**4. Overview of Metabolic Adaptation in** *Candida albicans*

A previous study suggested that *C. albicans* might be capable of using more than one carbon source at the same time during growth in specific niches in the host [34]. This study was based on the analysis of specific promoter-green fluorescent protein (GFP) fusions during systemic candidiasis. Almost all *C. albicans* cells infecting the kidney expressed GFP fusions with glycolytic promoters (*PYK1*, *PFK2*). Meanwhile one third to one-half of cells infecting the kidney also expressed *ICL1*- and *PCK1*-GFP fusions, suggesting that anabolic and catabolic pathways might be expressed at the same time. If this is the case, in principle, this would allow this pathogenic yeast to better utilise the complex mixture of available carbon sources in host niches. This working hypothesis would be consistent with earlier studies, suggesting that *C. albicans* is a glucose Crabtree-negative yeast. In other words, this pathogen retains respiratory activity even following exposure to glucose [44]. During growth on glucose, *ADH1* mRNA levels rise to maximum levels during late exponential growth phase and then decline to low levels in stationary phase [75]. The *ADH1* mRNA is relatively abundant during growth on galactose, glycerol, pyruvate, lactate or succinate, and less abundant during growth on glucose or ethanol. However, alcohol dehydrogenase levels do not correlate closely with *ADH1* mRNA levels. This locus may be controlled at both transcriptional and post-transcriptional levels, or other differentially regulated *ADH* loci may exist in *C. albicans* [75].

the CaIcl1-Ubi-Myc3 protein was rapidly degraded following glucose addition to cells grown on lactate or oleic acid. In conclusion, the addition of a ubiquitination site to CaIcl1 accelerates

The above observations strongly suggest that specific proteins can be targeted for degradation in *C. albicans* following exposure to glucose, and these proteins are degraded via ubiquitina‐ tion. If this is the case, it should be theoretically possible to detect ubiquitinated forms of these proteins. Hence immuno-precipitation experiments were then performed in an attempt to demonstrate ubiquitinated forms of the CaIcl1-Myc3 protein in *C. albicans.* Proteins were extracted from *C. albicans ICL1-UBI-*site-*MYC3-URA3* cells 20, 40 and 120 minutes after glucose addition. Analogous control extracts were also prepared from *S. cerevisiae* and *C. albicans* cells expressing ScIcl1+Myc3, CaIcl1+Myc3 and untagged parental strains grown on lactate plus glucose and lactate alone. These extracts were immunoprecipitated with an anti-Myc antibody that was predicted to precipitate Icl1 proteins having carboxyl-terminal Myc3 tags. These immunoprecipitates were then subjected to Western blotting with an anti-ubiquitin antibody to test whether any of these Myc3-tagged Icl1 proteins carry ubiquitin sequences. The Western blots were also probed with the anti-Myc antibody to confirm that Myc-tagged Icl1 proteins had been immunoprecipitated. This was the case. Interestingly, a weak ubiquitin-containing band of a length consistent with Icl1-Myc3 proteins was detected. Such bands were observed in three replicate experiments. These weak bands were observed for the ScIcl1-Myc3 CaIcl1- Ubi-Myc3 proteins following glucose addition (both proteins carry ubiquitination sites). However, no ubiquitination of these proteins was observed in the absence of glucose, or for the CaIcl1-Myc3 protein (which lacks a strong ubiquitination site) or for the untagged control

These data suggest that when *S.cerevisiae* Icl1 or an artificial *C. albicans* Icl1 carrying a ubiquiti‐ nation signal is expressed in *C. albicans*, it becomes ubiquitinated and destabilised in response to glucose. However, the native *C. albicans* Icl1 protein is not destabilised by glucose. There‐ fore, in response to glucose, target proteins became ubiquitinated and then degraded in *C.*

What natural *C. albicans* proteins might be subjected to ubiquitin-mediated, glucose-acceler‐ ated protein degradation? To address this, bioinformatic tools were used to predict possible ubiquitination sites in glycolytic, gluconeogenic and glyoxylate cycle enzymes in *S. cerevisiae* and *C. albicans.* Five enzymes were selected for analysis: Fbp1, Pck1, Mdh1, Eno1 and Mls1. Based on this analysis, *S. cerevisiae* Fbp1, Pck1 and Eno1 appear to carry strong ubiquitination sites, while only Eno1 *C. albicans* appears to have a high confidence ubiquitination site. In conclusion, while these central metabolic pathways are highly conserved between *S. cerevi‐ siae* and *C. albicans,* these organisms appear to display significant differences in the presence

A previous study suggested that *C. albicans* might be capable of using more than one carbon source at the same time during growth in specific niches in the host [34]. This study was based

its degradation in response to glucose in *C. albicans* [63].

of ubiquitination sites in the orthologous enzymes [63].

**4. Overview of Metabolic Adaptation in** *Candida albicans*

cells [63].

214 Genital Infections and Infertility

*albicans* [63].

Interestingly, a significantly smaller proportion of glucose is fermented to ethanol by *C. albicans* than by *S. cerevisiae* [75]. This is consistent with the low amounts of ethanol produced by *C. albicans* observed in this study.

*S. cerevisiae* is not able to assimilate both non-fermentable carbon sources and glucose at the same time because of glucose repression. Hence, we predicted that these yeasts have evolved different responses to glucose. Therefore, in this study, I analysed the regulation of carbon assimilation in *C. albicans* focussing on genes/enzymes involved in gluconeogenesis and the glyoxylate cycle. I tested *ICL1* and *PCK1* gene expression, Icl1 and Pck1 protein stability and the impact of glucose on the assimilation of non-fermentable carbon sources. The author compared their *C. albicans* responses to those of *S. cerevisiae* under equivalent conditions. The following conclusions can be drawn from these findings.

First, gluconeogenic and glyoxylate cycle mRNAs are sensitive to glucose in both *C. albicans* and *S. cerevisiae*. This reconfirmed previous findings from Aberdeen Fungal Group Laboratory [54, 55] and other laboratories [76]. Dramatic decreases in *ICL1* and *PCK1* mRNA levels were observed in *C. albicans* cells after exposure to 2% glucose. This glucose concentration is higher than the levels of glucose homeostatically maintained in human blood (about 0.1%). However, it is already known that *C. albicans* responds to lower glucose concentrations within the physiological range of blood glucose [54, 55]. Therefore, *C. albicans* is able to respond to blood glucose levels during disseminated haematological infections. Interestingly, patients with diabetes who often have elevated blood glucose levels, have a higher risk of systemic *Candida* infections [14], and dietary glucose enhances *C. albicans* colonisation and invasion [77].

The second main observation was that the Icl1 and Pck1 proteins are stable in *C. albicans* following glucose exposure. The addition of 2% glucose to *C. albicans* cells growing on lactate or oleic acid did not trigger the degradation of the Icl1 and Pck1 proteins, at least within the 4 hours examined. This is in contrast to the situation in *S. cerevisiae*, where the addition of 2% glucose triggered the rapid degradation of the Icl1 and Pck1 proteins. The estimated half-lives for these proteins in *S. cerevisiae* are more than 20 hours [78] indicating that these proteins are 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 same time as glucose in these carbon-rich niches.

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 increase the fitness of this pathogen.

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 degradation in response to glucose.

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, inactivation of the polyubiquitin gene slowed ScIcl1 degradation in *C. albicans* in response to glucose. This was consistent with the idea that ubiquitination contributes to glucose-acceler‐ ated protein degradation in *C. albicans.*

If this was the case, the addition of a ubiquitination signal should confer glucose-accelerated degradation upon the stable CaIcl1 protein. This was tested, showing that a carboxyl-terminal ScIcl1 ubiquitination signal was sufficient to trigger the rapid degradation of CaIcl1 following glucose addition to *C. albicans* cells [63].

Finally, direct biochemical evidence for the involvement of ubiquitination in glucose-acceler‐ ated protein degradation in *C. albicans* was obtained by showing that ubiquitin co-immuno‐ precipitated with the ScIcl1 and CaIcl1-Ubi proteins in *C. albicans*, but only when cells were exposed to glucose [63].

In conclusion, *C. albicans*is capable of destabilising target proteins in response to glucose, and this destabilisation is mediated by ubiquitination. The lack of ubiquitination sites on the *C. albicans* Icl1 and Pck1 proteins probably accounts for the observation that these glyoxylate cycle and gluconeogenic enzymes are not destabilised by glucose in *C. albicans.*
