**5. Conclusion and Future Perspectives**

very stable. This probably represents a significant difference in the physiological responses of these pathogenic and benign yeasts to glucose. *C. albicans* is able to establish infections in complex niches, many of which contain a rich mixture of alternative carbon sources [34]. The stability of the Icl1 and Pck1 proteins in *C. albicans*, even in the presence of glucose, provided the first clue that this pathogen might be able to assimilate alternative carbon sources at the

The third conclusion was that *C. albicans* is able to assimilate both glucose and alternative carbon sources at the same time. It was shown that glucose addition has no major impact on the assimilation of the alternative carbon sources (lactate and oleic acid). The maintenance of gluconeogenic and glyoxylate cycle enzymes, therefore, appears to allow *C. albicans* to continue to assimilate alternative carbon sources, even following glucose addition. Therefore, during a transient exposure to glucose in the bloodstream, for example, *C. albicans* would be able to maintain anabolic metabolism. Also, after phagocytosis, when the genes of glyoxylate cycle and gluconeogenesis have been induced [34], these pathways probably remain active some time afterwards because of the stability of their enzymes. It was reported that the glyoxylate cycle helps to protect *C. albicans* against host anti-microbial defences by facilitating anabolic metabolism in the absence of fermentable carbon sources [79]. Barelle *et al*. [34] indicated that the pathogen *C. albicans* regulates central carbon metabolism in a niche-specific manner during disease establishment and progression. These authors reported two stages *C. albicans* activate the glyoxylate cycle and gluconeogenesis in response to phagocytosis during the early stage of infection and this is followed by glycolytic metabolism when the fungus colonises tissue. This metabolic flexibility is thought to increase the biological fitness of this pathogen within its host. It is conceivable that the prolonged activity of the anabolic pathways might further

In conclusion, the regulation of central carbon metabolism in *S. cerevisiae* and *C. albicans* seems to have evolved in ways that reflect their different biological niches. *S. cerevisiae* has adapted to grow rapidly when high concentrations of sugars become available from fruit (*from feasts to famine*) [10], whereas *C. albicans* appears to have adapted to utilise the complex mixtures of carbon sources that are available in the GI tract or the bloodstream for example. The next step in this study is to test whether *C. albicans* has retained the ability to target accelerated protein

Next, the reverse cloning was done by replacing the *S. cerevisiae ICL1* ORF in *S. cerevisiae* with a Myc3-tagged *C. albicans ICL1* ORF. The aim was to test whether the CaIcl1 protein was destabilised by glucose when expressed in *S. cerevisiae.* This revealed that the CaIcl1 protein was not destabilised in response to glucose when expressed in *S. cerevisiae*. This indicated that the *C. albicans* Icl1 protein has lost the signal that triggers destabilisation in response to glucose.

The *S. cerevisiae* Fbp1 protein is destabilised by glucose via ubiquitination [64]. Therefore, we reasoned that the CaIcl1 protein might have lost ubiquitination signals, which could account for the stability of the CaIcl1 protein in *S. cerevisiae* following glucose addition. Indeed, a bioinformatic analysis revealed that while ScIcl1 carries ubiquitination sites, CaIcl1 does not.

To test whether ubiquitination might play a role in glucose-accelerated protein degradation in *C. albicans*, the impact of *UBI4* inactivation upon ScIcl1 degradation was tested. Interestingly,

same time as glucose in these carbon-rich niches.

216 Genital Infections and Infertility

increase the fitness of this pathogen.

degradation in response to glucose.

Overall, the topic was to explore the impact of glucose on the assimilation of alternative carbon sources and catabolite inactivation in *C. albicans.* To achieve this, the effects of glucose on the transcriptional and post-transcriptional regulation of key genes and enzymes were studied, and the molecular mechanisms that trigger protein destabilisation in response to glucose were examined. These effects were then compared with glucose responses in *S. cerevisiae.*

Gene expression in both *C. albicans* and *S. cerevisiae*is sensitive to glucose. In both yeasts, *ICL1* and *PCK1* mRNA expression levels were down-regulated in response to glucose. This confirmed the previous microarray studies in *S. cerevisiae* [54] and in *C. albicans* [55].

*ICL1* and *PCK1* are involved in the utilisation of alternative carbon sources such as organic and fatty acids, and these genes are repressed by glucose in *S. cerevisiae*. Indeed gluconeogenic and glyoxylate cycle genes have been shown to be repressed by glucose concentrations as low as 0.01% in *S. cerevisiae* [54]. Similarly, *C. albicans* genes involved in central carbon metabolism respond rapidly to the addition of glucose. Indeed the *PCK1* and *ICL1* genes have been shown to be repressed by glucose at levels as low as 0.1% [55]. These observations imply that the utilisation of alternative carbon sources could be repressed by glucose in *C. albicans*. Given that if *C. albicans* genes involved in the utilisation of alternative carbon sources are glucose- regulated in a similar fashion to those in *S. cerevisiae*, then it might be expected that these genes would be repressed during systemic infections. However, during phagocytosis by macrophages, *C. albicans* cells appear to up-regulate the glyoxylate cycle, as the expression of the isocitrate lyase (*ICL1*) and malate synthase (*MLS1*) genes is induced [80]. The same applies to the gluconeogen‐ ic gene, *PCK1* [34]. Furthermore both *ICL1* and *PCK1* appear to be expressed in some *C. albicans* cells during systemic kidney infections [34]. Various studies have shown that genes involved in gluconeogenic and glyoxylate cycle contribute to fungal virulence [34, 62, 79].

*C. albicans* often occupies niches that contain complex mixtures of carbon sources. Our initial working hypothesis, therefore, was that *C. albicans* might exploit many of these diverse carbon sources rather than focussing on glucose alone. Therefore, the tight glucose repression of the *PCK1* and *ICL1* genes seemed somewhat surprising [54, 55, 76] because this was inconsistent with our working hypothesis. Our subsequent discovery that the Pck1 and Icl1 enzymes were not destabilised by glucose was more consistent with this working hypothesis. Catabolite inactivation – the rapid degradation of specific enzymes following glucose exposure – is a welldefined phenomenon in *S. cerevisiae.* In the yeast, the gluconeogenic enzyme Fbp1 is rapidly degraded when cells are shifted from media with poor carbon sources to rich media containing glucose [59]. Consistent with this, in this project it was found that both the Icl1 and Pck1 proteins were rapidly degraded in *S. cerevisiae* in response to glucose. This was the case when cells were pre-grown on lactic acid or oleic acid. In contrast, the *C. albicans* Icl1 and Pck1 proteins were not destabilised in response to glucose when cells were exposed to glucose. The stability of the CaIcl1 and CaPck1 enzymes in the presence of glucose suggested that *C. albicans* might be capable of continuing to utilise alternative carbon sources even when glucose became available. This suggestion was more consistent with our working hypothesis that *C. albicans* has evolved to utilise carbon sources simultaneously in complex niches.

To examine this, the impact of glucose on the ability of *C. albicans* to assimilate radiolabelled lactic acid or oleic acid was tested and compared with that of *S. cerevisiae.* These incorporation studies confirmed the divergent behaviour of these yeasts with respect to their patterns of carbon assimilation. In *S. cerevisiae*, the assimilation of lactic and oleic acid was repressed by glucose and subsequent growth was reliant on glucose. In contrast, in *C. albicans*, the utilisation of lactic and oleic acid was not repressed by glucose. Hence, the utilisation of alternative carbon sources by *C. albicans* was not repressed by glucose, and thus the subsequent growth of this pathogen was supported by assimilating both glucose and alternative carbon sources [63].

In summary, both *C. albicans* and *S. cerevisiae* displayed similar responses to glucose at the transcriptional level, but their responses at post-transcriptional and metabolic levels differed significantly. Therefore, *C. albicans* can assimilate both glucose and alternative carbon sources at the same time, whereas *S. cerevisiae* is not able to do so (Fig. 1). This is predicted to play a significant role in the growth of this fungus during infection in the human host [41]. This could be tested by reprogramming the Icl1 and Pck1 enzymes to be glucose- sensitive in *C. albi‐ cans.* The prediction is that this engineered *C. albicans* strain would grow less well on mixed carbon sources containing glucose, and hence would display attenuated virulence. Additional experiments that could be carried should test the impact of carbon sources other than glucose, (such as lactose, galactose or fructose) [Ting and Sandai, unpublished] on the stability of gluconeogenic and glyoxylate cycle enzymes in *C. albicans.* Fructose, lactose and galactose are commonly found in the diet, and significant differences are thought to exist between *C. albicans* and *S. cerevisiae* with regard to the regulation of galactose utilisation [81, 82].

Another additional experiment could involve the broad screening of central metabolic enzymes in *C. albicans* for consensus ubiquitination target sites, beyond the preliminary screen performed here. This would help to extend the predicted impact of glucose on central metabolic pathways. This preliminary bioinformatic screen of gluconeogenic and glyoxylate cycle enzymes has indicated that the *S. cerevisiae* Icl1, Fbp1, Eno1 and Pck1 proteins carry high confidence ubiquitination sites (Fig. 2). Previous studies have shown that ScFbp1 is catabolite inactivated in a glucose-dependent manner via ubiqutination and proteasomal degradation [59]. In contrast, in *C. albicans*, only Eno1 carries a high confidence ubiquitination site. This bioinformatic analysis is consistent with the suggestion that gluconeogenesis and the glyoxy‐ late cycle are insensitive to glucose or at least glucose stimulated, ubiquitin-mediated protein degradation in *C. albicans* [63].

cells during systemic kidney infections [34]. Various studies have shown that genes involved

*C. albicans* often occupies niches that contain complex mixtures of carbon sources. Our initial working hypothesis, therefore, was that *C. albicans* might exploit many of these diverse carbon sources rather than focussing on glucose alone. Therefore, the tight glucose repression of the *PCK1* and *ICL1* genes seemed somewhat surprising [54, 55, 76] because this was inconsistent with our working hypothesis. Our subsequent discovery that the Pck1 and Icl1 enzymes were not destabilised by glucose was more consistent with this working hypothesis. Catabolite inactivation – the rapid degradation of specific enzymes following glucose exposure – is a welldefined phenomenon in *S. cerevisiae.* In the yeast, the gluconeogenic enzyme Fbp1 is rapidly degraded when cells are shifted from media with poor carbon sources to rich media containing glucose [59]. Consistent with this, in this project it was found that both the Icl1 and Pck1 proteins were rapidly degraded in *S. cerevisiae* in response to glucose. This was the case when cells were pre-grown on lactic acid or oleic acid. In contrast, the *C. albicans* Icl1 and Pck1 proteins were not destabilised in response to glucose when cells were exposed to glucose. The stability of the CaIcl1 and CaPck1 enzymes in the presence of glucose suggested that *C. albicans* might be capable of continuing to utilise alternative carbon sources even when glucose became available. This suggestion was more consistent with our working hypothesis that *C.*

in gluconeogenic and glyoxylate cycle contribute to fungal virulence [34, 62, 79].

218 Genital Infections and Infertility

*albicans* has evolved to utilise carbon sources simultaneously in complex niches.

*albicans* and *S. cerevisiae* with regard to the regulation of galactose utilisation [81, 82].

Another additional experiment could involve the broad screening of central metabolic enzymes in *C. albicans* for consensus ubiquitination target sites, beyond the preliminary screen

To examine this, the impact of glucose on the ability of *C. albicans* to assimilate radiolabelled lactic acid or oleic acid was tested and compared with that of *S. cerevisiae.* These incorporation studies confirmed the divergent behaviour of these yeasts with respect to their patterns of carbon assimilation. In *S. cerevisiae*, the assimilation of lactic and oleic acid was repressed by glucose and subsequent growth was reliant on glucose. In contrast, in *C. albicans*, the utilisation of lactic and oleic acid was not repressed by glucose. Hence, the utilisation of alternative carbon sources by *C. albicans* was not repressed by glucose, and thus the subsequent growth of this pathogen was supported by assimilating both glucose and alternative carbon sources [63]. In summary, both *C. albicans* and *S. cerevisiae* displayed similar responses to glucose at the transcriptional level, but their responses at post-transcriptional and metabolic levels differed significantly. Therefore, *C. albicans* can assimilate both glucose and alternative carbon sources at the same time, whereas *S. cerevisiae* is not able to do so (Fig. 1). This is predicted to play a significant role in the growth of this fungus during infection in the human host [41]. This could be tested by reprogramming the Icl1 and Pck1 enzymes to be glucose- sensitive in *C. albi‐ cans.* The prediction is that this engineered *C. albicans* strain would grow less well on mixed carbon sources containing glucose, and hence would display attenuated virulence. Additional experiments that could be carried should test the impact of carbon sources other than glucose, (such as lactose, galactose or fructose) [Ting and Sandai, unpublished] on the stability of gluconeogenic and glyoxylate cycle enzymes in *C. albicans.* Fructose, lactose and galactose are commonly found in the diet, and significant differences are thought to exist between *C.*

The next main step in this project involved testing whether *C. albicans* has retained the molecular capability of destabilising target proteins in response to glucose. Examinations of Icl1 protein levels in both *S. cerevisiae* and *C. albicans* revealed that while Icl1 is rapidly degraded in response to glucose in *S. cerevisiae*, Icl1 remains stable in *C. albicans*. To test whether the *C. albicans* Icl1 protein has lost the signal that triggers destabilisation in response to glucose, the *C. albicans ICL1* ORF was expressed in *S. cerevisiae.* Interestingly, the *C. albicans* Icl1 protein was not destabilised in response to glucose when it was expressed in *S. cerevisiae*. This was consistent with the idea that the *C. albicans* Icl1 protein has lost the signal required to trigger glucose-accelerated degradation [66].

The next step is to test whether *C. albicans* has retained the ability to degrade target proteins in response to glucose. To investigate this, the tagged *S. cerevisiae* Icl1 protein was expressed in *C. albicans.* This tagged *S. cerevisiae* Icl1 protein was rapidly degraded when the *C. albicans* cells were exposed to glucose. This indicated that *C. albicans* has retained the molecular apparatus that mediates glucose-accelerated protein decay (or "catabolite inactivation").

What apparatus mediates this glucose-accelerated degradation? In *S. cerevisiae,* ubiquitination plays an important role in the rapid proteasome-mediated degradation of Fbp1 in response to glucose [57]. Therefore, the amino acid sequences of ScIcl1 and CaIcl1 were screened for consensus ubiquitination sites. This revealed that while the ScIcl1 sequence has strong ubiquitination sites, the CaIcl1 sequence does not (Fig. 3). Therefore, to examine whether ubiquitination plays a role in glucose-accelerated protein degradation in *C. albicans*, the impact of a polyubiquitin (*ubi4/ubi4*) null mutation [74] on the degradation of ScIcl1 in *C. albicans* was tested. The tagged *ScICL1* ORF was expressed in this *C. albicans ubi4/ubi4* mutant and its decay rate was measured in the presence and absence of glucose. Compared to the controls, the degradation of ScIcl1 was relatively slow in the *C. albicans ubi4/ubi4* mutant. This suggested that ubiquitination plays a role in glucose-accelerated protein degradation in *C. albicans.*

To test this further, we investigated the effects of introducing a *S. cerevisiae* ubiquitination site on to the carboxyl-terminus of the stable CaIcl1 protein. This CaIcl1-Ubi protein was destabi‐ lised in response to glucose in *C. albicans* (Fig. 3). This confirmed that ubiquitination plays a key role in glucose-accelerated protein degradation in *C. albicans.* It is also confirmed that *C. albicans* has retained the molecular apparatus that destabilises target proteins in response to glucose. In summary, *C. albicans* cells have retained the molecular apparatus that degrades target proteins in response to glucose, but CaIcl1 has lost the signal that triggers this destabi‐ lisation. As a result, the stability of CaIcl1 enzyme in the presence of glucose allows *C.* *albicans* cells to continue to use alternative carbon sources such as lactic and oleic acid rather than switching to glycolysis.

What is the apparatus required to trigger glucose accelerated decay? Ubiquitin- mediated protein degradation occurs via the proteasome [83]. This is a conserved molecular machine comprised of numerous protein subunits. Proteins are targeted to the proteasome via ubiqui‐ tination, which involves several steps (Fig. 4). Ubiquitin is activated in a two-step process involving the E1 and E2 enzymes and the hydrolysis of ATP. The primed ubiquitin molecule, once attached to an E2 enzyme via a thioester linkage, is then ligated to the target protein via an E3 ligase which provides the substrate specificity. A previous PhD student in the Aberdeen Fungal Group Laboratory [84] has already shown that proteasomal subunits are highly conserved across the fungal kingdom and are conserved in *C. albicans* (Fig. 4). Furthermore, E1, E2 and E3 enzymes are conserved in *C. albicans* (Fig 4) [85], which are consistent with the ubiquitination apparatus being retained in this pathogen. Presumably, the inactivation of components of this system could block glucose-accelerated protein degradation in *C. albicans.*

In *S. cerevisiae*, Ubc8 appears to be the ubiquitin-conjugating enzyme involved in glucoseaccelerated protein degradation. Ubc8 has been described as a ubiquitin-conjugating enzyme that negatively regulates gluconeogenesis by mediating the glucose-induced ubiquitination of Fbp1 [57, 64, 85, 86]. Interestingly the Ubc8 protein appears to be conserved in *C. albicans* [87]. Additional ubiquitin-conjugating enzymes exist in *C. albicans,* such as Ubc4 and Ubc6, and these might also be involved. However, all these *UBC* genes remain uncharacterised in *C. albicans*, and therefore it is not yet known whether these genes play a role in glucose-accelerated protein degradation. Interestingly however, *UBC8* is the only ubiquitin-related gene that is up-regulated at the transcriptional level following glucose exposure in *C. albicans* (Table 1). Therefore, Ubc8 is an excellent candidate for the ubiquitin-conjugating enzyme that mediates glucose-accelerated protein degradation in *C. albicans*. Clearly, a future experiment that could be carried out would be to create a *C. albicans ubc8/ubc8* mutant and to test whether this mutation blocks glucose-accelerated protein degradation in *C. albicans*. Other *UBC* genes might also be inactivated to test the possibility that they are not involved in glucose-accelerated protein degradation [63].

These findings are relevant to our understanding of *C. albicans* growth and survival in the host, and hence to *C. albicans* pathogenicity. *C. albicans* persists as commensal during colonisation in the healthy human GI tract where monosaccharides (such as glucose, fructose and galactose) and disaccharides (such as sucrose and lactose) can be abundant [88]. These sugars probably help to sustain the fungus, for example, during the first 6 months of a host's life when new born infants consume milk and hence lactose [89]. Both disaccharides and monosaccharides may supply a carbon-rich environment for microbes such as *C. albicans*, and they are also absorbed into the bloodstream [82]. However, during systemic infections *C. albicans* cells invade the bloodstream and often internal organs such as kidney. Glucose is present in the bloodstream but appears to become limited in systemic microenvironments that are colonised during infection of organs. At this point, the fungal cells probably switch to gluconeogenesis and glyoxylate cycle metabolism to utilise the available alternative carbon sources [34]. Also, during phagocytosis, by macrophages and neutrophils, *C. albicans* cells switch to the assimi‐ lation of alternative carbon sources, activating genes such as *ICL1* [34, 62, 79, 89] that are required for full pathogenicity [34, 79]. It would appear from the findings in this chapter, that *C. albicans* has evolved in such a manner that this fungus can continue to assimilate these alternative carbon sources, even after exposure to glucose. The presumption is that this ability to use several of carbon sources in these complex and carbon-rich microenvironments contributes to the growth and pathogenicity of *C. albicans* in these microenvironments. Possibly the greatest challenge for the future is to elucidate exactly what carbon sources individual *C. albicans* cells assimilate *in vivo* during commensalism, mucosal infection and systemic candi‐ diasis and to elucidate the contribution of transcriptional and post-transcriptional control mechanisms to this regulation.

This carbon metabolism of *C. albicans* might be investigated further by examining the ability of mutant *C. albicans* cells that carry the *ICL1-ubi* allele to grow and assimilate carbon *in vivo*. These *C. albicans* cells express Icl1 that is destabilised by glucose because of the addition of the carboxyl-terminal ubiquitination site. In principle, Icl1 would be degraded and cells no longer able to metabolise via the glyoxylate cycle following glucose addition. As a result, those cells would presumably be less able to course infections and would be less able to compete for available nutrients against other microorganisms such as endogenous bacteria in the GI tract compared to their wild type. It is likely to show less successful colonisation, virulence and fitness due to this defect in its ability to assimilate both glucose and alternative carbon sources at the same time [63].

**Growth on alternative carbon sources**

*albicans* cells to continue to use alternative carbon sources such as lactic and oleic acid rather

What is the apparatus required to trigger glucose accelerated decay? Ubiquitin- mediated protein degradation occurs via the proteasome [83]. This is a conserved molecular machine comprised of numerous protein subunits. Proteins are targeted to the proteasome via ubiqui‐ tination, which involves several steps (Fig. 4). Ubiquitin is activated in a two-step process involving the E1 and E2 enzymes and the hydrolysis of ATP. The primed ubiquitin molecule, once attached to an E2 enzyme via a thioester linkage, is then ligated to the target protein via an E3 ligase which provides the substrate specificity. A previous PhD student in the Aberdeen Fungal Group Laboratory [84] has already shown that proteasomal subunits are highly conserved across the fungal kingdom and are conserved in *C. albicans* (Fig. 4). Furthermore, E1, E2 and E3 enzymes are conserved in *C. albicans* (Fig 4) [85], which are consistent with the ubiquitination apparatus being retained in this pathogen. Presumably, the inactivation of components of this system could block glucose-accelerated protein degradation in *C. albicans.* In *S. cerevisiae*, Ubc8 appears to be the ubiquitin-conjugating enzyme involved in glucoseaccelerated protein degradation. Ubc8 has been described as a ubiquitin-conjugating enzyme that negatively regulates gluconeogenesis by mediating the glucose-induced ubiquitination of Fbp1 [57, 64, 85, 86]. Interestingly the Ubc8 protein appears to be conserved in *C. albicans* [87]. Additional ubiquitin-conjugating enzymes exist in *C. albicans,* such as Ubc4 and Ubc6, and these might also be involved. However, all these *UBC* genes remain uncharacterised in *C. albicans*, and therefore it is not yet known whether these genes play a role in glucose-accelerated protein degradation. Interestingly however, *UBC8* is the only ubiquitin-related gene that is up-regulated at the transcriptional level following glucose exposure in *C. albicans* (Table 1). Therefore, Ubc8 is an excellent candidate for the ubiquitin-conjugating enzyme that mediates glucose-accelerated protein degradation in *C. albicans*. Clearly, a future experiment that could be carried out would be to create a *C. albicans ubc8/ubc8* mutant and to test whether this mutation blocks glucose-accelerated protein degradation in *C. albicans*. Other *UBC* genes might also be inactivated to test the possibility that they are not involved in glucose-accelerated

These findings are relevant to our understanding of *C. albicans* growth and survival in the host, and hence to *C. albicans* pathogenicity. *C. albicans* persists as commensal during colonisation in the healthy human GI tract where monosaccharides (such as glucose, fructose and galactose) and disaccharides (such as sucrose and lactose) can be abundant [88]. These sugars probably help to sustain the fungus, for example, during the first 6 months of a host's life when new born infants consume milk and hence lactose [89]. Both disaccharides and monosaccharides may supply a carbon-rich environment for microbes such as *C. albicans*, and they are also absorbed into the bloodstream [82]. However, during systemic infections *C. albicans* cells invade the bloodstream and often internal organs such as kidney. Glucose is present in the bloodstream but appears to become limited in systemic microenvironments that are colonised during infection of organs. At this point, the fungal cells probably switch to gluconeogenesis and glyoxylate cycle metabolism to utilise the available alternative carbon sources [34]. Also, during phagocytosis, by macrophages and neutrophils, *C. albicans* cells switch to the assimi‐ lation of alternative carbon sources, activating genes such as *ICL1* [34, 62, 79, 89] that are required for full pathogenicity [34, 79]. It would appear from the findings in this chapter, that

than switching to glycolysis.

220 Genital Infections and Infertility

protein degradation [63].

**Growth on alternative carbon sources and pathogenesis**

**Figure 1.** Proposed model of the impact of glucose on the assimilation of alternative carbon sources by *S. cerevisiae* and *C. albicans.* In *C. albicans* and *S. cerevisiae* transcription of both *ICL1* and *PCK1* are repressed by glucose. However, their regulation at post-transcriptional levels and metabolism diverge significantly. The *S. cerevisiae* Icl1 and Pck1 proteins are rapidly destabilised in response to glucose and the assimilation of alternative carbon sources is repressed by glu‐ cose. In contrast, in *C. albicans* the Icl1 and Pck1 proteins decay slowly in response to glucose and continue to assimi‐ late both glucose and alternative carbon sources at the same time.

In addition, it would be interesting to conduct further investigations of the impact of other carbon sources such as galactose, fructose, sucrose, fatty acid and amino acid on carbon assimilation in *C. albicans*. For example galactose might also trigger glucose-like repression in *C. albicans* [82]. The behaviour of *C. albicans* towards other carbon sources differs from *S. cerevisiae.* It would be interesting to define the effects of these carbon sources on the levels and activities of glycolytic, gluconeogenic, glyoxylate cycle and fatty acid β-oxidation enzymes. Which other carbon sources trigger the ubiquitination-mediated degradation of these proteins in *C. albicans*? How do these mechanisms affect the ability of *C. albicans* to assimilate lipid, carbohydrate, protein and other organic acids in their human host during commensalism and infection? Such studies will provide a more complete physiological understanding of *C. albicans* for the future research.

**Figure 2.** Bioinformatic prediction of ubiquitination sites in glycolytic, gluconeogenic and glyoxylate cycle enzymes from *S. cerevisiae* and *C. albicans.* Icl1, Pck1, Eno1 and Fbp1 were examined. Those carrying high confidence ubiquitina‐ tion sites are highlighted in red.

Metabolic Adaptation of Isocitrate Lyase in the Yeast Pathogen *Candida albicans* http://dx.doi.org/10.5772/62406 223

In addition, it would be interesting to conduct further investigations of the impact of other carbon sources such as galactose, fructose, sucrose, fatty acid and amino acid on carbon assimilation in *C. albicans*. For example galactose might also trigger glucose-like repression in *C. albicans* [82]. The behaviour of *C. albicans* towards other carbon sources differs from *S. cerevisiae.* It would be interesting to define the effects of these carbon sources on the levels and activities of glycolytic, gluconeogenic, glyoxylate cycle and fatty acid β-oxidation enzymes. Which other carbon sources trigger the ubiquitination-mediated degradation of these proteins in *C. albicans*? How do these mechanisms affect the ability of *C. albicans* to assimilate lipid, carbohydrate, protein and other organic acids in their human host during commensalism and infection? Such studies will provide a more complete physiological understanding of *C.*

*S. CEREVISIAE C. ALBICANS*

**Glucose**

**HXK2 PGI1 PFK1 FBP1 FBA1 GAP1 TPI1**

Phosphoenolpyruvate

**PCK1**

**gluconeogenesis**

**Oleic acid**

Glycerate-3P Glycerate-2P

Glycerate-1,3P2

Fructose-1,6P2

G1P

**UGP1**

Mannose-6P

Glycerone-P **DAK2**

Ac-CoA

Succinate

Fumarate

Brown, 2005

Malate **FUM12**

Oxaloacetate

**MDH11**

Pyruvate

**PDA1 PDB1 LAT1 PDX1**

**LAT1**

G6P

Fructose-6P

**PMI40** Inositol-1P

**INO1**

**PGK1 GPM1 ENO1 CDC19**

Glyceraldehyde-3P

**PDC11** Ethanol

**glycolysis**

**ADH2**

**lactate**

Isocitrate

**IDH2**

2-Oxoglutarate

Acetate

**ACS1**

**CIT1**

**glyoxylate cycle**

Glyoxylate

**KGD1 SDH12**

Acetaldehyde **IPF16300**

Citrate

**ICL1**

**ACO1**

**LSC2** Succinyl-CoA

**TCA cycle**

*albicans* for the future research.

222 Genital Infections and Infertility

**Glucose**

**HXK2 PGI1 PFK1 FBP1 FBA1**

Phosphoenolpyruvate

**PCK1**

Glycerate-3P Glycerate-2P

Glycerate-1,3P2

Fructose-1,6P2

G1P

**UGP1**

Mannose-6P

Glycerone-P **DAK2**

Ac-CoA

Pyruvate

**PDB1ADH2 gluconeogenesis**

Succinate

Fumarate

tion sites are highlighted in red.

Brown, 2005

**Oleic acid**

Malate **FUM12**

Oxaloacetate

**MDH11**

**PDA1 PDB1 LAT1 PDX1**

**LAT1**

G6P

Fructose-6P

**PMI40** Inositol-1P

**INO1**

**GAP1 TPI1**

> **PGK1 GPM1 ENO1 CDC19**

Glyceraldehyde-3P

**PDC11** Ethanol

**glycolysis**

**lactate**

Isocitrate

**IDH2**

2-Oxoglutarate

**Figure 2.** Bioinformatic prediction of ubiquitination sites in glycolytic, gluconeogenic and glyoxylate cycle enzymes from *S. cerevisiae* and *C. albicans.* Icl1, Pck1, Eno1 and Fbp1 were examined. Those carrying high confidence ubiquitina‐

Acetate

**ACS1**

**CIT1**

**glyoxylate cycle**

Glyoxylate

**KGD1 SDH12**

Acetaldehyde **IPF16300**

Citrate

**ICL1**

**ACO1**

**LSC2** Succinyl-CoA

**TCA cycle**

**Figure 3. Role of ubiquitin-mediated protein degradation in enzyme destabilisation in C. albicans in response to glucose**. Bioinformatics comparison of CaIcl1 and ScIcl1 reveals a lack of putative ubiquitination target in CaIcl1. Intro‐ duction of carboxyl-terminal ScUbi-site in CaIcl1 plus Myc-tagging protein and testing the impact of glucose on the turnover of CaIcl1 with C-terminal ScUbi-site. Adding back a Ubi-site accelerates degradation in response to glucose.




**Table 1.** Expression of *C. albicans* and *S. cerevisiae* homologues involved in protein ubiquitination [55].

*Candida dataset Candida* gene

common

Systematic orf19Number

224 Genital Infections and Infertility

Expression Ratio glucose Concentration (%) 0.0 0.01 0.1 1.0

CA5648 orf19.7347 *UBC6* -1.0 1.0 1.1 -1.0 YER100W *UBC6* 1.0 1.0 1.2 1.6

CA4199 orf19.4540 *UBC8* **1.3 2.1 3.6 2.5** YEL012W *UBC8* 1.0 1.1 -1.0 -1.1

CA5109 orf19.6424 *UBC9* 1.0 1.2 1.1 1.0 YDL064W *UBC9* 1.1 1.3 1.2 -1.1

CA5769 orf19.5411 *UBC12* -1.1 1.0 -1.1 1.1 YLR306W *UBC12* 1.0 -1.6 -1.3 -1.5

CA0417 orf19.2225 *UBC13* 1.2 1.1 1.1 1.1 YDR092W *UBC13* 1.1 -1.4 -1.3 -1.2

CA3263 orf19.2697 *UBR12* -1.2 1.0 -1.1 -1.1 YLR024C *UBR2* 1.0 -1.6 -1.7 -1.6

CA3262 orf19.2695 *UBR11.3* -1.1 1.0 -1.1 -1.1 YGR184C *UBR1* 1.0 -1.4 -1.4 -1.3

CA1279 orf19.3628 *RSP5* -1.0 1.1 1.2 1.2 YER125W *RSP5* 1.0 1.1 1.0 -1.1

CA5435 orf19.3237 *UFD4* -1.1 1.3 1.3 1.4 YKL010C *UFD4* 1.0 -1.4 -1.4 -1.3

*Saccharomyces dataset* S. cerevisiae gene homology Systematic common

Expression Ratio glucose Concentration (%) 0.0 0.01 0.1 1.0

Description

enzyme, 3-prime end

> E2 ubiquitinconjugating enzyme (by homology)

Ubiquitinconjugating enzyme (by homology)

E2 ubiquitinconjugating enzyme (by homology)

E2 ubiquitinconjugating enzyme (by homology)

E2 ubiquitinconjugating enzyme (by homology)

Ubiquitin-protein ligase (by homology)

Ubiquitin-protein ligase, 3´ end ( by homology)

Ubiquitin-protein ligase (by homology)

Ubiquitin fusion degradation protein (by homology)

**Figure 4. Protein conservation in the proteasome.** Each protein is colour-coded according to its mean distance to *Sc*REF scores (scale bottom right: high *Sc*REF scores represent low sequence conservation). Each subunit of the actual machine (20S core particle and 19S cap) has been colour -coded based on the mean of the mean distance to *Sc*REF val‐ ues for the proteins that form it in all species. Rpn13 (*Sc*REF distance = 82) is not included in the picture. Ubiquitinrelated enzymes have been reviewed by Hochstrasser (1996), and protein component of the machine was identified based on the review by Sharon and co-workers (2006). In the ubiquitin-ligating enzymes, except for the Ubr1 and Ubr2 enzymes, all the others are hect-domain proteins [84].

**low**
