft1 was elevated in pulmonary infection suggesting that C. neoformans is experiencing iron deprivation. We recently showed that iron acquisition during infection is partially dependent on Cft1 and that this permease is required for transferrin utilization (Jung et al., 2008).

Snf1 and virulence-related gene expression

In yeast, the Snf1 protein kinase is required for the metabolic shift that occurs upon glucose depletion, for growth on alternative carbon sources, and for the response to a variety of environmental stresses such as sodium and lithium salts, alkaline pH, heat shock and hyperosmolarity (Alepuz et al., 1997; Hardie et al., 1998; Kemp et al., 1999; 2003; Vyas et al., 2001; Portillo et al., 2005; Ye et al., 2006; Hong and Carlson, 2007). We found that Snf1 in C. neoformans plays a role in the growth response to acetate, ethanol and sucrose, and that sensitivity of the snf1 mutant was noted for nitrosative stress and for the drugs rapamycin and amphotericin B. We found that all of the snf1 phenotypes in C. neoformans were manifested at the host temperature of 37°C, but not at 30°C. In S. cerevisiae, a snf1 mutant has reduced thermotolerance, and carbon source utilization mutants in C. albicans have more severe defects at 37°C (Thompson-Jaeger et al., 1991; Ramirez and Lorenz, 2007). In C. neoformans, snf1 mutant strains grew as well as the WT strain on media (either YPD or YNB) with glucose (or glycerol, galactose, raffinose, arabinose) as the sole carbon source at 37°C, indicating that loss of SNF1 did not generally reduce thermotolerance, but rather influenced specific phenotypes.

SNF1 has been implicated in the virulence of plant pathogenic fungi. For example, the SNF1 gene in C. carbonum is required for the expression of cell wall-degrading enzymes and for disease symptom formation on maize (Tonukari et al., 2000). The SNF1 gene in Fusarium oxysporum, a pathogen that causes vascular wilt disease in over 100 cultivated plant species, regulates the transcription of genes encoding cell wall-degrading enzymes and virulence on both Arabidopsis thaliana and Brassica oleracea (Ospina-Giraldo et al., 2003). So far there has been no description of a role for SNF1 in the virulence of fungal pathogens of animals. A SNF1 homologue in C. albicans is essential for viability, and disruption of one allele resulted in morphological changes and decreased growth, but did not influence virulence (Petter et al., 1997). The discovery of a role for SNF1 in the virulence of C. neoformans reveals a new regulatory connection between carbon source utilization and growth in mammalian hosts. Part of the virulence defect may be due to reduced melanin production at 37°C. Laccase expression in C. neoformans is repressed by elevated temperature and enhanced by glucose starvation as well as copper, iron and calcium (Zhu and Williamson, 2004). Laccase expression also occurs during early pulmonary infection (Garcia-Rivera et al., 2005). Both LAC1 and LAC2 contribute to melanin production and both are regulated by the cAMP pathway; differences have been noted for the two genes, however (Missall et al., 2005; Pukkila-Worley et al., 2005). Notably, LAC2 but not LAC1 is regulated in response to oxidative and nitrosative stresses in a manner that is influenced by the TSA1 gene encoding a thiol peroxidase (Missall et al., 2004;2005). Missall et al. (2005) presented a model for the regulation of LAC2 expression in response to nitric oxide stress and proposed that a stress-activated protein kinase might function downstream of Tsa1.

In terms of contributions to virulence, the laccases produced by the LAC1 and LAC2 have different but overlapping substrate specificities that may allow the utilization of different diphenolic substrates in brain tissue. The LAC1 gene appears to make the larger contribution to virulence in a mouse model of cryptococcosis, although mice still succumb to infection with a lac1 mutant (Pukkila-Worley et al., 2005). Given these results, it is likely that the Snf1 protein influences other aspects of virulence because a snf1 mutant fails to cause disease. It is possible that the melanin defect contributes to the observed poor dissemination of the snf1 mutant from the lung to the brain because melanin formation is known to influence this process (Noverr et al., 2004). However, the snf1 mutant also failed to persist in brain tissue, perhaps indicating that Snf1 influences the expression of additional virulence factors. For example, Snf1 might regulate processes that are important for virulence in C. neoformans such as gluconeogenesis (Panepinto et al., 2005), as well as control the expression of cell surface factors that mediate interactions with the host and/or influence the response to host defences. Overall, the identification of a role for Snf1 presents opportunities to further characterize the role of the protein in the regulation of central metabolism, the utilization of alternative carbon sources and the response of the fungal cells to the stressful conditions of the host environment.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Strains, plasmids and media

The serotype A strain H99 (C. neoformans var. grubii) and the S. cerevisiae strains W303-1A (MATa ade2-1 trp1-1 his3-11, 15 can1-100 ura3-1 leu2-3, 112) and MCY4908 (W303-1A snf1Δ10) were used in the study. The strains were maintained on YPD medium (1% yeast extract, 2% peptone, 2% dextrose and 2% agar). Selectable markers for the transformation of C. neoformans were from plasmid pCH233 (nourseothricin resistance) and pJAF1 (neomycin resistance). Plasmid BPH618 was used for yeast transformation. YPD plates containing neomycin (200 μg ml−1) were used to select C. neoformans transformants and YPD plates containing nourseothricin (100 μg ml−1) were used to select the SNF1-complemented transformants. S. cerevisiae transformants were selected on YNB agar (yeast nitrogen base without amino acids and uracil) supplemented with 2% glucose and the other required nutrients. YNB agar (without amino acids and uracil) was supplemented with 2% glucose, 2% sucrose, 2% raffinose or 2% maltose, and used to examine the growth of the yeast snf1 mutant. YPD and/or YNB plates (YNB with amino acids) supplemented with different inhibitors or chemicals were used for phenotypic experiments. Escherichia coli was grown on LB broth or agar supplemented with 100 μg ml−1 of ampicillin at 37°C.

Isolation of C. neoformans cells from mouse lungs for SAGE analysis

The C. neoformans cells for mouse inoculation were grown overnight in YPD in a 30°C shaker, washed with phosphate-buffered saline (PBS) and re-suspended in PBS at a concentration of 8.2 × 107 cells per 50 μl. A total of 20 female A/Jcr mice were anaesthetized with ketamine and xylazine, inoculated by nasal inhalation and subjected to bronchoalveolar lavage at 8 and 24 h. At each time point, the treated mice were euthanized, a small incision was made in the trachea and a capillary tube was inserted towards the lungs. The tube was secured by silk thread and a series of 0.5 ml aliquots of ice-cold water were flushed into the lungs. A total of 10 ml of water was used per mouse and the lavage fluids from each inoculation group were pooled. The cells were washed twice with ice-cold water, frozen at −80°C and lyophilized for RNA isolation. In total, 3.0 × 108 cells were obtained at 8 h and 2.6 × 108 cells were collected at 24 h.

SAGE library construction, sequencing and analysis

SAGE library construction, sequencing and analysis were as previously described (Steen et al., 2002, 2003; Hu et al., 2007). Briefly, RNA was isolated from lyophilized cells by vortexing with glass beads (3.0 mm, acid-washed and RNase-free) for 15 min in 15 ml of TRIZOL extraction buffer (Invitrogen, Carlsbad, CA, USA). The mixture was incubated for 15 min at room temperature, total RNA was isolated according to the manufacturer's instructions and RNA quality was assessed by agarose gel electrophoresis. Total RNA was used directly for SAGE library construction as described by Velculescu et al. (1995) using the I-Long SAGE kit (Invitrogen). The tagging enzyme for cDNA digestion was NlaIII and 29 PCR cycles were performed to amplify the ditags during library construction. Colonies were screened by PCR (M13F and M13R primers) to assess the average clone insert size and the percentage of recombinants. Clones from the libraries were sequenced by BigDye primer cycle sequencing on an ABI PRISM 3700 DNA analyser. Sequence chromatograms were processed using PHRED (Ewing and Green, 1998), and vector sequence was detected using Cross_match (Gordon et al., 1998). Fourteen-base-pair tags were extracted from the vector-clipped sequence, and an overall quality score for each tag was derived based on the cumulative PHRED score. Duplicate ditags and linker sequences were removed as described previously (Steen et al., 2002). Only tags with a predicted accuracy of ≥ 99% were used, and statistical differences between tag abundance in different libraries were determined using the methods of Audic and Claverie (1997).

The libraries yielded 21 510 (8 h) and 20 129 (24 h) tags. An overview of the abundance classes for both SAGE libraries is presented in Table S1, with both the number of different tag sequences and the total number of tags present in each class for the cells from lung tissue at 8 or 24 h after infection. An overview of pair-wise analyses of differential expression for all of the libraries is presented in Table S2, and the 100 most abundant tags in each library are listed in Table S3. All libraries were normalized to 20 000 to allow direct comparisons, and the tags that appeared less than once in any given library were removed. The EST database available for strain H99 at the University of Okalahoma's Advanced Center for Genome Technology (http://www.genome.ou.edu/cneo.html) was used for the preliminary assignment of tags to genes. When an EST sequence could not be identified for a particular tag, the genomic sequence for H99 at the Duke University Center for Genome Technology (http://cgt.genetics.duke.edu/data/index.html) and the Broad Institute (http://cneo.genetics.duke.edu/) was used to identify contigs with unambiguous tag assignments. Note that a limitation of the SAGE approach is that some transcripts are not detected because of low abundance and/or the absence of an NlaIII site for transcript processing.

Complementation of a S. cerevisiae snf1 mutation with the SNF1 gene of C. neoformans

To obtain a cDNA for SNF1, total RNA was isolated from frozen C. neoformans cells. The cDNA was synthesized using random hexamer priming and Superscript transcriptase II (Invitrogen Canada). A 2270 bp PCR product was obtained using primers SNF1-cDNA-5 and SNF1-cDNA-6, cloned into TOPO-TA vector (pSNF1c-topo) and then subcloned into pBPH618 to obtain pCnSNF1c. The same pair of primers was used to amplify the SNF1 gene from genomic DNA of strain H99 and a PCR product of 2950 bp was obtained. This product was cloned into a TOPO-TA vector (pSNF1g-TOPO) and subcloned into pBPH618 to create pCnSNF1g. Both SNF1 inserts in pCnSNF1c and pCnSNF1g were sequenced to confirm the presence of the C. neoformans gene. To complement yeast snf1 mutant with C. neoformans SNF1 homologue, pCnSNF1c and pBPH618 (empty vector) were transformed into strain MCY4908 by PEG and lithium acetate treatment. Transformants were selected on SCD (dexose)-URA and 5-FOA plates for uracil prototrophy, and on SCS (sucrose) for sucrose utilization. The sequences of the primers for this study are given in Table S6.

Deletion of the SNF1 gene in C. neoformans

A snf1::NEO deletion allele was constructed using a modified overlap PCR procedure (Davidson et al., 2002; Yu et al., 2004). Briefly, the primers SNF1-1/SNF1-3 and SNF1-4/SNF1-6 (Table S6) were used with genomic DNA to obtain the left and right arms for the deletion construct. The NEO selectable marker was amplified using primers SNF1-2/SNF1-5 and the plasmid pJAF1. The snf1:NEO allele results in the deletion of the complete open reading frame of SNF1 (2926 bp). The resulting PCR product (3854 bp) was used to transform strain H99 by biolistic transformation (Davidson et al., 2000). Transformants were grown on YPD plates containing neomycin and screened by colony PCR with Extaq polymerase (Takara) using primer pairs SNF1-7/SNF1-8 and SNF1-9/hug-Neo. Primer SNF1-9 was designed from the region upstream of SNF1 and hug-Neo was designed for the NEO gene. Transformants in which WT allele was replaced were confirmed by genomic hybridization as described (Hu and Kronstad, 2006). One mutant designated SNF1-22 contained the deletion allele and was studied further. For complementation of the deletion mutation, the SNF1 gene was amplified by PCR using primers SNF1-rec-for and SNF1-rec-rev, and genomic DNA from strain H99. The 4627 bp product was digested with XbaI and cloned into the XbaI site of pCH233, creating the plasmid pSNF1rec. The strain SNF1-22 was transformed with pSNF1rec by biolistic transformation, and transformants were selected on YPD containing nourseothricin (100 μg ml−1). Re-introduction of SNF1 was confirmed by colony PCR and genomic hybridization.

Quantification of gene expression during pulmonary infection or during growth in culture

Total RNA from frozen cells collected from the lungs of infected mice was obtained as described above (from an independent experiment), and DNA was removed by treatment with DNase I for 30 min at 25°C. Subsequently, cDNA was synthesized using random hexamers and Superscript transcriptase II (Invitrogen Canada). The resulting cDNA was used for real-time PCR with primers targeted to the 3′ regions of transcripts. Primers were designed using PrimerExpress v3 (Applied Biosystems). The Power SYBR Green PCR mix (Applied Biosystems) was used according to the manufacture's recommendations. An Applied Biosystems 7500 Fast Real-time PCR system was used to detect and quantify the PCR products with the following conditions: incubation at 95°C for 10 min followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. The cDNA of the ACT1 gene was used to normalize the data. Dissociation analysis on all PCR reactions confirmed the amplification of a single product for each primer pair and the absence of primer dimerization. Relative gene expression was quantified using SDS software 1.3.1 (Applied Biosystems).

Growth conditions for examining the influence of reduced glucose levels, acetate and loss of SNF1 were as follows. Cells (WT, snf1 and snf1::SNF1) were grown in YNB + 2% glucose at 37°C to mid-log phase, and washed with the 37°C pre-warmed medium. Equal numbers of cells (1.5 × 108 cells) were transferred to either YNB + 2% glucose or YNB + 0.2% glucose or YNB + 2% acetate, and cultured for 5 h at 37°C prior to RNA isolation. The sequences of the primers for the PCR analysis are given in Table S7.

Stress and drug response assays

To examine the response of C. neoformans WT, snf1 and snf1::SNF1 strains to various stress conditions, exponentially growing cultures were washed, re-suspended in H2O and adjusted to a concentration of 0.2 × 105 cells μl−1. The cell suspensions were diluted 10-fold serially, and 5 μl of each dilution was spotted onto YPD and/or YNB plates supplemented with different chemicals. Plates were incubated for 2–5 days (depending on the conditions) at 30°C or 37°C, and photographed. The responses of strains to oxidative, nitrosative, osmotic stress and to agents that challenged cell wall integrity were examined. The specific assays were performed on YPD and/or YNB plates supplemented with or without 1.2 M KCl, 1.0 or 1.5 M NaCl, 75 mM LiCl, 0.1% SDS, 0.5 mg ml−1 Congo red, 3 μg ml−1 menadione, 0.5 mM H2O2 and 2, 4 or 8 mM sodium nitrite (NaNO2). For carbon source utilization experiments, YNB agar (yeast nitrogen base, 6.7%) was supplemented with one of the following carbon sources: 0.5% or 2% glucose, 0.5% or 2% sucrose, 2% raffinose, 2% maltose, 2% galactose, 0.5% or 2% sodium acetate, 0.5% or 2% ethanol, 0.5% or 2% glycerol, 0.5% or 2% lactic acid. Sensitivity to inhibitors of calcineurin and TOR signalling were examined by spotting the cell dilutions on YNB plates containing 2 or 4 μg ml−1 FK506, 125 or 250 μg ml−1 cyclosporine A, or 1 or 10 μg ml−1 rapamycin. The antifungal drugs amphotericin B (0.5 μg ml−1) and fluconazole (0.5 or 1 μg ml−1) were also tested. The figures show only the conditions where the mutant gave a different response compared with the WT strain.

Capsule formation and melanin production

Low-iron medium was used to examine capsule formation. A single colony from a YPD plate for each strain was cultured overnight at 30°C in liquid YPD medium. Cells were harvested and diluted in low-iron water, and 106 cells were added into 3 ml of LIM for further incubation at 30°C for 48 h. After incubation, the capsule was stained by India ink and examined by differential interference microscopy (DIC). To examine melanin production, a single colony of each strain was incubated overnight at 30°C in liquid YPD medium, washed and diluted to 2 × 104 cells ml−1. Five microlitres of serial dilutions from this stock were spotted onto L-DOPA plates containing 0.1% glucose. The plates were incubated for 3 days at 30°C or 37°C, and melanin production was monitored and photographed daily.

Virulence assays

For virulence assays, female A/Jcr mice (4–6 weeks old) were obtained from Jackson Laboratories (Bar Harbor, ME, USA). The C. neoformans cells for inoculation were grown in YPD medium overnight at 30°C, washed in PBS and re-suspended at 1.0 × 106 cells ml−1 in PBS. Inoculation was by intranasal instillation with 50 μl of cell suspension (5.0 × 104). The status of the mice was monitored twice per day post inoculation. Differences in virulence were statistically assessed with the Krustal–Wallis test. For histopathology, infected mice were euthanized by CO2 inhalation, and organs were excised and placed in 10% buffered formalin. Fixed organs were sent to Wax-It Histology Services (Vancouver, BC, Canada) for sectioning and staining with Mayer's Mucicarmine. For determination of the fungal load in organs, infected mice were euthanized by CO2 inhalation and organs were excised, weighed and homogenized in 1 ml of PBS using a MixerMill (Retsch). Serial dilutions of the homogenates were plated on Sabouraud dextrose agar plates containing 35 μg ml−1 chloramphenicol and colony-forming units were counted after an incubation for 48 h at 30°C.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Dr Marian Carlson for providing yeast strains, Dr Philip Hieter for providing plasmid pBPH618 and Dr Joseph Heitman for providing plasmids pJAF1 and pCH233. Dr Gary Cox provided excellent advice on performing the pulmonary infections. We also thank the staff of Canada's Michael Smith Genome Sciences Centre for SAGE advice and sequencing. This work was supported by the National Institute of Allergy and Infectious Disease (R01 AI053721), the Canadian Institutes of Health Research and the British Columbia Lung Association. J.W.K. is a Burroughs Wellcome Fund Scholar in Molecular Pathogenic Mycology.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
MMI_6374_sm_Figures_S1-S4_and_Tables_S1-S7.pdf8143KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.