Glucose Sensing Network in Candida albicans: a Sweet Spot for Fungal Morphogenesis

Glucose, a ubiquitous carbon source preferred by most cells (96), was first identified by Andreas Marggraf in 1747 (67). Even though it is the most abundant hexose on earth (33), glucose is often a limiting nutrient in biological systems (2); it is a precious resource for which organisms fiercely compete (54). An elegant genetic illustration of this is the fact that organisms from bacteria to humans have highly evolved sensing and signaling machinery dedicated to the detection, acquisition, and utilization of glucose (38, 76, 80, 108).

The framework for understanding sugar sensing in fungi began with studies of Saccharomyces cerevisiae (baker's yeast). This model eukaryote is a voracious glucophile that rapidly metabolizes glucose to produce massive amounts of CO₂ and ethanol, a trait refined over at least 4,000 years of human civilization (104). The distantly related human pathogen Candida albicans has become an organism of choice for fungal genetics, motivated by its huge impact on human health and thanks to recently developed genetic and biochemical tools for its analysis (4, 11, 68, 91). In the 200 million years since C. albicans and S. cerevisiae last shared a common ancestor (27, 84), genetic drift combined with adaptation to differing environments has drastically “rewired” the regulatory circuitry of the sugar response network (12, 63, 83). For C. albicans, glucose is also a morphogen that influences yeast-to-hypha transitions (37), and this trait is critical for optimal virulence in the host (reviewed in reference 7). Both fungi sense glucose mainly through three pathways, none of which operates in isolation (Fig. 1): (i) the SRR (sugar receptor-repressor) pathway, (ii) the glucose repression pathway, and (iii) the adenylate cyclase pathway. Each system utilizes a distinct signal transduction cascade, but extensive cross-regulation weaves them together into a single sugar response network.

FIG. 1.

A working model of the C. albicans glucose-sensing network. Three independent yet interconnected signal transduction cascades are triggered by glucose in C. albicans to produce a coordinated response. The asterisks indicate mechanisms inferred from work with S. cerevisiae. Glucose is indicated by blue hexagons, galactose by a gray hexagon, and sucrose by a disaccharide (linked hexagon and pentagon). Components and processes activated in the presence of glucose are shown in black, while those that are inactivated by glucose are shown in gray. Kinases are indicated as stars. The red lines indicate repression, and the green lines indicate activation; certain genes are derepressed by low levels of glucose but repressed under high-glucose conditions. The SRR pathway (center) is as follows. Hgt4 senses extracellular glucose or galactose, which initiates Yck2-mediated phosphorylation, and SCF^Grr1-mediated ubiquitylation, of the transcriptional corepressor Std1, resulting in its degradation (13, 85). Cellular depletion of Std1 is followed by phosphorylation of the Rgt1 transcriptional repressor by PKA, rendering it inactive (46). Expression of Rgt1 target genes, including those involved in alternative respiration, fermentation, and glucose repression, and the HGT genes for hexose import, is derepressed (3, 13, 85). The glucose repression pathway (left) is as follows. Upon hexose influx through the Hgt transporters, intracellular glucose is phosphorylated to glucose-6-phosphate, primarily through the action of the hexokinase Hxk2 (55). Subsequent glycolysis raises cellular ATP levels, and under conditions of glucose abundance (>0.2% for C. albicans), this instigates the dephosphorylation/inactivation of the Snf1 kinase through the action of the Reg1/Glc7 phosphatase complex (48, 81, 103). The absence of Snf1 activity enables the Mig1/Mig2 transcriptional repressors to mediate the repression of genes required for the use of alternative (nonfermentable) carbon sources, genes involved in the adenylate cyclase pathway, and high-affinity HGT genes needed only when glucose is scarce (24, 58, 66, 85). The adenylate cyclase pathway (right) is as follows. Either activated Gpr1/Gpa2-GTP (61, 64, 79) or activated Ras1-GTP (29) (or both) stimulates the Cyr1/Cdc35 adenylate cyclase through mechanisms that appear to require intracellular phosphorylated glucose (19). The noncanonical G_β protein Asc1 antagonizes the Gpr1 branch of this pathway (107). Activated Cyr1/Cdc35 requires the cyclase-associated protein Cap1 to generate cAMP from ATP (6, 30), and the cellular abundance of cAMP is downregulated mainly through the action of the phosphodiesterase Pde2 (5, 39). Increased cAMP levels inactivate the Bcy1 inhibitory subunit of PKA (18). Once activated, PKA phosphorylates the Efg1 transcription factor (10), which functions as an activator for hypha-specific genes and for genes that promote virulence (32, 34) but also acts as a feedback repressor for EFG1 (93).

SRR pathway.

Hexose transporters, which are 12 transmembrane-spanning proteins that form substrate-selective pores in the plasma membrane, facilitate glucose uptake into eukaryotic cells (42, 53). The transporters are part of a very large protein family called the major facilitator superfamily whose members transport solutes by facilitated diffusion (not to be confused with symporting or antiporting) of molecules (28, 42, 98, 108). They are encoded by large gene families (up to 17 members) and can harbor species-specific protein signatures that make them novel drug targets in eukaryotic pathogens (31). In fungi, select members of the large hexose transporter family have evolved into sensors of glucose and other structurally related hexoses.

S. cerevisiae employs both a high-affinity (Snf3) and a low-affinity (Rgt2) glucose sensor poised in the plasma membrane to monitor glucose levels in the environment (70). Although direct binding to hexoses has not been demonstrated, Snf3 and Rgt2 are almost certainly glucose receptors because they are highly similar to hexose transporters throughout their sugar-binding domains, and they are critical for the cell's response to glucose (70). Sensors of this type are incapable of importing glucose, and what insight can be gleaned from work on functional transporters (i.e., human GLUT1 or yeast Hxt/Hgt proteins) does not yet resolve the molecular basis underlying a sensors' inability to transport (22, 43, 44, 77). Instead, sensors generate an intracellular signal that induces the expression of appropriate hexose transporter (HXT) genes. Each sensor has a C-terminal cytoplasmic tail (absent from transporters) that is neither necessary nor sufficient for signal generation and that does not physically occlude transport (65). Therefore, it is believed that the determinants for hexose sensing are encoded in the 12-transmembrane domain, while the tail serves to amplify transduction of the glucose signal to the transcriptional repressor Rgt1, the master regulator of the HXT genes (40). Sensor tails form part of a protein complex that includes the yeast casein kinase Yck1 (encoded by either S. cerevisiae YCK1 [ScYCK1] or ScYCK2) and two paralogous transcriptional corepressors, Std1 and Mth1. C. albicans harbors a single ortholog of Std1 and Mth1 (12) and has two genes (C. albicans YCK2 [CaYCK2] and CaYCK3) homologous to ScYCK2 that encode a Yck1 isoform, but only CaYCK2 is predicted to encode the C-terminal Cys-Cys palmitoylation motif necessary for participation in glucose sensing at the plasma membrane (65) (Fig. 1, center).

Sensor stimulation by glucose activates Yck1, which catalyzes the phosphorylation of Std1 and Mth1, marking them for ubiquitylation by the SCF^Grr1 ubiquitin-protein ligase, and results in their subsequent degradation (65). Depletion of the cellular pool of Std1 and Mth1 exposes the Rgt1 repressor to phosphorylation by protein kinase A (PKA), which renders Rgt1 unable to bind DNA, thereby derepressing transcription of its target genes (46). In this way, the abundance of Std1 and Mth1 acts as a molecular dial, transforming the strength of the glucose stimulus into an appropriate transcriptional response. The Snf3/Rgt2 glucose sensors and the Std1/Mth1-Rgt1 repressor complex are fungus specific (95), making them unique targets for antifungal development. One study with S. cerevisiae implicated the Snf3/Rgt2 pathway in yeast morphogenesis: insertion of a Tn3 transposon into the ScMTH1 gene completely negated invasive growth by haploid Σ1278b cells, in which invasive (pseudohyphal) growth is controlled by the PKA and mitogen-activated protein kinase pathways (92). Snf3/Rgt2-type sensors have also been characterized in Kluyveromyces lactis (Rag4) (9), Hansenula polymorpha (Hxs1) (89), Neurospora crassa (60), and C. albicans (Hgt4) (13), and BLAST searching readily identifies novel candidate orthologs throughout the Ascomycetes.

In C. albicans, the signal from Hgt4 is transduced through CaStd1 and ultimately to CaRgt1, which derepresses transcription of genes for sugar import, including NGT1 for N-acetylglucosamine transport and many other HGT genes, an effect that is antagonized through glucose repression under high-sugar conditions (see below) (3, 13, 85). The Hgt4 signaling pathway controls the expression of genes associated with fermentation (the HGT genes, HXK2, TYE7, and PFK26) and, in contrast to the pathway in S. cerevisiae, also regulates the expression of genes involved in alternative respiration (AOX2 and NDE1) (13). Alternative respiration is found in plants, some protozoa, and most fungi but is absent from S. cerevisiae and from mammals (reviewed in reference 99); therefore, components of the pathway present novel, nonhost targets for antifungal development. Although alternative respiration has been recognized in plants and fungi for over 30 years, its biological role is not known, though it appears to be important for production of the penicillin cephalosporin C by Acremonium chrysogenum (41) and for ethanol production in the xylose fermenter Pichia stipitus (86). Further study of alternative respiration in C. albicans with regard to morphogenesis or host-pathogen interactions may yield insight into the biological role of this ancient yet enigmatic mode of metabolism.

Glucose, galactose, fructose, or sucrose (a glucose-fructose disaccharide hydrolyzed in the periplasm by the Suc2 enzyme) all stimulate morphogenesis of true hyphae (62), which is regarded as a virulence trait that contributes to mortality in C. albicans infections (16, 82). Mutations in each component of the SRR pathway affect hyphal morphogenesis. Constitutively signaling mutants are hyperfilamentous (HGT4-1, Δstd1, or Δrgt1), whereas nonsignaling Δhgt4 null mutants are hypofilamented (13, 85), and the Δgrr1 null mutation causes dramatic pseudohyphal growth (14) (Fig. 2). Indeed, Δhgt4 null mutants show retarded virulence in a mouse model of disseminated candidiasis (13). Since S. cerevisiae neither forms true hyphae nor often colonizes or becomes virulent within hosts, further studies of C. albicans will more directly address how its sugar-sensing network reacts to the host environment, yielding insight into both colonization and pathogenesis.

FIG. 2.

Glucose-sensing genes that affect fungal cell morphology. Shown are mutations in the glucose-sensing pathways that have been shown to impact yeast-hypha morphogenesis in C. albicans (see the text and references therein). The asterisk indicates overexpression of a wild-type PDE2 allele.

Glucose and galactose sensing were believed to be mutually exclusive in fungi, a paradigm set by studies conducted with S. cerevisiae, which cannot utilize both sugars concurrently. In baker's yeast, sensing glucose through Snf3/Rgt2 initiates “glucose repression” of GAL genes required for galactose metabolism, while galactose detection by Gal3 reduces glucose influx by inducing MTH1 expression, which reinforces Rgt1-mediated squelching of the HXT genes (12). In stark contrast, C. albicans has no canonical Gal3-type sensor and senses both glucose and galactose through Hgt4 (12, 63). Examining multiple fungi in the S. cerevisiae-K. lactis clade sheds light on the evolution of this signaling network during the ∼200 million years since the divergence of S. cerevisiae and C. albicans. Hgt4-type sensors appear to be “ancestral” hexose sensors, while the Gal3-based galactose-specific sensing mechanism seems to have arisen after S. cerevisiae and C. albicans diverged but before the whole-genome duplication event (12). The C. albicans Hgt4 sensor responds to glucose levels as low as 0.01% (0.56 mM), which makes it a very high-affinity glucose sensor, active at physiological levels of glucose. It is also a low-affinity galactose sensor that begins to respond to galactose at 0.6% (33 mM) (13).

C. albicans colonizes the human gastrointestinal (GI) tract at birth or quickly thereafter (21, 51) and persists as a benign commensal (>50% of healthy adults are colonized) (45). The GI tract is a Candida niche replete with monosaccharide glucose and galactose, especially during the first 6 months of life, when infants, for whom candidemia can be particularly debilitating and deadly (21, 51), are sustained by milk. The primary sugar in milk, lactose (present at 6 to 7%), is a galactose-glucose disaccharide hydrolyzed by lactase in the gut. A priori, this environment seems ideal for microbes that utilize both monosaccharides concurrently, and indeed, competitive-colonization experiments indicate that Hgt4 is critical for C. albicans persistence in the mouse GI tract (V. Brown, unpublished data), which suggests a biological role for this pathway in human colonization. Glucose and galactose in the gut are not only used by GI microbes, but are also absorbed into the bloodstream, and thus are two of the most abundant monosaccharides in human blood and urine (James Shoemaker, personal communication; data available for download at http://biochemweb.slu.edu/msl/index.html), and these are two niches in which C. albicans infections are pathogenic.

Glucose repression.

The glucose repression pathway monitors the intracellular energy charge, which is sensitive to the rate of the first step of glucose metabolism—its phosphorylation (Fig. 1, left). This pathway is conserved among fungi and appears to be triggered at 10 mM (∼0.2%) glucose levels (13). In S. cerevisiae, three kinases catalyze the phosphorylation of glucose: Hxk1 (hexokinase 1), Hxk2 (hexokinase 2), and Glk1 (glucokinase 1), with Hxk2 providing the bulk of the activity toward glucose (55). C. albicans HXK2 expression, like that of its ortholog in S. cerevisiae, is regulated by the SRR pathway (via Hgt4 and Rgt1) (13, 85). It is also upregulated in cells in white phase compared to opaque phase cells (97) and is downregulated during deep-seated infections in mice (100), but Δhxk2 null mutants have not been characterized in this pathogen.

In the absence of glucose, when the AMP/ATP ratio is high, the yeast ortholog of mammalian AMP-activated protein kinase, Snf1, is active (103) and phosphorylates the transcriptional repressor Mig1. This phosphorylation inactivates Mig1 by causing it to be sequestered in the cytoplasm (24). Snf1 can also phosphorylate Rgt1, enabling it to repress at least one of its target genes (HXK2 [71]), though the general applicability of this mode of Rgt1 regulation has not been established. As glucose is imported via the SRR pathway, glucose phosphorylation, at the initial expense of ATP but coupled with concurrent changes in the rates of adenine biosynthesis (48) and ATP generation through glycolysis, results in a decreased cellular AMP/ATP ratio. This change in the energy balance promotes the dephosphorylation and inactivation of Snf1 (103), carried out by the Reg1/Glc7 protein phosphatase I complex, consisting of a regulatory subunit (Reg1) that directs the phosphatase (Glc7) to Snf1 (81). Unlike in S. cerevisiae, null mutations of snf1 are lethal in C. albicans (75), and thus, further characterization of the role of Snf1 in filamentation or virulence has not been pursued, though evidence from fungal plant pathogens does suggest that Snf1 may play a part in these processes: Δsnf1 mutants of Gibberella zeae (anamorph, Fusarium graminearum) (50) and Fusarium oxysporum (69) show severely reduced mycelial growth and defects in virulence.

In response to glucose and the inactivation of Snf1, dephosphorylated Mig1 enters the nucleus to effect the repression of genes required for the metabolism of alternative carbon sources and gluconeogenesis (24). Hxk2, the hexokinase most active toward glucose (55), also acts as a transcriptional corepressor, binding to Mig1 immediately upon glucose exposure and blocking its phosphorylation by Snf1 (1). C. albicans has two orthologs of ScMIG1 (CaMIG1 and CaMIG2) that may be at least partially redundant in function, as are Mig1 and Mig2 in S. cerevisiae (58). The CaMig1 protein lacks the Snf1 phosphorylation sites identified in ScMig1 (106) that are required for regulation (25), and like ScMIG2, the CaMIG1 gene is transcriptionally regulated by CaRgt1 (13, 85). Therefore, how CaMig1 and CaMig2 are regulated to enact glucose repression remains uncertain in C. albicans.

Gene expression profiling of C. albicans Δmig1 null mutants indicated that CaMig1 represses the expression of a set of genes similar to the set repressed by Mig1 in S. cerevisiae (66, 85). Knocking out CaMIG1 yielded no morphological or virulence phenotypes (106). It was concluded from these studies that the glucose repression pathway has no effect on morphogenesis or virulence, but this assertion is perhaps premature, because hypomorphic snf1 alleles, as well as Δhxk2, Δmig2, and Δmig1 Δmig2 mutants have yet to be fully examined. The issue is further complicated by the fact that although low concentrations of glucose (0.01 to 0.25%) stimulate filamentation, very high levels of glucose (>0.5%) inhibit hyphal morphogenesis by an unknown mechanism (62).

Adenylate cyclase pathway.

Certain classes of G-protein-coupled receptors that sense nutrients (i.e., sugars and amino acids) appear to be unique to fungi, representing additional targets for antifungal drug development (reviewed in reference 26). Gpr1-type receptors have been characterized in Schizosaccharomyces pombe (Git3) (reviewed in reference 36) and C. albicans (61). This pathway begins at the cell surface with the G-protein-coupled receptor Gpr1 and its cognate G_α protein, Gpa2 (57) (Fig. 1, right). Gpr1 binds glucose with a 50% effective concentration of 20 mM (∼0.4%) and binds the disaccharide sucrose (glucose-fructose) even more avidly (50% effective concentration, 0.5 mM, or ∼0.01%) (52). Because sucrose is hydrolyzed to the monosaccharides glucose and fructose in the periplasmic space (between the fungal cell wall and the plasma membrane) by the Suc2 invertase (17), sucrose, along with its degradation products, probably concurrently activates both Gpr1 and the high-affinity SRR receptor (Snf3 in S. cerevisiae or Hgt4 in C. albicans). Sugar stimulation of Gpr1 promotes the GTP-bound (active) form of Gpa2 by promoting GDP release, a process antagonized by the noncanonical G_β protein Asc1 (107). Null mutations in either CaGPR1 or CaGPA2 abrogate yeast-hypha transitions, causing reduced virulence during blood infections in mice (61, 64, 79), while a constitutively active GPA2^Q355L allele was shown to promote filamentation in a Δgpr1 null mutant (61, 79) (Fig. 2). Gpa2-GTP activates adenylate cyclase (Cyr1), which catalyzes the production of cyclic AMP (cAMP) from ATP (74). Depletion of ATP by this route may contribute to fluctuating AMP/ATP ratios upon glucose exposure, thereby influencing the glucose repression pathway through changes in the cellular energy charge.

Adenylate cyclase appears to require intracellular phosphorylated glucose in order to be activated through Gpr1-Gpa2, but the mechanism remains elusive (19). In the absence of CaGPR1, levels of cAMP still spike in response to serum or large amounts of glucose (100 mM, or 1.8%), suggesting that CaCyr1 can be activated by Gpr1-independent means (61). While other ligands can directly bind/activate adenylate cyclase in C. albicans, including bicarbonate (47), muramyl dipeptides (105), and perhaps the quorum-sensing molecule farnesol (23), the most likely Gpr1-independent route in response to glucose is through direct CaCyr1 binding by activated CaRas1 (29). The Ras GTPases are activated in response to phosphorylated glucose, either through its activation of Cdc25 (a guanine nucleotide exchange factor) (29) or through its inhibition of Ira2 (a GTPase-activating protein) (20, 73). CaCyr1 (also called Cdc35) requires Cap1 (also called Srv2) for activity in response to activated Ras1 (6, 30). Functional Ras1 is necessary for the formation of true hyphae (Δras1 null mutants can form only pseudohyphae), while constitutively active RAS1^G13V results in hyperfilamentous growth (49) (Fig. 2).

As the cellular level of cAMP rises, it binds to and inactivates CaBcy1, the inhibitory subunit of the PKAs encoded by CaTPK1 and CaTPK2 (18). With this inhibition relieved, active PKA phosphorylates several transcription factors to promote robust yeast-to-hypha transitions. PKA is activated in S. cerevisiae through a similar process (15, 94) and promotes pseudohyphal growth (72). cAMP accumulation in S. cerevisiae is downregulated by two phosphodiesterases, Pde1 (low affinity) and Pde2 (high affinity). Pde1 is a substrate for PKA phosphorylation, and thus activated, Pde1 provides negative feedback regulation for glucose signaling (59), but Pde2 in particular has been shown to directly affect morphogenesis in S. cerevisiae (56, 101) and in C. albicans (39), where pde2 null mutants show increased pseudohyphal growth and PDE2 overexpression suppresses true filamentation (5) (Fig. 2). For C. albicans, Pde2 is also required for optimal virulence (5).

Activated PKA phosphorylates Efg1 in C. albicans and its S. cerevisiae homologs (Sok2 and Phd1), all of which are regulators of cellular morphogenesis (10). In C. albicans, PKA-activated Efg1 turns on genes encoding hypha-specific cell wall proteins (HWP1 and HWP2) (87), adhesins (e.g., ALS1) (34), and hypha-secreted aspartyl proteases (SAP4 to -6) (32), all of which are factors enabling host colonization and virulence. Activated Efg1 also downregulates its own (EFG1) expression to enact a feedback regulatory circuit for this pathway (93). Mutations in CaEFG1 cause reduced hyphal morphogenesis in vitro (90), and Δefg1 null mutants show reduced virulence during blood infections (78). In a study of intestinal colonization by C. albicans, null mutations in efg1 caused increased (and conversely, EFG1 overexpression decreased) the fungal burden in the mammalian GI tract (102). Thus, further study of this pathway promises to illuminate the mechanisms underlying commensal colonization, as well as virulence.

Communications between sensing pathways.

The three main glucose-sensing mechanisms (shown in Fig. 1) engage in extensive cross talk through the activities of kinases and through the downstream genes regulated by each pathway, but many of these connections await study in C. albicans. For example, Rgt1 in S. cerevisiae is a target of activated PKA, and recent work has shown that CaRgt1 represses hyphal morphogenesis, though the mechanism has not been elucidated (12, 85). If experiments on S. cerevisiae lend insight into C. albicans biology, the data suggest that PKA, through its phosphorylation of both Rgt1 and Efg1, acts as a central hub integrating glucose sensing, uptake, and metabolism with cellular morphogenesis (10, 46, 85, 90, 92). Other environmental sensing pathways intersect with the glucose-sensing network: the ScSsy1 amino acid-sensing pathway (orthologous to the C. albicans Csy1 pathway) requires components of the SRR pathway (Yck1 kinase and the SCF^Grr1 ubiquitin ligase complex) to function (88). In addition, the Tor1 kinase that governs nitrogen catabolism also regulates HXT1 expression in S. cerevisiae at the transcriptional and posttranslational levels (reviewed in reference 35), and in C. albicans, Tor1 appears to affect cellular filamentation in part through Efg1 (8). Therefore, the entire glucose-sensing network (Fig. 1) is linked to other nutrient-sensing pathways as part of a coordinated environmental response system.

Both C. albicans and S. cerevisiae possess highly evolved, exquisitely tuned sugar response networks that integrate three main signaling pathways to ensure proper cellular responses to dynamic environments. Sugar sensing has profound effects on cell morphology in both S. cerevisiae and C. albicans, and for C. albicans, sugar sensing is vital for host colonization and for optimal virulence. A future quest is to understand how the sugar-sensing network in C. albicans orchestrates the appropriate metabolic and morphological responses in native habitats to enable fungal survival or promote virulence within the host. Such studies have the potential to illuminate other host-fungal interactions, as well, and ultimately to suggest novel ways to thwart pathogens.