**7. Mitochondrial Metabolism Is Sensitive to Glucose Availability in** *C. albicans*

Amino acid metabolism can be directly or indirectly regulated by glucose. Direct control is exerted by Mig1 and Mig2, well-studied factors that bind promoters and repress transcription when glucose is abundant [115]. Indirectly, glucose can negatively and pleiotropically regulate amino acid metabolism by controlling the function of mitochondria. For example, we and others have shown that *C. albicans* mitochondrial activity can be downregulated by glucose in a manner similar to *S. cerevisiae,* albeit to a lesser extent [59,116]. Our data indicate that the repressing effect of glucose is clearly evident in *C. albicans* at 0.2% or higher [59], and more sensitive transcriptomic studies have noted effects of 0.01% glucose, a very low level of glucose [116]. The more pronounced repressive effect of glucose on mitochondrial respiration in *S. cerevisiae* is likely due to the limited capacity to oxidize NADH when glycolytic flux is high [117]. This is expected to be similar in *C. albicans*; however, since *C. albicans* has a functional complex I with a higher capacity to oxidize NADH, the threshold level for glucose's repression of mitochondrial respiration is higher than that in *S. cerevisiae*. Consistently, we observed that the level of ATP is higher in 0.2% glucose than in 2% glucose [59]. Consistently with the model that ATP-dependent Ras1 activation drives filamentous growth [118], filamentation is more robust when glucose is <0.2% [39,59]. The lower ATP level observed for cells grown in the presence of 1% glycerol is likely due to the lower levels of reduced NADH (low NADH/NAD<sup>+</sup> ratio) that can be oxidized to generate the membrane potential needed to generate ATP [59].

The pleiotropic effect of glucose on mitochondrial activity and amino acid catabolism is nicely illustrated by arginine catabolism. Arginine catabolism occurs in a bifurcated manner that generates two products that are independently catabolized either in the cytosol (urea) or mitochondria (proline). When glucose is absent, the proline catabolic pathway becomes essential for arginine catabolism as cells lacking *PUT1* or *PUT2* failed to grow in the presence of arginine as the sole nitrogen and carbon source, whereas cells lacking *DUR1,2* grew [59]. However, when high glucose was added as the main carbon source, the *put1*−/− defect was rescued as the metabolism shifted from pure respiratory to mixed types of growth (respiratory/fermentative), shifting the metabolic burden of nitrogen assimilation to the cytosolic Dur1,2. Strikingly, the enzymes required to catabolize proline were still expressed; however, since the mitochondria were repressed by high glucose levels, they were unable to carry out their catabolic functions of converting proline to glutamate.

### **8. Ammonia Generation and Excretion**

In the human body, amino acids are an abundant source of nitrogen for *C. albicans.* However, the utilization of amino acids in excess of the amount necessary to support growth and basic cellular functions must be controlled due to the accumulation of ammonia as a metabolic byproduct. Excess ammonia is toxic to cells. When cells are grown using amino acids as energy sources, excess ammonia exits into the extracellular medium, resulting

in environmental alkalization (Figure 6A). Interestingly, this capacity to increase environmental pH via ammonia extrusion is believed to support the pathogenic growth of fungal pathogens such as *C. albicans* in certain acidic microenvironments, e.g., the phagosome of macrophages (reviewed in [119,120]).

**Figure 6. Ammonia extrusion in** *C. albicans***.** (**A**) When *C. albicans* utilizes amino acids or Nacetylglucosamine (GlcNac) as nitrogen sources for growth, ammonia is produced, promoting the alkalization of the extracellular pH. Amino acids can be either free or derived internally from the proteolytic degradation of oligopeptides, which are then converted to glutamate either in the cytosol (by specific aminotransferases) or mitochondria (proline catabolism), creating a glutamate pool in the cytosol (Glucyto). The NAD+-dependent glutamate dehydrogenase (Gdh2) catalyzes the conversion of Glucyto to α-ketoglutarate, releasing ammonia in the process. Urea generated via arginine catabolism (or from the extracellular environment) can also be degraded to ammonia via the urea amidolyase (Dur1,2) enzyme. The deamination of GlcNac is dependent on glucosamine-6-phosphate isomerase (Nag1), which catalyzes the conversion of glucosamine-5-phosphate (GN5P) to fructose 6-phosphate (F6P). Excess ammonia produced in the cytosol must be removed in order to avoid its toxic effects; due to the cytosolic pH of around 6.5, most of the free ammonia (NH3) is converted to ammonium (NH4 +) (see graph below), which can be directly assimilated via central nitrogen metabolism (i.e., Gdh3 and Gln1), whereas a small fraction is released into the environment, where it could neutralize the acidic pH, producing ammonium (NH4 +). Whether ammonia (or even ammonium) is exported through simple diffusion or via exporters (Ato) requires further study. (**B**) At the normal cytosolic pH of ≈6.5, ammonia (NH3) is converted to ammonium (NH4 +). We present a plot showing the relative concentrations of NH3 and NH4 <sup>+</sup> in aqueous solution based on pH and temperature. Image adapted and redrawn from Huang, J; *Handbook of Environmental Engineering*) [121]. The ratio of NH4 + to NH3 in this equilibrium is highly pH-dependent. At low acidic pH, the ammonium form (NH4 +) dominates. As the pH increases, the ammonia (NH3) form also increases, and the proportion becomes equal at the pKa value. Higher temperatures favor the NH3 gas side of the equilibrium balance with NH4 +.

It has been proposed that ammonia derived from amino acid catabolism enables *C. albicans* cells to neutralize the acidic luminal pH of the phagosome, reducing the activities of hydrolytic enzymes with low pH optima and inducing *C. albicans* to switch

morphologies, resulting in hyphal growth, thus facilitating macrophage evasion [31]. This model was largely premised on studies using a strain lacking *STP2* (*stp2*Δ/Δ), the SPS transcription factor that positively regulates the expression of amino acid permeases required for amino acid uptake [31,32]. Strains carrying *stp2*Δ/Δ exhibit defects in both environmental alkalization and the capacity to escape the phagosome of the engulfing macrophage [31,32]. This model assigned the critical ammonia-generating event to the urea amidolyase (Dur1,2), which catalyzes the conversion of urea to ammonia and CO2, as cells lacking this enzyme (*dur1,2*Δ/Δ) show alkalization defects when cells are grown in media with high glucose [32]. However, Dur1,2 has only been linked to ammonia generation in the presence of its substrate urea, and *DUR1,2* expression is under tight NCR control [44,56]. Thus, it is unlikely that Dur1,2 contributes to the alkalization of a growth medium with abundant preferred nitrogen sources such as amino acids and even ammonium sulfate.

There is mounting evidence that alkalization of the phagosomal compartment is not requisite for *C. albicans* cells to evade macrophages. Results obtained using dual-wavelength ratiometric fluorescence imaging to quantify the pH in the phagosome revealed that increased phagosomal pH is the consequence of elongating hyphal cells, physically stretching the phagosomal membrane, causing transient leaks [122]. The induction of hyphal growth was observed to precede alkalization. In addition, the proton-pumping activity of V-ATPase exceeds the rate of ammonia extrusion by several orders of magnitude [122]. Recently, we reported that Gdh2, the enzyme that catalyzes the conversion of glutamate to α-KG, is responsible for the bulk of ammonia produced from amino acid metabolism [61]; a *gdh2* null strain is unable to alkalize a medium containing amino acids as the sole nitrogen and carbon source. Surprisingly, the capacity of *gdh2*−/− mutants to escape the macrophage phagosome or its virulence in murine systemic infection model was not affected, indicating that amino acid-dependent environmental alkalization is not essential for the virulence of *C. albicans*. Consistently with a previous report [122], we observed that viable wildtype cells pre-stained with a pH-sensitive dye (pHrodo), of which the fluorescence intensity varied inversely to pH, were retained in acidic phagosomes, and this observation is was even when a high MOI (more ammonia-extruding cells) was used [61].

In *C. albicans*, Gdh2 is a cytosolic component [61] and its expression is independent of NCR; Gdh2 is well expressed in cells grown in a medium with high levels of amino acids, even when supplemented with high levels of ammonium sulfate [61]. Consistently, a strain lacking *GLN3* and *GAT1*, which encode for the GATA transcription factors activating NCRsensitive genes, remain alkalization-competent (our unpublished data). Despite its cytosolic localization, Gdh2-dependent alkalization is tightly linked to mitochondrial function as acute inhibition of the mitochondria with a sublethal dose of antimycin, a potent respiratory complex III inhibitor, virtually abolished alkalization in wildtype cells even when a very high starting cell density was used (OD600 ≈ 5) [61]. Since proline catabolism is a major source of glutamate in the mitochondria and since this alkalization is partially dependent on proline catabolism [61], it is likely that pharmacological inhibition of the mitochondria pleiotropically prevented either the generation or export of mitochondrial glutamate. In a similar way, the inability of *C. albicans* to alkalinize the extracellular environment when grown in the presence of high glucose (2%) is likely due to the capacity of glucose to pleiotropically inhibit or downregulate mitochondrial function [59,116]. Gdh2 levels appear to be regulated by pH as the protein levels decrease as the pH of the growth medium approaches neutrality, which is consistent with the observed dependency of alkalization on the starting cell density [61]. In addition to amino acids, growth on N-acetylglucosamine (GlcNac) can also raise extracellular pH via ammonia extrusion. However, the origin of alkalinizing ammonia is distinct as it is catalyzed by the enzyme glucosamine-6-phosphate isomerase (Nag1), which deaminates glucosamine-5-phosphate (GN5P), converting it to fructose 6-phosphate (F6P) [123].

Regarding the fate of intracellular ammonia, in aqueous solution, ammonia can exist either as a gas (NH3, ammonia) or as a cationic (NH4 +, ammonium) species; the ratio (ammonia/ammonium) increases with pH (pKa = 9.25) (Figure 6B). Since the pH of the cytosol

in actively growing *C. albicans* wildtype cells is maintained at ~6.5 [124], the protonated form NH4 <sup>+</sup> predominates and can be directly assimilated by the NADPH-dependent glutamate dehydrogenase (Gdh3) or glutamine synthetase (Gln1). Due to its being positively charged, ammonium cannot readily diffuse out of cells, but rather requires transporters or channels to traverse the phospholipid bilayer of biomembranes. A small fraction of the total ammonia species exists in the unprotonated form (NH3) that can be released into the extracellular space, where it could exert its neutralizing effect by reacting to the hydrogen ions (H+), generating ammonium (NH4 +). How ammonia traverses the plasma membrane from the cytosol is unclear, as proposed earlier, because ammonia extrusion requires the ammonia transport outward (Ato) proteins, a family of plasma-membrane-bound proteins thought to facilitate ammonia export [32,125]. Strains lacking *ATO5* (*ato5*Δ/Δ) or a dominant point mutation in *ATO1* (*ATO1G53D*) show strong alkalization defects, and consistently, the overexpression of *ATO* genes accelerates alkalization [32,125]. However, it is also known that ammonia (NH3) is membrane-permeable and can easily diffuse out of yeast cells [126–128]. Whether ammonia (or even ammonium) is exported through simple diffusion or via exporters (Ato) remains to be clarified.

In addition to ammonia extrusion, yeast cells have an alternative mechanism to minimize the toxic effects of ammonia. *S. cerevisiae* can indirectly limit the production of ammonia by excreting cytosolic amino acids such as glutamate to the extracellular space via proteins that belong to the multidrug resistance transporter family that are thought to function as H<sup>+</sup> antiporters (e.g., Aqr1) [129]. Whether the same amino acid extrusion process, limiting intracellular ammonia production, operate in *C. albicans* is not yet known but a putative *AQR1* homolog has been identified in the *C. albicans* genome (*QDR2*/C3\_05570W). Qdr2 may perform the same function, constituting a rudimentary ammonia detoxification mechanism in *C. albicans*.

### **9. Conclusions and Outlook**

*C. albicans* is an opportunistic fungal pathogen that is intimately linked to its human hosts. Since *C. albicans* grows in symbiosis with humans, fungal cells must survive and propagate under identical physiological conditions as human cells. The capacity of *C. albicans* to establish persistent infections relies heavily on their capacity to assimilate nutrients in a competitive landscape where both hosts cells and even other members of the microbiome compete for nutrients. Amino acids are among the most versatile nutrients available in the hosts; they can be assimilated as both nitrogen and carbon precursors, transformed to key metabolic intermediates or utilized to modulate extracellular pH via ammonia formation. Although *S. cerevisiae* paved the way for most of our understanding of nutrient assimilation and metabolic processes in yeasts, there are clearly significant differences that exist in *C. albicans* that must be taken into account as they are crucial to our understanding of how this fungal pathogen assimilate nutrients in the host, especially in the context of infectious growth.

Some of the so-called poor or non-preferred nitrogen sources in *S. cerevisiae*, such as proline, are efficiently utilized by *C. albicans*. This observation is in alignment with recent findings that the enzymes required to utilize proline in *C. albicans* are independent of NCR, allowing the unrestricted utilization of proline regardless of whether other nitrogen sources are available [59,60]. Proline constitutes some of the most abundant proteins in humans (e.g., collagen, mucin); thus, given that *C. albicans* possesses a multi-subunit respiratory complex I (NADH dehydrogenase), similar to human cells, it is not surprising if *C. albicans* evolved to prefer this amino acid as an energy source for growth. Interestingly, proline has long been known as one of the most potent inducers of yeast-to-hyphal transitions, a key virulence factor in *C. albicans* [59,130–133]. We have shown that the induction of morphogenesis occurs via ATP-dependent Ras1 activation [59]. One molecule of proline can be completely oxidized to generate approximately 30 ATP equivalents [134,135], reinforcing the idea that proline is an important energy source for many types of cells, especially under nutrient-limited conditions. We have shown that *C. albicans* growth in

the phagosome of the macrophage is dependent on proline catabolism to obtain energy to survive despite a multitude of environmental stresses [59]. The inadvertent replication of our previous data [59] in the corrected paper [61] highlights the idea that proline is sensed by *C. albicans* in the phagosome of macrophages. Our data are also consistent with recent transcriptomic data showing that proline induced the expression of *ICL1*, a gene encoding the key glyoxylate cycle enzyme isocitrate lyase 1 (Icl1), which is known to be derepressed in *C. albicans*, being engulfed by macrophages [60]. Consequently, strains lacking the capacity to utilize proline have defects in escaping the phagosome of macrophages [59]. In terms of environmental alkalization, proline catabolism plays a major role by virtue of glutamate production (Put2 product). In the presence of arginine as the sole nitrogen and carbon source, proline catabolism is essential as it is the primary catabolic route to generate glutamate, which can then be subsequently catabolized by Gdh2 to ammonia and α-KG, a key TCA cycle intermediate. However, in the presence of other amino acids, the proline catabolic pathway becomes less essential for alkalization as other amino acids can be transaminated to generate glutamate (Figure 5). Proline utilization in *C. albicans* provides a clear example of how evolution influences and fine-tunes metabolism, leading to unique capabilities, in this case to the utilization of nutrients in a manner not relevant for other related yeasts. Consequently, a thorough examination of other amino acid catabolic pathways in *C. albicans* is warranted, the premise being that many important regulatory differences may exist, and that these may be specifically linked to the evolution of *C. albicans* within mammalian hosts.

A major challenge to correctly interpret experimental results derived from studies examining nutrient sensing and assimilation in *C. albicans* is understanding how laboratory growth conditions influence the results. Many of the standard laboratory conditions do not reflect the mammalian micro-niches in which *C. albicans* resides. For example, many host–pathogen interaction experiments involving innate immune cells are carried out in cell culture medium (RPMI or DMEM) containing 5–10% fetal bovine serum. These media readily trigger filamentous growth in *C. albicans*, independently of host cell interactions (e.g., with macrophages), resulting in the false impression that certain genes are not important for the survival of *C. albicans* during co-culture with innate immune cells. This is especially crucial when looking at the role of specific genes that are required for nitrogen acquisition. For example, there is a possibility that the importance of certain genes under NCR control will be erroneously dismissed as they are not expressed under nitrogen-replete conditions such as those in cell culture media. In addition, it is common practice to use strains pregrown in YPD, a complex medium that is high in glucose (2%) and rich in nitrogen (amino acids, peptides), prior to shifting cells to desired experimental test conditions. In humans, the level of blood glucose is maintained within homeostatic limits (0.05–0.1%; 3–5 mM glucose) that are well below the level used in YPD [116]. The dramatic reorientation of metabolism resulting from merely shifting conditions is likely to influence the response, and in many instances may provide a conflicting readout. For example, yeast cells grown in YPD build up an extensive reservoir of amino acids with vacuolar pools during growth in nitrogen-rich conditions [136,137]. This influences nutrient-based signals. Furthermore, many studies have relied on fixed-point microscopy coupled with differential staining to observe and deduce the role of specific mutations on filamentous growth. This approach relies heavily on observing obvious growth defects that may not be readily apparent on strains lacking genes relevant to nutrient acquisition. Although useful information has been obtained, many of these results often reflect "general" rather than "niche-specific" hyphal defects, highlighting the need to identify more suitable laboratory conditions that better mimic mammalian microenvironments.

Although a great deal of information regarding nutrient-induced processes in *C. albicans* is accumulating, there are major gaps in our knowledge with respect to the contribution of the host. The availability of assimilable nitrogen sources, i.e., the abundance of amino acids released as a result of host activities, is often overlooked. The contribution of host-derived activities to the degradation of the extracellular matrix (ECM) during stress due to the

proteolytic activities of proteases secreted by different cell types is not fully understood. For example, in people of advanced age, who due to medical advances represent a growing population, often suffer from sarcopenia or muscle wasting. A hallmark of sarcopenia is that the amino acid proline is elevated in the blood, indicating the degradation of structural proteins rich in proline such as collagen [84]. Indeed, the elevation of free amino acids in the blood is linked to other pathological states in humans, including cancer [83,84]. It is likely that amino acid limitation influences the capacity of cancer cells to establish malignant forms of growth; cancer cells have been found to exhibit enhanced rates of amino acid uptake [138]. Furthermore, amino acid metabolism is an important factor during wasting in cancer patients (cachexia) and in aging individuals [139,140]. Clearly, illuminating the entire repertoire of regulatory mechanisms associated with amino acid signaling is crucial to understanding life processes in both healthy and disease states, and studies in *C. albicans* may provide important insights with clear therapeutic applications.

**Author Contributions:** Conceptualization, F.G.S.S. and P.O.L.; writing—original draft preparation, F.G.S.S.; writing—review and editing, F.G.S.S. and P.O.L.; visualization, F.G.S.S.; funding acquisition, P.O.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** Original research in our laboratory is supported by the Swedish Research Council (P.O.L.) VR-M 2019-01547.

**Acknowledgments:** We would like to thank the members of the Ljungdahl laboratory for their patience and constructive comments throughout the course of this work. Many colleagues have contributed with strains and important insights and are collectively acknowledged. Furthermore, the vastness of the subject matter and space limitations have precluded the referencing of all relevant papers, and undoubtedly we have failed to cite some papers of equal or greater value than the ones cited; we apologize for the inadvertent omission of uncited work.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

