**2. Effect of glucose on alternative carbon sources in** *Candida albicans*

The assimilation of carbon sources is fundamentally important for the growth of *C. albicans* and for the establishment of infections in the human host. As described earlier, to grow, a microbe must be able to assimilate carbon [34]. For most yeasts, glucose is generally a preferred carbon source and for *S. cerevisiae* the chosen mode of metabolism is often fermentative in the presence of excess glucose. This uses the Embden-Meyerhof or glycolytic pathway, resulting in the formation of ethanol. In the absence of glucose, *S. cerevisiae* adapts to utilise the alter‐ native carbon sources that are available and switching to non-fermentable carbon metabolism. Likewise, *C. albicans* alters the expression of its metabolic functions to facilitate cell survival [35]. *C. albicans* adjusts its metabolism to growth in biofilms by up-regulating amino acid biosynthesis genes [36]. When exposed to human neutrophils or cultured macrophages, *C. albicans* also up-regulates amino acid biosynthesis genes and displays a shift from fermentative to non-fermentative metabolism [35, 37, 38]. Importantly, the utilisation of non-fermentable carbon sources requires gluconeogenesis and the glyoxylate cycle [39]. Furthermore, the glyoxylate cycle is required for fungal virulence [40]. These examples illustrate the metabolic flexibility of this pathogen and the relevance of metabolic adaptation pathogenicity [41].

Our understanding of the physiology of *C. albicans* has been largely based on presumptions that the central carbon metabolism in *S. cerevisiae* and *C. albicans* is similar. In these fungi, the pathways of central carbon metabolism, including glycolysis, gluconeogenesis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle and glyoxylate cycle, are highly conserved [14, 42]. However, metabolic differences do exist between *C. albicans* and *S. cerevisiae*, the most obvious of which relates to their patterns of sugar utilisation [41]. For example, *S. cerevisiae* belongs to the group that is called Crabtree-positive yeasts which have the ability to produce ethanol even in the presence of oxygen. In contrast, *C. albicans* is designated as a Crabtree-negative yeast [43] because it retains respiratory capacity in the presence of excess glucose [44].

As described earlier, pathways of alternative carbon assimilation in *S. cerevisiae* are subject of glucose repression study. The genetics of glucose repression have been studied, and the regulatory elements that drive this regulation have been described in *S. cerevisiae* [10, 12, 45, 46, 47]. These include co-regulatory mechanisms that act on common elements within the promoter sequences of gluconeogenic and glyoxylate cycle genes. The carbon source regulator elements (CSRE) in the promoters of the *S. cerevisiae PCK1, FBP1, MLS1* and *ACR1* are required for their transcriptional induction in the absence of glucose [48 - 52]. In addition the promoters of the *S. cerevisiae ICL1, MLS1* and *FBP1* genes contain binding sites for the transcription repressor Mig1 [53]. Mig1 represses the transcription of these genes in the presence of glucose, and the activity of Mig1 is being regulated by Snf1 (AMP kinase) signalling.

Transcript profiling of glucose responses in *C. albicans* and *S. cerevisiae* has shown that both yeasts are sensitive to very low levels of glucose [54, 55]. In *S. cerevisiae* and *C. albicans*, glycolytic genes were up-regulated and gluconeogenic and TCA cycle genes were down-regulated even when only 0.01% glucose was added to the growth medium. Yin *et al*. [54] also showed that *S. cerevisiae* ribosomal protein genes also respond to glucose but that they were less sensitive to glucose than the metabolic genes mentioned above. In *S. cerevisiae*, ribosomal protein gene expression was up-regulated following glucose addition at concentrations above 0.1% [54, 56].

Therefore addition of glucose to *S. cerevisiae* cells growing on alternative carbon sources causes a rapid shift from non-fermentative to fermentative metabolism, in part through tight regula‐ tion of gene transcription. Glucose also regulates metabolic activity in *S. cerevisiae* at posttranscriptional levels. Glucose triggers the accelerated decay of gluconeogenic mRNA (*PCK1, FBP1*) [54]. Furthermore, glucose triggers the catabolite inactivation and degradation of gluconeogenic and glyoxylate cycle enzymes in *S. cerevisiae.* Fructose-1,6-bisphophatase (FBPase) is expressed when yeast cells are grown on non-fermentable carbon sources. When the cells are then transferred to a glucose-containing medium, the cells rapidly degrade FBPase to inactivate gluconeogenic activity. This was shown by immunoprecipitation and Western blotting [57]. These authors found that the ubiquitin-conjugating enzyme Ubc8p contributes to glucose-induced ubiquitination of FBPase and that this ubiquitination proceeds the catabolite degradation of the enzyme via the proteasome [58], three other gluconeogenic and glyoxylate cycle enzymes were identified as additional targets of the catabolite inactivation machinery [59]. In addition, it was discovered that an amino-terminal proline residue is essential for the rapid degradation of FBPase in response to glucose. FBPase phosphorylation was not necessary for degradation to occur [59]. This amino-terminal ubiquitination target site on FBPase essentially functions as an autonomous, primary degradation signal.

Likewise, *C. albicans* alters the expression of its metabolic functions to facilitate cell survival [35]. *C. albicans* adjusts its metabolism to growth in biofilms by up-regulating amino acid biosynthesis genes [36]. When exposed to human neutrophils or cultured macrophages, *C. albicans* also up-regulates amino acid biosynthesis genes and displays a shift from fermentative to non-fermentative metabolism [35, 37, 38]. Importantly, the utilisation of non-fermentable carbon sources requires gluconeogenesis and the glyoxylate cycle [39]. Furthermore, the glyoxylate cycle is required for fungal virulence [40]. These examples illustrate the metabolic flexibility of this pathogen and the relevance of metabolic adaptation pathogenicity [41].

Our understanding of the physiology of *C. albicans* has been largely based on presumptions that the central carbon metabolism in *S. cerevisiae* and *C. albicans* is similar. In these fungi, the pathways of central carbon metabolism, including glycolysis, gluconeogenesis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle and glyoxylate cycle, are highly conserved [14, 42]. However, metabolic differences do exist between *C. albicans* and *S. cerevisiae*, the most obvious of which relates to their patterns of sugar utilisation [41]. For example, *S. cerevisiae* belongs to the group that is called Crabtree-positive yeasts which have the ability to produce ethanol even in the presence of oxygen. In contrast, *C. albicans* is designated as a Crabtree-negative yeast [43] because it retains respiratory capacity in the

As described earlier, pathways of alternative carbon assimilation in *S. cerevisiae* are subject of glucose repression study. The genetics of glucose repression have been studied, and the regulatory elements that drive this regulation have been described in *S. cerevisiae* [10, 12, 45, 46, 47]. These include co-regulatory mechanisms that act on common elements within the promoter sequences of gluconeogenic and glyoxylate cycle genes. The carbon source regulator elements (CSRE) in the promoters of the *S. cerevisiae PCK1, FBP1, MLS1* and *ACR1* are required for their transcriptional induction in the absence of glucose [48 - 52]. In addition the promoters of the *S. cerevisiae ICL1, MLS1* and *FBP1* genes contain binding sites for the transcription repressor Mig1 [53]. Mig1 represses the transcription of these genes in the presence of glucose,

Transcript profiling of glucose responses in *C. albicans* and *S. cerevisiae* has shown that both yeasts are sensitive to very low levels of glucose [54, 55]. In *S. cerevisiae* and *C. albicans*, glycolytic genes were up-regulated and gluconeogenic and TCA cycle genes were down-regulated even when only 0.01% glucose was added to the growth medium. Yin *et al*. [54] also showed that *S. cerevisiae* ribosomal protein genes also respond to glucose but that they were less sensitive to glucose than the metabolic genes mentioned above. In *S. cerevisiae*, ribosomal protein gene expression was up-regulated following glucose addition at concentrations above 0.1% [54, 56]. Therefore addition of glucose to *S. cerevisiae* cells growing on alternative carbon sources causes a rapid shift from non-fermentative to fermentative metabolism, in part through tight regula‐ tion of gene transcription. Glucose also regulates metabolic activity in *S. cerevisiae* at posttranscriptional levels. Glucose triggers the accelerated decay of gluconeogenic mRNA (*PCK1, FBP1*) [54]. Furthermore, glucose triggers the catabolite inactivation and degradation of gluconeogenic and glyoxylate cycle enzymes in *S. cerevisiae.* Fructose-1,6-bisphophatase (FBPase) is expressed when yeast cells are grown on non-fermentable carbon sources. When

and the activity of Mig1 is being regulated by Snf1 (AMP kinase) signalling.

presence of excess glucose [44].

204 Genital Infections and Infertility

We reasoned that glucose responses might have diverged significantly between *C. albicans* and *S. cerevisiae*. Our rationale was that the relaxation of glucose repression would confer an evolutionary advantage upon a yeast such as *C. albicans* by allowing this pathogen to continue to assimilate alternative carbon sources even when small amounts of glucose are present *in vivo.* This section describes the testing of this working hypothesis through comparison of the effects of glucose upon gluconeogenic and glyoxylate cycle gene expression in *C. albicans* and *S. cerevisiae.*

To understand carbon assimilation in *C. albicans*, growth on selected alternative carbon sources was first defined. Therefore analogous growth experiments were carried out for both *S. cerevisiae* and *C. albicans*in media containing glucose or alternative carbon sources. Lactic acid was chosen as one alternative carbon source because it is a three-carbon molecule of physio‐ logical relevance found in various host niches and in the bloodstream after exercise [60]. Also Aberdeen Fungal Group Laboratory has generated a considerable body of data on cells grown on lactate [54, 55].

Yeast cells were grown overnight in media containing 2% lactate or 2% (2% each or 1% + 1%) both glucose and lactate. These cells were then harvested and used to inoculate fresh media of the same composition and grown for 10 hours. Glucose and ethanol levels and growth absorbance (at OD340) were measured.

As expected, both yeasts grew better on media containing glucose plus lactate, than on lactate alone. *C. albicans* grew faster than *S. cerevisiae* on both media under these conditions. However, both yeasts displayed similar rates of glucose consumption and different ethanol accumulation under these conditions. Glucose was utilised rapidly by both *S. cerevisiae* and *C. albicans*. Ethanol levels in glucose plus lactate cultures significantly increased in *S. cerevisiae*, but they remained similar during glucose assimilation by *C. albicans*. This indicated that most glucose was not fermented to ethanol under the experimental conditions examined in *C. albicans* (minimal medium; 30°C; 200 rpm).

The fatty acid i.e. oleic acid was chosen as the second alternative carbon source for analysis. A fatty acid was chosen because lipids represent a rich source of carbon in the host, *C. albicans* is known to secrete lipases [61], and *C. albicans* is known to induce fatty acid β-oxidation genes following phagocytosis by macrophages [62].

Once again, analogous growth experiments were carried out for both *S. cerevisiae* and *C. albicans* in media containing oleic acid or glucose plus oleic acid. Yeasts cells were grown overnight in media containing 0.2% oleic acid or 2% glucose plus oleic acid, and these cells were used to inoculate fresh media containing the same amount of carbon sources. Growth was monitored for 10 hours. Once again, glucose and ethanol levels and absorbance (OD340) were measured.

Both yeasts grew on oleic acid or on oleic acid plus glucose. As expected more growth was observed for both yeasts on the glucose containing medium compared with the medium containing oleic acid alone. *C. albicans* grew more efficiently than *S. cerevisiae* on both media. Once again, the rates of glucose consumption and ethanol production were different for both yeasts. Both yeasts consumed glucose rapidly, but ethanol levels were accumulated signifi‐ cantly throughout the experiment in *S. cerevisiae*. This suggested that most glucose was not fermented to ethanol under these conditions in *C. albicans*. Less ethanol was generated during growth on oleic acid (about 2 mg/ml or 0.02%) compared to during growth on lactic acid (about 4 mg/ml or 0.04%).

Previous work by Yin *et al*. [54] using Northern blotting and transcriptomic analyses showed that transcripts encoding the gluconeogenic enzymes (*FBP1* and *PCK1*) are repressed by glucose in *S. cerevisiae*. To reconfirm this report and to compare it with the glucose responses of *C. albicans* more directly in this study, we first examined the responses of *S. cerevisiae* glyoxylate cycle (*ScICL1*) and gluconeogenic mRNAs (*ScPCK1*) using the following experi‐ mental approach.

*S. cerevisiae* cells were grown to mid-exponential phase in a minimal medium containing lactate or oleic acid as the sole carbon source and lactate + glucose and oleic acid + glucose. The levels of the *S. cerevisiae ICL1* and *PCK1* mRNAs were measured relative to the housekeeping β-actin gene (*ScACT1*), following the addition of glucose to a final concentration of 2%. Samples were collected and frozen immediately in liquid nitrogen for RNA extraction. *S. cerevisiae ICL1, PCK1* and *ACT1* primers [63] were then designed, and the expression of these genes was quantified using Syber green quantitative real-time polymerase chain reaction (qRT-PCR).

*ScICL1* mRNA levels showed a dramatic decrease within 30 minutes of glucose addition to cells growing on lactate or oleic acid. Similarly, *ScPCK1* mRNA levels declined after glucose addition to cells growing on lactate or oleic acid media. This strong repression occurred 30 minutes after glucose addition. These results confirmed that in *S. cerevisiae*, the *ICL1* and *PCK1* transcripts are strongly repressed by glucose [54]. At least for the *PCK1* mRNA, this repression is mediated by transcriptional repression and accelerated mRNA degradation [54].

Aberdeen Fungal Group Laboratory has also described the global transcriptional responses of *C. albicans* to low (0.01%), medium (0.1%) and high (1%) glucose concentrations by microarray analysis [55]. The data indicated that a total of 347 *C. albicans* genes were up-regulated, and 344 genes were down-regulated in response to at least one of the glucose concentrations examined. There are170 of these genes that were up-regulated and 180 genes that were downregulated by 0.01% glucose, indicating that about half of glucose-regulated genes are respon‐ sive to low glucose levels. Therefore, the authors concluded that like *S. cerevisiae*, *C. albicans* is exquisitely sensitive to glucose, responding to concentrations as low as 0.01%. Hence, at the start of this study, an aim was to confirm the impact of glucose on specific mRNAs that encode enzymes required for the assimilation of alternative carbon sources. The transcripts encoding the glyoxylate cycle enzyme isocitrate lyase (*CaICL1*) and the gluconeogenic enzyme phos‐ phoenolpyruvate carboxykinase (*CaPCK1*) were the main focus here.

Once again, analogous growth experiments were carried out for both *S. cerevisiae* and *C. albicans* in media containing oleic acid or glucose plus oleic acid. Yeasts cells were grown overnight in media containing 0.2% oleic acid or 2% glucose plus oleic acid, and these cells were used to inoculate fresh media containing the same amount of carbon sources. Growth was monitored for 10 hours. Once again, glucose and ethanol levels and absorbance (OD340)

Both yeasts grew on oleic acid or on oleic acid plus glucose. As expected more growth was observed for both yeasts on the glucose containing medium compared with the medium containing oleic acid alone. *C. albicans* grew more efficiently than *S. cerevisiae* on both media. Once again, the rates of glucose consumption and ethanol production were different for both yeasts. Both yeasts consumed glucose rapidly, but ethanol levels were accumulated signifi‐ cantly throughout the experiment in *S. cerevisiae*. This suggested that most glucose was not fermented to ethanol under these conditions in *C. albicans*. Less ethanol was generated during growth on oleic acid (about 2 mg/ml or 0.02%) compared to during growth on lactic acid (about

Previous work by Yin *et al*. [54] using Northern blotting and transcriptomic analyses showed that transcripts encoding the gluconeogenic enzymes (*FBP1* and *PCK1*) are repressed by glucose in *S. cerevisiae*. To reconfirm this report and to compare it with the glucose responses of *C. albicans* more directly in this study, we first examined the responses of *S. cerevisiae* glyoxylate cycle (*ScICL1*) and gluconeogenic mRNAs (*ScPCK1*) using the following experi‐

*S. cerevisiae* cells were grown to mid-exponential phase in a minimal medium containing lactate or oleic acid as the sole carbon source and lactate + glucose and oleic acid + glucose. The levels of the *S. cerevisiae ICL1* and *PCK1* mRNAs were measured relative to the housekeeping β-actin gene (*ScACT1*), following the addition of glucose to a final concentration of 2%. Samples were collected and frozen immediately in liquid nitrogen for RNA extraction. *S. cerevisiae ICL1, PCK1* and *ACT1* primers [63] were then designed, and the expression of these genes was quantified using Syber green quantitative real-time polymerase chain reaction (qRT-PCR).

*ScICL1* mRNA levels showed a dramatic decrease within 30 minutes of glucose addition to cells growing on lactate or oleic acid. Similarly, *ScPCK1* mRNA levels declined after glucose addition to cells growing on lactate or oleic acid media. This strong repression occurred 30 minutes after glucose addition. These results confirmed that in *S. cerevisiae*, the *ICL1* and *PCK1* transcripts are strongly repressed by glucose [54]. At least for the *PCK1* mRNA, this repression

Aberdeen Fungal Group Laboratory has also described the global transcriptional responses of *C. albicans* to low (0.01%), medium (0.1%) and high (1%) glucose concentrations by microarray analysis [55]. The data indicated that a total of 347 *C. albicans* genes were up-regulated, and 344 genes were down-regulated in response to at least one of the glucose concentrations examined. There are170 of these genes that were up-regulated and 180 genes that were downregulated by 0.01% glucose, indicating that about half of glucose-regulated genes are respon‐ sive to low glucose levels. Therefore, the authors concluded that like *S. cerevisiae*, *C. albicans* is

is mediated by transcriptional repression and accelerated mRNA degradation [54].

were measured.

206 Genital Infections and Infertility

4 mg/ml or 0.04%).

mental approach.

*C. albicans* cells were grown to mid-exponential phase in media containing lactate or oleic acid as the sole carbon source using the same procedures described for *S. cerevisiae* by Yin *et al*. [54]. Glucose was then added to a final concentration of 2%; samples were taken for RNA analysis at various times thereafter; the levels of the *CaICL1, CaPCK1* and *CaACT1* mRNAs were measured by qRT-PCR. The relative expression of *CaICL1* (compared with the internal *CaACT1* control) was high in lactate- and oleic acid-grown cells compared with the cells that were exposed to glucose. The *CaICL1* mRNA was strongly down-regulated within 60 minutes after glucose addition under both of these growth conditions. The *CaPCK1* mRNA was expressed at relatively high levels in lactate- and oleic acid-grown cells, and was also strongly downregulated within 60 minutes of glucose addition. These results confirmed that in both *S. cerevisiae* and *C. albicans*, the *ICL1* and *PCK1* genes are strongly repressed by glucose [63].

The next step is to test the effects of glucose on the expression levels of the CaIcl1 and CaPck1 proteins in *C. albicans* and to compare this response with the corresponding situation *S. cerevisiae.* In *S. cerevisiae*, the effects of glucose on fructose-1,6-bisphosphatase (FBPase) have been intensively studied and it was reported that the FBPase protein (ScFbp1) is rapidly degraded upon the addition of glucose [58]. Also it has been reported that the levels of cytosolic malate dehydrogenase, fructose-1,6-bisphosphatase, isocitrate lyase and phosphoenolpyru‐ vate carboxykinase are all low in *S. cerevisiae* after glucose addition [59]. Therefore, as a starting point, we tested for ourselves whether Icl1 and Pck1 decline in *S. cerevisiae* upon glucose addition.

To achieve this, the *S. cerevisiae ICL1* coding region was tagged at its 3´-end with Myc9 and the *PCK1* coding region was tagged with HA6. Control Western blots with these tagged strains and their untagged parental strains demonstrated that ScIcl1-Myc9 and ScPck1-HA6 were expressed during growth on non-fermentable carbon sources [63].

After confirming the validity of the ScIcl1-Myc9 and ScPck1-HA6 tagging, the next step is to examine the effects of glucose upon the stability of these proteins following glucose addition to *S. cerevisiae* cells. Therefore, the epitope-tagged *S. cerevisiae* strains were grown on lactate or oleic acid, and then glucose was added to a final concentration of 2%. All experiments were performed on exponentially growing cells. Samples were prepared at various times thereafter, and the levels of the ScIcl1-Myc9 and ScPck1-HA6 proteins were measured by Western blotting. Clearly, glucose addition led to the degradation of ScIcl1 and ScPck1. These results confirmed that in *S. cerevisiae* Icl1 and Pck1 are degraded in response to glucose. Interestingly, the degradation of ScPck1 appears to start about 2 hours after glucose addition, whereas ScIcl1 degradation starts earlier. This might reflect differences in the mechanisms of glucoseactivated degradation of these proteins, via ubiquitin-mediated or vacuole-mediated path‐ ways, as described by Regelmann *et al*. [64].

Having confirmed that glucose addition to cells growing on non-fermentable carbon sources leads to the degradation of Icl1 and Pck1 in *S. cerevisiae*, the next step is to test the effects of glucose upon the corresponding enzymes in *C. albicans.*

*C. albicans* strains expressing Myc3-tagged Icl1 or Myc3-tagged Pck1 were then used in an analogous experimental design to our previous *S. cerevisiae* protein analysis. *C. albicans* cells were grown in media containing non-fermentable carbon sources (lactate or oleic acid) as sole carbon sources, and then 2% glucose was added while cells were in the exponential growth phase. Cells were then harvested at various time periods, and their proteins were extracted for Western blotting. Proteins were loaded in equal amounts onto the SDS/PAGE gels, and expression of the Myc3-tagged CaIcl1 and CaPck1 proteins was detected with anti-Myc antibodies and the images quantified using a phosphorimager.

The *C. albicans* Icl1 protein was expressed during growth on lactate or oleic acid. Interestingly, CaIcl1 was not destabilised by the addition of 2% glucose. Indeed, CaIcl1 protein levels were not significantly different from the control even after 4 hours. Likewise, CaPck1 expression levels were relatively high during growth on lactate or oleic acid, and CaPck1 was also not destabilised by glucose addition. These data suggested that glucose does not affect the stability of the CaIcl1 and CaPck1 proteins. This behaviour of *C. albicans* was in direct contrast to that observed in *S. cerevisiae*, in which the Icl1 and Pck1 proteins were destabilised by glucose [63].

Carbon assimilation is essential for the generation of new biomass (i.e. growth). Therefore, the growth of *C. albicans* in the immunocompromised host depends upon the assimilation of available carbon sources *in vivo*, and the fungus must adjust its metabolism to the microen‐ vironments it occupies in the host [41].

Previous reports have suggested that *S. cerevisiae* might provide a reasonable metabolic paradigm for *C. albicans* as reviewed by Brown [41]. Certainly, many of the pathways of central metabolism are conserved in fungi, including the glycolytic, gluconeogenic and pentose phosphate pathways and the TCA and glyoxylate cycles [14, 42]. Pathways for the generation of storage and cell wall carbohydrates are also conserved. Furthermore, the pathways of amino acid, lipid and nucleotide catabolism and anabolism appear to be conserved. However, significant metabolic differences do exist between *C. albicans* and *S. cerevisiae*, as revealed by their different patterns of sugar utilisation. In fact, differences in the patterns of carbohydrate assimilation are used routinely to distinguish *C. albicans* from other microbes in the clinic [65, 66]. Also, significant differences in the regulation of carbon metabolism are emerging for *C. albicans* and *S. cerevisiae* [67, 68]. Based on these observations, we tested whether there are metabolic differences in carbon assimilation between these fungi. The approach here was to measure the effects of glucose upon the assimilation by *S. cerevisiae* and *C. albicans* of radiola‐ belled lactic or oleic acid into large molecular weight compounds.

To achieve this, *S. cerevisiae* cells were grown to exponential phase on media containing lactate or oleic acid as sole carbon source. These cells were then harvested and resuspended in equivalent media containing radiolabelled lactic or oleic acid. Glucose (2%) was added to test samples, and no glucose was added to control samples. The assimilation of 14C-lactic acid or 3 H-oleic acid by *S. cerevisiae* cells into large molecular weight TCA-precipitable material was then measured. In *S. cerevisiae*, both lactate and oleic acid assimilation were rapidly repressed by glucose. Therefore the *S. cerevisiae* cells stopped the assimilation of these secondary carbon sources and apparently switched quickly to glucose assimilation. This confirmed the generally held view that *S. cerevisiae* does not assimilate both glucose and secondary carbon sources at the same time.

Having confirmed that glucose addition to cells growing on non-fermentable carbon sources leads to the degradation of Icl1 and Pck1 in *S. cerevisiae*, the next step is to test the effects of

*C. albicans* strains expressing Myc3-tagged Icl1 or Myc3-tagged Pck1 were then used in an analogous experimental design to our previous *S. cerevisiae* protein analysis. *C. albicans* cells were grown in media containing non-fermentable carbon sources (lactate or oleic acid) as sole carbon sources, and then 2% glucose was added while cells were in the exponential growth phase. Cells were then harvested at various time periods, and their proteins were extracted for Western blotting. Proteins were loaded in equal amounts onto the SDS/PAGE gels, and expression of the Myc3-tagged CaIcl1 and CaPck1 proteins was detected with anti-Myc

The *C. albicans* Icl1 protein was expressed during growth on lactate or oleic acid. Interestingly, CaIcl1 was not destabilised by the addition of 2% glucose. Indeed, CaIcl1 protein levels were not significantly different from the control even after 4 hours. Likewise, CaPck1 expression levels were relatively high during growth on lactate or oleic acid, and CaPck1 was also not destabilised by glucose addition. These data suggested that glucose does not affect the stability of the CaIcl1 and CaPck1 proteins. This behaviour of *C. albicans* was in direct contrast to that observed in *S. cerevisiae*, in which the Icl1 and Pck1 proteins were destabilised by glucose [63].

Carbon assimilation is essential for the generation of new biomass (i.e. growth). Therefore, the growth of *C. albicans* in the immunocompromised host depends upon the assimilation of available carbon sources *in vivo*, and the fungus must adjust its metabolism to the microen‐

Previous reports have suggested that *S. cerevisiae* might provide a reasonable metabolic paradigm for *C. albicans* as reviewed by Brown [41]. Certainly, many of the pathways of central metabolism are conserved in fungi, including the glycolytic, gluconeogenic and pentose phosphate pathways and the TCA and glyoxylate cycles [14, 42]. Pathways for the generation of storage and cell wall carbohydrates are also conserved. Furthermore, the pathways of amino acid, lipid and nucleotide catabolism and anabolism appear to be conserved. However, significant metabolic differences do exist between *C. albicans* and *S. cerevisiae*, as revealed by their different patterns of sugar utilisation. In fact, differences in the patterns of carbohydrate assimilation are used routinely to distinguish *C. albicans* from other microbes in the clinic [65, 66]. Also, significant differences in the regulation of carbon metabolism are emerging for *C. albicans* and *S. cerevisiae* [67, 68]. Based on these observations, we tested whether there are metabolic differences in carbon assimilation between these fungi. The approach here was to measure the effects of glucose upon the assimilation by *S. cerevisiae* and *C. albicans* of radiola‐

To achieve this, *S. cerevisiae* cells were grown to exponential phase on media containing lactate or oleic acid as sole carbon source. These cells were then harvested and resuspended in equivalent media containing radiolabelled lactic or oleic acid. Glucose (2%) was added to test samples, and no glucose was added to control samples. The assimilation of 14C-lactic acid or

H-oleic acid by *S. cerevisiae* cells into large molecular weight TCA-precipitable material was

glucose upon the corresponding enzymes in *C. albicans.*

208 Genital Infections and Infertility

antibodies and the images quantified using a phosphorimager.

belled lactic or oleic acid into large molecular weight compounds.

vironments it occupies in the host [41].

3

The next step is to investigate the effects of glucose on carbon assimilation in *C. albicans*. Once again, the approach was to test the assimilation of radiolabelled carbon sources by the cell. The same procedures were followed as for *S. cerevisiae*, with *C. albicans* cells being grown on lactate or oleic acid and then the assimilation of 14C-lactic acid or 3 H-oleic acid into TCA-precipitable material was measured after glucose addition [63].

Unlike *S. cerevisiae*, *C. albicans* was still able to assimilate both lactate and oleic acid for some hours after addition of glucose. Lactate metabolism appeared to continue relatively normal because only minor effects were observed in the lactate uptake following glucose addition. Tests with the other secondary carbon source (oleic acid) showed similar results. Therefore, *C. albicans* is able to assimilate lactate and glucose at the same time. Similarly, both oleic acid and glucose can be assimilated by *C. albicans* at the same time. This suggests that glucose has a minimal immediate impact on the ability of *C. albicans* to assimilate secondary carbon sources at least over the timescales examined [63].

Therefore, there are significant differences between *C. albicans* and *S. cerevisiae* with respect to the regulation of their assimilation of alternative carbon sources. Apparently in *S. cerevisiae*, glucose and secondary carbon sources are not assimilated at the same time. In contrast, in *C. albicans*, the continued stability of gluconeogenic and glyoxylate cycle enzymes after glucose exposure correlates with the ability of *C. albicans* cells to continue to assimilate alternative carbon sources, even after glucose addition. Hence, the hypothesis that significant metabolic differences exist between *C. albicans* and *S. cerevisiae* was confirmed [63].
