*Review* **Amino Acid Sensing and Assimilation by the Fungal Pathogen** *Candida albicans* **in the Human Host**

**Fitz Gerald S. Silao and Per O. Ljungdahl \***

Department of Molecular Biosciences, Wenner-Gren Institute, SciLifeLab, Stockholm University, 114 19 Stockholm, Sweden; fitzgerald.silao@scilifelab.se

**\*** Correspondence: per.ljungdahl@scilifelab.se

**Abstract:** Nutrient uptake is essential for cellular life and the capacity to perceive extracellular nutrients is critical for coordinating their uptake and metabolism. Commensal fungal pathogens, e.g., *Candida albicans*, have evolved in close association with human hosts and are well-adapted to using diverse nutrients found in discrete host niches. Human cells that cannot synthesize all amino acids require the uptake of the "essential amino acids" to remain viable. Consistently, high levels of amino acids circulate in the blood. Host proteins are rich sources of amino acids but their use depends on proteases to cleave them into smaller peptides and free amino acids. *C. albicans* responds to extracellular amino acids by pleiotropically enhancing their uptake and derive energy from their catabolism to power opportunistic virulent growth. Studies using *Saccharomyces cerevisiae* have established paradigms to understand metabolic processes in *C. albicans*; however, fundamental differences exist. The advent of CRISPR/Cas9-based methods facilitate genetic analysis in *C. albicans*, and state-of-the-art molecular biological techniques are being applied to directly examine growth requirements in vivo and in situ in infected hosts. The combination of divergent approaches can illuminate the biological roles of individual cellular components. Here we discuss recent findings regarding nutrient sensing with a focus on amino acid uptake and metabolism, processes that underlie the virulence of *C. albicans*.

**Citation:** Silao, F.G.S.; Ljungdahl, P.O. Amino Acid Sensing and Assimilation by the Fungal Pathogen *Candida albicans* in the Human Host. *Pathogens* **2022**, *11*, 5. https:// doi.org/10.3390/pathogens11010005

Academic Editor: Jonathan Richardson

Received: 17 November 2021 Accepted: 19 December 2021 Published: 22 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Keywords:** *Candida albicans*; human fungal pathogen; nutrient sensing; amino acid metabolism; proline catabolism; mitochondria; SPS-sensor; nitrogen catabolite repression; glucose repression

### **1. Introduction**

All organisms require nutrients to live, grow and successfully reproduce. The ability of an organism to assimilate nutrients in a given ecological niche is dependent on its ability to sense and respond to the availability of nutrients and on intrinsic cellular properties. Defining the key signaling events activated by nutrient sensing systems and the metabolic capacities of an organism provides a compelling description that reflects the organism's role in the niche. For opportunistic human pathogens, acquiring nutrients to support commensal or pathogenic growth is not a trivial task as the availability of key nutrients is dependent on several extrinsic host-specific factors. Such factors include host defense activities that often are linked to substantial and rapid changes in biophysical parameters, e.g., extracellular pH and the generation of reactive oxygen species (ROS), different nutrient and metabolic activities of host tissues at sites of fungal cell colonization, and the presence of competing microorganisms.

Of the fungal pathogens capable of infecting humans, *Candida albicans* is considered to be the most important, and arguably the most successful. *C. albicans* is a natural commensal of humans, capable of colonizing virtually all anatomical sites (Figure 1). This fungus can switch from harmless commensal to pathogenic growth and thereby cause a spectrum of pathologies, ranging from mucosal to life-threatening systemic infections, collectively termed candidiasis. It is imperative to distinguish the difference between commensal and invasive virulent growth; invasion is distinct from superficial colonization as the former is accompanied by inflammatory signals, resulting from the activated immune response. Clinical cases presenting *C. albicans* infections of the urogenital tract [1–8], kidney [1,7,9–13], liver [14,15], lungs [16–18], spleen [19,20] and even the heart [21–26] have been reported. In rare cases, mostly in neonates, *C. albicans* can traverse the blood– brain barrier, resulting in infections of the brain [27–30]. These observations indicate that *C. albicans* can successfully establish and grow in different host niches. Consequently, *C. albicans* must possess the means to successfully adapt in order to obtain and use a wide range of host-derived nutrients. Given their opportunistic character, the question remains open as to how *C. albicans* cells fine-tune their nutrient acquisition machineries to support commensal and pathogenic growth under apparently disparate environmental conditions.

**Figure 1.** *C. albicans* **can infect virtually all anatomical sites in the body.** Infections can either be superficial, mainly affecting the skin or mucous membrane, or invasive, involving fungal entry into the blood (candidemia) and dissemination to other internal organs. Disseminated growth happens when *C. albicans*, colonizing an anatomical site (usually the gut), catheters or other medical implants, enter the blood and then disseminate to other organs such as the lungs (pulmonary), liver (hepatic), spleen (splenic), pancreas (pancreatic) and kidney (renal). These infections can be localized or can re-enter the bloodstream again, allowing them to reach additional anatomical sites, and in rare cases the brain (cerebral). *C. albicans* in the blood can enter the urine via the kidney, resulting in candiduria (yeast in the urine). Other complications of *Candida* infections include the appearance of fungus balls in certain sites, resulting in obstruction in these areas and the formation of abscesses. In women, vaginal candidiasis is an important concern since the vagina serves as a main reservoir for *C. albicans*. In addition, infection of the genitourinary tract is more often diagnosed in women than in men due to anatomical structural differences such as a shorter urethra and the close proximity of the vagina and anus.

*C. albicans* requires a source of nitrogen to synthesize proteins needed to carry out necessary cellular functions and to generate nucleotides for DNA and RNA synthesis. There is a plethora of nitrogen sources that *C. albicans* can theoretically utilize in the host, for example, amino acids, urea, peptides, proteins and N-acetyl glucosamine (GlcNAc) and even ammonia. Of the nitrogen sources available in the host, amino acids are preferred as they can be easily assimilated and used as both nitrogen and carbon sources [31,32]. Most of the current understanding of nutrient assimilation in *C. albicans* is inferred from extensive studies in the non-pathogenic yeast *Saccharomyces cerevisiae*. However, although many of the regulatory mechanisms operating between the two species are conserved, it is becoming clear that substantial differences exist, likely reflecting the different environments in which these fungi evolved. Although *S. cerevisiae* is readily found freely in nature, *C. albicans* has evolved in close association with mammalian hosts as a commensal. Furthermore, although *S. cerevisiae* prioritizes the ability to ferment sugar even in the presence of oxygen, *C. albicans* relies on mitochondrial oxidative phosphorylation to generate the energy necessary to survive in hosts. Aside from being able to thrive better at higher temperatures, i.e., 37 ◦C, a striking and important difference between *C. albicans* and *S. cerevisiae* is that *C. albicans* possesses mitochondria with all four proton-pumping and energy-conserving complexes of the respiratory chain, including NADH dehydrogenase (Complex I). In *S. cerevisiae* mitochondria, NADH is oxidized by matrix NADH dehydrogenases that are not coupled to energy-conserving proton-pumping mechanisms; hence, the oxidation of NADH in yeast does not directly contribute to ATP production [33].

The clear differences in metabolism between the established yeast model and the fungal pathogen *C. albicans* need to be considered when analyzing its virulence properties. In this review we focus on amino acid sensing and metabolism with an emphasis on proline catabolism. We begin by introducing some basic concepts regarding nitrogen source utilization and assimilation and then present a more focused discussion regarding amino acid metabolism, the generation of ammonia and associated consequences, and the central role of mitochondria in the production of energy for virulent growth.

### **2. Amino Acids as Nitrogen Sources in Host Environments**

As an opportunistic pathogen, *C. albicans* can sense a multitude of environmental signals, including changes in the availability of diverse nitrogen sources, including amino acids. The signaling pathways that are induced regulate the activity of downstream effector transcription factors that engage programs of gene expression, some that are required for virulent growth (reviewed in [34,35]). A limited number of amino acid sensors have been characterized in *C. albicans* (Figure 2), the best understood being the plasma membrane-localized SPS sensor of extracellular amino acids [36–38]. Strains lacking a functional SPS sensor have a diminished capacity to take up amino acids, do not filament in the presence of serum and are less virulent during systemic infection in mice. These results provide a clear example of how an ordinary basic physiological process, such as nitrogen (amino acid) acquisition, can become an "accidental" but important virulence trait of an opportunistic human pathogen. Additional sensors of external amino acids in *C. albicans* have been reported, such as Gpr1, a G-protein-coupled receptor proposed to sense methionine [39], and Gap2, a general amino acid transporter that is the functional ortholog of Gap1 in *S. cerevisiae* (ScGap1) that could function as a transceptor [40]. Proteins that sense the availability of other well-characterized nitrogen sources, e.g., Mep2 that responds to ammonium [41], can also provide important regulatory signals governing amino acid use. Although *C. albicans* cells have been shown to respond to the presence of GlcNAc [42,43] and urea [44], active sensing mechanisms for these nitrogen sources have not been described. Nitrogen-containing compounds (amino acids) are taken up from the extracellular environment through a number of distinct transporters localized at the plasma membrane (Figure 2). Activation of the SPS sensor enhances the capacity of cells to take up and assimilate diverse nitrogen substances. This is accomplished as the signals derived from the activated SPS sensor induce the expression of several genes encoding amino acid permeases, secreted aspartyl protease *SAP2*, peptide and oligopeptide transport proteins [36,45,46].

**Figure 2. Amino acid sensors and transporters localized at the plasma membrane of** *C. albicans*. (**Left**) the SPS sensor responds to the presence of extracellular amino acids. The main SPS sensor is composed of Ssy1, a plasma membrane-bound receptor homologous to amino acid permeases but without a capacity to transport amino acids; Ptr3, a scaffold protein that mediates intracomplex protein–protein interactions; and Ssy5, an endoprotease that proteolytically activates downstream transcription factors. The G-protein-coupled receptor 1 (Gpr1), together with intracellular cognate protein Gpa2, has been implicated in both amino acid (methionine) and lactate sensing. Gap2 is an ortholog of *S. cerevisiae* Gap1, which is thought to function as a transceptor, i.e., a functional transporter capable of initiating downstream signaling events independently of transport. (**Right**) uptake of extracellular amino acids is facilitated by a number of genetically distinct amino acid permeases (AAP) that have either broad or narrow substrate specificities. Amino acids can also enter the cell as oligopeptides taken up by Ptr2 for di-/tri-peptides, or a family of oligopeptide transporters (OPT) for oligopeptides comprising between 4 and 8 residues.

### **3. Nitrogen Catabolite Repression (NCR)**

In addition to sensing the availability of extracellular sources of nitrogen, yeast cells can gauge the quality of internalized sources of nitrogen and respond appropriately to adjust metabolism. Nitrogen catabolite repression (NCR) is a supra-pathway that controls nitrogen source utilization through the repression of genes required for the utilization of secondary nitrogen sources when preferred ones are available. Most of the assumptions with respect to NCR in *C. albicans* are derived from extensive studies examining NCR in *S. cerevisiae*. Understanding the differences between these organisms is essential to accurately describing the hierarchy of nitrogen source assimilation and use by *C. albicans* during pathogenic growth.

In *S. cerevisiae*, NCR is controlled by four GATA transcription factors: Gln3, Gat1, Dal80 and Gzf3, all of which possess zinc-finger DNA-binding motifs that recognize a conserved GATAAG consensus sequence present in the promoters of target genes (reviewed in [47]) (Figure 3). Gln3 and Gat1 act as positive regulators of gene expression, whereas Dal80 and Gzf3 act in a negative manner to repress target gene expression. The ability of the GATA factors to compete for binding GATAAG sequences is influenced by nitrogen source availability and is even modulated by events within the nucleus [48,49]. In the presence of preferred nitrogen sources, such as ammonium and certain amino acids, Gln3 and Gat1 are tethered in the cytosol, restricting their translocation into the nucleus. For Gln3, nuclear exclusion is maintained by binding to the phosphorylated species of its interacting partner, Ure2. Gln3 and likely Gat1 can be phosphorylated, but the phosphorylation status of Gln3 does not affect its capacity to bind Ure2. Gln3 targets to and is retained in the nucleus in cells carrying deletion or mutationally inactivated alleles of Ure2, resulting in the constitutive expression of NCR-sensitive genes. Unlike Gln3, Gat1 is not entirely dependent on Ure2 for retention in the cytosol, and therefore other regulatory components apparently contribute to controlling Gat1 movement and inducing activity. Contrary to Gln3 and Gat1, Dal80 and Gzf3 are constitutively localized in the nucleus. Furthermore, in contrast to *GLN3*, *GAT1*, *GZF3* and *DAL80* are expressed under the control of promoters containing multiple

GATAAG sequences, placing their expression under NCR [50–53]. Consequently, these factors participate in regulating each other's expression (cross-regulation) and in certain instances exhibit partial autogenous regulation [48,52–54] (Figure 3).

**Figure 3. Schematic diagram of nitrogen catabolite repression (NCR) in yeast** (adapted from Ljungdahl and Daignan-Fornier, 2012 [47]). Gln3 and Gat1 are two positive GATA effectors of NCR that are normally excluded from the nucleus under preferred nitrogen replete conditions. Nuclear exclusion is thought to occur via the interaction of Gln3 with the phosphorylated version of the Ure2 protein.

NCR has been described in *C. albicans*; however, the information available is limited to studies using strains lacking Gln3 and/or Gat1 [55–57]. Strains lacking either or both of these GATA transcription factors are unable to efficiently utilize a number of alternative nitrogen sources. This has been shown to be linked to the lack of derepression of genes necessary for their catabolism. Results from the Fonzi laboratory have shown that Gln3 and Gat1 appear to exert both specific and overlapping functions, depending on the available nitrogen sources [56]. Certain amino acids traditionally classified as poor, such as proline in *S. cerevisiae* [47,58], are readily utilized by *C. albicans* lacking Gln3 and Gat1, clearly indicating that proline utilization is not subject to tight NCR control [56]. Consistently, recent work in our group and others have shown that enzymes of the proline catabolic pathway (e.g., Put1 and Put2) can be induced in the presence of preferred nitrogen sources (e.g., ammonium or amino acids) [59–61] and even in a strain lacking Gln3 and Gat1 [59]. In addition, the gene encoding glutamate dehydrogenase (*GDH2*), a key player in central nitrogen metabolism, is robustly expressed when there is an abundance of preferred nitrogen sources such as ammonium and amino acids, indicating that its expression is independent of NCR [61]. This latter finding is in striking contrast to *S. cerevisiae*, with its *GDH2* subject to tight NCR control (reviewed in [47]).

These clear differences between *C. albicans* and *S. cerevisiae* are not trivial, and clearly reflect divergent evolutionary trajectories and the need for *C. albicans* to rapidly respond to distinct host environments. For example, as *C. albicans* cells breach epithelial barriers and reach the blood, they are exposed to high concentrations of amino acids, a condition that likely represses NCR-controlled genes, including those required for the assimilation of nitrogen derived from the degradation of host proteins. The transcription factor *STP1* is NCR-controlled and under these conditions is not expressed, which limits the expression of the Stp1-dependent secreted protease Sap2 and oligopeptide transporters.

### **4. Extracellular Amino Acid Sensing and Uptake—The SPS Sensing System**

The plasma-membrane-localized SPS (Ssy1-Ptr3-Ssy5) sensor of *C. albicans* has been characterized [36,38,59,62]. The SPS sensor enables cells to sense and respond to the presence of extracellular amino acids (Figure 4). Again, progress has largely been guided by ongoing studies using *S. cerevisiae* as a model. In *S. cerevisiae,* the SPS signaling pathway controls the expression of a distinct set of amino acid permease (AAP) genes encoding transporters catalyzing proton-driven amino acid uptake. Two homologous effector transcription factors, Stp1 and Stp2, are synthesized as inactive precursors that localize to the cytoplasm due to N-terminal regulatory domains. The regulatory domains possess cytoplasmic retention and nuclear degron motifs, both of which are required to maintain the "off-state" of SPS-sensor-regulated gene expression. The cytoplasmic retention motifs prevent these factors from efficiently entering the nucleus, and the degron motif targets the low levels of full-length Stp1 and Stp2 that escape cytoplasmic retention for degradation by means of a novel inner-nuclear-membrane-associated degradation (INMAD) pathway. The INMAD pathway is defined by the E3 ubiquitin ligase Asi complex (Asi1-Asi2-Asi3) [63–65]. Extracellular amino acids activate the SPS sensor by binding to the receptor component Ssy1, which undergoes a conformational change that activates the Ssy5 protease in a Ptr3 dependent manner: Ptr3 functions as a scaffold that mediates intracomplex protein–protein interactions. Activated Ssy5 cleaves the N-terminal regulatory domains of Stp1 and Stp2, a processing event that enables the cleaved factors, lacking cytoplasmic retention and degron motifs, to efficiently translocate to the nucleus and bind to upstream activating sequences (UASaa) in the promoters of AAP genes. AAPs are co-translationally inserted into the endoplasmic reticulum (ER) membrane, contiguous with the outer nuclear membrane. The movement of AAPs to the PM requires the ER membrane-localized chaperone Shr3, which facilitates their folding and packaging into ER-derived secretory vesicles, a requisite for their functional expression [66,67]. The SPS sensing system enables amino acids to induce their own uptake.

Orthologs of the SPS sensing system are present in *C. albicans* ([36–38,59,62]; reviewed in [35]) (Figure 4). There is, however, a major difference. In contrast to *S. cerevisiae*, Stp1 and Stp2 in *C. albicans* activate different sets of genes that express proteins facilitating the assimilation of distinct external nitrogen sources [36,62]. Stp1 regulates the expression of *SAP2*, encoding the major and broad-spectrum secreted aspartyl proteinase (Sap) and multiple oligopeptide transporters (Opts) [36]. *STP1* expression is subject to NCR and controlled by the GATA transcription factors Gln3 and Gat1 ([68], reviewed in [69]). Accordingly, *STP1* expression is repressed when preferred nitrogen sources, i.e., ammonium sulfate and amino acids, are available and is derepressed when these nitrogen sources become limiting or absent. *STP2* is constitutively expressed and functions analogously to Stp1/Stp2 in *S. cerevisiae* and derepresses the expression of multiple amino acid permeases. *C. albicans* strains lacking either Ssy1 or Csh3, the latter an ortholog of yeast Shr3, fail to efficiently respond to the presence of extracellular amino acids and have impaired an capacity to filament in amino acid-based media [37,38]. Not all amino acids activate the SPS sensor, as can be observed by monitoring the proteolytic processing of Stp2 [36,59]. The capacity to activate the sensor and induce Stp2 processing is limited to a subset of amino acids; the presence of glutamine and arginine leads to robust SPS sensor activation. Stp2 processing is observed 5 min post-induction, indicating that the SPS sensing system rapidly responds to the presence of extracellular amino acids.

**Figure 4. The SPS sensing system of** *C. albicans***.** Ssy1 is the primary amino acid sensor that functions with the scaffold protein Ptr3 and the protease Ssy5 as a multimeric receptor complex. Stp1 and Stp2 are the effector transcription factors of this pathway. ((**Left panel**), OFF state) In the absence of extracellular amino acids, Stp1 and Stp2 are produced as latent cytoplasmic precursors that are retained in the cytosol due to N-terminal regulatory domains that possess both a cytoplasmic retention motif and a nuclear degron, the latter recognized by the E3-ubiquitin ligase, Asi3. ((**Right panel**), ON state) In the presence of amino acids, Ssy1 is stabilized in a signaling conformation, which initiates downstream events, resulting in the activation of the Ssy5 protease. Activated Ssy5 endoproteolytically cleaves the N-terminal regulatory domains of Stp1 and Stp2. The shorter, cleaved forms efficiently translocate into the nucleus, where they bind upstream activating sequences (UASaa) and induce the expression of SPS-regulated genes (SRG). Importantly, Stp1 and Stp2 induce divergent subsets of genes, and *STP1* expression is under NCR control; *STP1* is repressed in cells grown in the presence of millimolar concentrations of amino acids, whereas Stp2 is constitutively expressed. Activated Stp2 induces the expression of amino acid permease (AAP) genes. AAPs are translated and initially inserted in the ER membrane, where they require the assistance of the ERmembrane-localized chaperone Csh3, the ortholog of yeast Shr3, to attain native structures. In the absence of Csh3, AAPs aggregate and are retained in the ER. Activated Stp1 derepresses the expression of *SAP2*, a secreted protease, and multiple oligopeptide transporter genes that facilitate peptide uptake. Stp1 triggers responses required for host protein utilization, whereas Stp2 induces amino acid utilization.

Additional sensing systems in *C. albicans*, capable of transmitting signals regarding extracellular amino acid availability, have been reported. The G-protein-coupled receptor (Gpr1) has been proposed to sense extracellular methionine [70] or even glucose, similarly to *S. cerevisiae* [71]. The idea that methionine is the primary activating ligand for Gpr1 stems from the fact that the addition of methionine could trigger the rapid internalization of Gpr1 in a manner consistent with ligand-mediated receptor internalization ([70]; reviewed in [72]). More recently, however, lactate has been proposed to be the primary activating ligand for Gpr1 [73]. Hence, the role of Gpr1 in amino-acid-induced morphogenesis remains to be defined. What is known is that ligand activation of Gpr1 stimulates GTP-GDP exchange in its effector Gα protein Gpa2; the active GTP-bound form of Gpa2 subsequently binds to the Gα-binding domain in the N-terminal region of the adenylate cyclase Cyr1, leading to enhanced cAMP production (reviewed in [74,75]). Null mutations of the *GPR1* or *GPA2* in *C. albicans* diminish filamentous growth on solid media, and consistently, filamentation can be restored via the addition of exogenous cAMP [71]. Interestingly, although Gpr1 and Gpa2 were initially characterized on the basis of increased cAMP synthesis in response to glucose, deletions of *GPR1* or *GPA2* do not affect glucose-induced cAMP signaling, and cells remain responsive to methionine and proline [39,70]. Furthermore, the levels of cAMP in *gpr1*-null mutants spike in response to serum or large amounts of glucose (100 mM, or 1.8%), suggesting that Cyr1 can be activated by Gpr1-independent processes [70].

### **5. Amino Acids from Proteins and Peptides**

Although free amino acids are preferred, as they can be rapidly utilized as both carbon and nitrogen sources, the bulk of amino acids in hosts are typically fixed in proteins, e.g., the extracellular matrix proteins collagen and mucin. Consequently, extracellular proteolytic enzymes are required to cleave host proteins to release amino acids and peptide fragments that can subsequently be taken up by the cell. It is important to note that the breakdown of host proteins that occurs at sites of infection can be due to proteases secreted either from the fungal or host cells [76,77]. In vitro, *C. albicans* can acquire peptides and amino acids derived from extracellular proteins, e.g., albumin, collagen and mucin. This requires the expression of secreted aspartyl proteases (Saps) [78–80] or the activity of matrix metalloproteinases (MMPs) [81,82]. The host can also trigger the proteolytic degradation of tissues as observed in some pathological conditions such as cancer [83] or sarcopenia (muscle wasting) [84]. Once internalized, peptides are then degraded to amino acids through the activity of several intracellular proteases, e.g., metallopeptidase, dipeptidase, carboxypetidases and serine proteases, which liberate free amino acids [85–88]. The induced expression of some of these enzymes is complex and depends on the release from strict regulatory processes, including NCR [68]. Although the overall effect on virulence remains to be clarified, the discovery of the fungal toxin candidalysin is important as it can also trigger the release of free amino acids by contributing to the lysis of host cells [89–91].
