**4. The Role of Metabolism during Host-***Candida* **Interactions**

Interactions of *C. albicans* with innate immune and epithelial cells have been extensively studied in the past [101,102]. Great strides have been made in our understanding of how host cells recognize this fungus via PRRs, although the relevance of each receptor varies depending on the infection context, either being systemic [103] or at the mucosal barriers [104]. Phagocytosis by immune cells [105], or attachment to and invasion of epithelia [29], are the next steps in the infectious process and are essential to promote immune responses in these cell types. Secretion of the peptide toxin candidalysin contributes to cell damage and activation, especially in the case of epithelial cells [106,107].

However, there are still a lot of gaps in our knowledge of how all these responses are regulated. As explained above, host cells undergo metabolic reprogramming upon interacting with microbes or microbial components, modulating how they respond to infections and competing over nutrients. In this section, we will discuss the current knowledge regarding the role of (immuno)metabolism during fungal interactions with epithelial and innate immune cells (Figure 3).

### *4.1. Impact of Metabolism during C. albicans Interactions with Immune Cells*

Following their first contact with *C. albicans* (i.e., recognition, phagocytosis, etc.), activated immune cells reprogram their metabolism to mount an effective response. Transcripto mics-based analysis of peripheral blood mononuclear cells (PBMCs) stimulated with *C. albicans* shows a consistent upregulation of glycolysis, whilst no change (TCA cycle) or even downregulation (PPP) was observed for other pathways [108]. Specific stimulation of monocytes by heat-killed yeast or hyphae drives upregulation of several glycolytic enzymes, along with increased lactate production and glucose consumption, suggesting a shift towards aerobic glycolysis. Like β-glucan-stimulated cells [14], heat-killed cells promoted both higher ECAR (extracellular acidification rate) and OCR (oxygen consumption rate) levels, showing that OxPhos is also upregulated. This increased glycolysis plays a key role in immune responses as inhibiting glycolysis (2-DG and dichloroacetate, DCA) and mTOR pathway signalling (Torin1) significantly downregulates cytokine production post-fungal challenge [108].

The induced shift in metabolic pathways of infected monocytes with *C. albicans* differs between yeast and hyphal stimulation and is mediated by C-type lectins (CLR) but not by Toll-like receptors (TLR), showing the heterogenicity of host receptors in fungal recognition and responses. The responses generated to different *Candida* morphotypes is also varied. Monocytes infected with yeast cells activate glycolysis, oxidative phosphorylation, and glutaminolysis, whilst those infected with hyphae activate only glycolysis. Thus, we can see that the mechanisms of glucose metabolism are central players in regulating anti-*C. albicans* immunity and cytokine production [108]. Similarly, *A. fumigatus* induces an increase in aerobic glycolysis that is involved in macrophage responses to this filamentous fungus [109]. Of note, induction of metabolic reprogramming is mediated by the phagosomal removal of *A. fumigatus* melanin and its detection by the recently discovered melanin receptor MelLec [110]. This recognition is involved in HIF-1α mobilisation and subsequent cytokine release [109].

**Figure 3.** Interplay between *Candida* and host cells lead to metabolic reprogramming in both organisms. (**A**) Recognition of *C. albicans* by macrophages/monocytes drive changes in metabolism towards aerobic glycolysis, leading to the production of lactate that can be used by the fungus to enhance its β-glucan masking to evade immune responses. In turn, phagocytosed fungal cells use their metabolic plasticity to adapt to the nutrient-poor environment inside phagolysosomes. After piercing cell membranes using hyphae, *C. albicans* switches to glycolysis and depletes glucose from the medium, leading to macrophage cell death. (**B**) Oral epithelial cells activate HIF-1α when challenged with *C. parapsilosis*, whilst *C. albicans* adapts its metabolism upon interaction with these cells. (**C**) Variable responses are observed in different *Candida* species after interacting with vaginal epithelial cells. However, host cells exert a common early response to all of them mediated by changes in mitochondrial activity and morphology, with higher release of mitochondrial reactive oxygen species (mtROS) and DNA (mtDNA). Created with BioRender.com (last accessed 12 January 2022).

The main interface of host-fungal interaction is the fungal cell wall, a highly flexible structure with ability to remodel itself in the presence of a variety of environmental pressures, such as antifungal drugs [111]. Immune cells activated by *C. albicans* infection generate metabolites that can be sensed by fungi, which then remodel their cell wall in response to improve their immune evasion/protection. The major component of fungal cell walls, β-glucan, is a major fungal PAMP involved in the activation of many of the host antifungal responses [112]. The increased lactate levels associated with the shift to aerobic glycolysis may lead to β-glucan masking, preventing recognition of this key PAMP [113,114]. This phenotype (observed in multiple pathogenic *Candida* species) is only activated by appropriate lactate concentrations, and not by other metabolites such as proline, acetate, and methionine. This phenomenon is facilitated by the activation of the Crz1 transcription factor by the G protein-coupled receptor, Gpr1. Crz1 modulates the expression of genes involved in lactate-induced β-glucan masking. The outcome of this masking is significantly reduced visibility of *Candida* cells in terms of immune responses and thus diminished levels of tumour necrosis factor-alpha (TNFα) release and neutrophils [113]. Thus, *Candida* can successfully escape from macrophage uptake by taking advantage of the carbon sources released during metabolic rewiring of host cells in response to infection.

Upon *C. albicans* infection, macrophages are recruited to the site of infection and engulf fungal cells to try to destroy them or inhibit their growth in the phagolysosome through oxidative and nitrosative mechanisms [115]. *C. albicans*, however, has developed mechanisms to survive inside macrophages through metabolism manipulation. Two successive reprograming events of macrophages in response to *Candida* have been identified as follows, using whole-genome arrays: the early and late responses [116]. Their transcriptional profiles show the enhancement of gluconeogenesis and the glyoxylate cycle by upregulation of all genes involved in the conversion of fatty acid to glucose and a massive down-regulation of translation-related genes during the early response, suggesting a switch from glycolysis to gluconeogenesis, the glyoxylate cycle, and FAO during this early response. In addition, *Candida* cells phagocytosed by either macrophages [116] or neutrophils [117] upregulate arginine biosynthetic genes in response to ROS, rather than nutrient starvation, with these genes being important for germ tube and hyphal formation [118]. In contrast, the late response includes the reactivation of protein translation machinery and glycolysis. The metabolic shift in *C. albicans* cells following interaction with macrophages is assumed to be driven by the poor nutrient availability inside macrophage phagolysosomes, in which glucose concentration for instance is extremely low. This metabolic remodelling is dependent on the pathogenicity of *C. albicans* and *C. glabrata* since the non-pathogenic fungus *Saccharomyces cerevisiae* fails to demonstrate this response [76,116,119–121]. Moreover, similar events occur in *C. albicans* when it is exposed to neutrophils or whole human blood [117,120,122].

As stated earlier, during infection, activated macrophages shift their metabolism to aerobic glycolysis to activate antimicrobial inflammation and host defences. This means that for their survival *Candida*-activated macrophages rely specifically on glucose as their carbon source and additionally cannot reactivate mitochondrial oxidative phosphorylation. At the same time, ingested *C. albicans* cells similarly switch to aerobic glycolysis in the later phase of infection. As a result, macrophages and their ingested *C. albicans* compete for the available glucose [123]. During this nutrient war, the combatants rapidly consume the local glucose, leading to glucose depletion and triggering the "starvation" death of macrophages. Unlike macrophages, *C. albicans* cells have enough metabolic plasticity to switch their carbon source to alternatives such as the glyoxylate pathway, and in doing so survive the loss of glucose. As described earlier, these events are regulated by Tye7 and Gal4 *C. albicans* transcription factors. In *tye7*Δ*/*Δ*gal4*Δ*/*Δ mutant strains, glycolysis and glucose consumption occurs at a far lower rate and, therefore, induction of macrophage starvation and cell death is lower. Using metformin to shut down the mitochondrial respiratory chain and drive faster glucose consumption ramps up the rate of death of activated macrophages by *C. albicans* with the knock-on effect of increasing mortality. In contrast, boosting local glucose levels by continuous administration of glucose improved these outcomes [123].

Glucose depletion not only leads to the rapid death of activated macrophages but also causes inflammasome activation by activating NLRP3, due to increased fungal burden [124]. NLRP3 has a protective role during infection, being a PRR that triggers processing and secretion of IL-1α. Therefore, the regulation of NLRP3 is crucial during *C. albicans* infection [125]. In a recent study, the mechanism behind NLRP3 activation during infection of macrophages was investigated, showing that inflammasome activation was broadly uniform among multiple clinical isolates of *C. albicans*, and rather than being dependent on hyphal formation, was purely down to glucose competition. Notably, reducing fungal ability to consume glucose (by using the *tye7*Δ*/*Δ*gal4*Δ*/*Δ mutant strains) or increasing the glucose levels both reduce NLRP3 activation and IL-1β production [124].

It was believed for a long time that hyphae are essential for the pathogenicity of *C. albicans* during infections. This hypothesis, however, was challenged with the discovery that metabolic adaptation during systemic infections can be as important as morphological plasticity [126]. In a murine model of systemic candidiasis using the yeast-locked *eed1*Δ*/*Δ mutant, virulence was retained, leading to rapid yeast proliferation, and higher fungal loads in organs such as the kidneys or liver. Phenotypic analyses of the mutant strain showed enhanced growth rates in physiologically relevant carbon sources, including lactate, acetate, and citrate. A few genes involved in carboxylic acid and citrate metabolism were upregulated, alongside with *GAT1* that promotes proliferation in casamino acid rich environments. Therefore, the metabolic flexibility of *C. albicans* yeast-locked *eed1*Δ*/*Δ mutant in using alternative carbon sources (such as fatty acids, carboxylic and amino acids) at lower concentrations or the absence of glucose enhances its colonization ability and pathogenicity. Hence, metabolic adaptation and fitness of *C. albicans* during infection not only supress the activity and recognition of immune cells, but also enhance the pathogenicity and mortality in systemic infection independently of hyphal formation.

### *4.2. Role of Metabolism during C. albicans Interactions with Epithelial Cells*

While metabolic changes in immune cells have been the subject of recent studies, our current knowledge of these changes in epithelial cells (ECs) following microbial infection is limited. ECs are not merely passive barriers to prevent the invasion of microbes at the body's exterior surfaces but are also important in maintaining the balance with resident microbial communities. There are few studies showing metabolic reprogramming in ECs during microbial infection, namely increased glycolytic activity during *Staphylococcus aureus* infection of skin keratinocytes [35].

Concerning ECs interactions with fungi, oral epithelial cells (OECs) have been found to upregulate metabolic reprogramming-related genes in response to fungal infections, including HIF1-α pathway during *C. albicans* oropharyngeal candidiasis in mice [127] or *C. parapsilosis* infection of human OECs [128]. Similar to what is observed in phagocytosed cells, *C. albicans* upregulates gluconeogenesis, the glyoxylate pathway and FAO in the late phase of interaction with OECs, which might be related to the invasion process [129]. However, further analyses should be performed to unravel the mechanisms underlying these metabolic shifts.

Additionally, vaginal EC responses to varied species of *Candida* (*C. albicans*; *C. glabrata*; *C. parapsilosis*; and *C. tropicalis*) have also been studied using dual RNA sequencing in a time course infection model for vaginal ECs, analysing both fungal and host transcriptomic profiles [130]. In this study, Pekmezovic and co-workers showed a biphasic response to *Candida* spp. in vaginal ECs. The initial response is highly uniform among *Candida* species and characterised by mitochondrial-associated type 1 interferon (IFN) signalling. Of note, most mitochondrial genes were upregulated in the early phase of *Candida* infection, and the morphology of mitochondria changed in response to the infection. Moreover, mitochondrial DNA (mtDNA) and ROS are released into the vaginal ECs cytoplasm in all *Candida* species, both acting as damage-associated molecular patterns (DAMPs). In terms of fungal transcriptome, at 3 h post-infection *C. albicans* and *C. glabrata* upregulated carbohydrate catabolic processes and stress response pathways, whilst *C. parapsilosis* upregulated, among others, genes related to amino acid metabolism, iron transport, ribosome assembly and translation. In contrast, *C. tropicalis* differentially expressed genes were mainly related to RNA processing, ribosome biogenesis and ergosterol biosynthetic processes. Unlike the early responses, the late damage-associated epithelial transcriptional response is morphology-dependent, with the hyphal-associated toxin candidalysin enhancing the host responses [130].

### **5. Conclusions and Future Perspectives**

Nutritional environment and metabolic adaptations in both host and fungal cells are key during their interactions. Further characterising and understanding host immunometabolic responses to *Candida* infections will potentially help developing novel therapeutic strategies to modulate these responses. In addition, identifying which metabolic enzymes are essential during the activation of anti-*Candida* immunity will lead to the detection of genetic variants associated with higher susceptibility in individuals suffering from recurrent or chronic fungal infections. Likewise, modulating nutrients in the infection environment could help enhance host responses and/or hamper fungal growth. Therefore, further research on these promising fields must be carried out to expand our knowledge and design new strategies to tackle fungal infections.

**Author Contributions:** Writing—original draft preparation, A.P., N.B., S.D.S.N., A.H.; writing review and editing, A.P., S.S., D.L.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This review was supported by the Engineering and Physical Sciences Research Council (EPSRC) EP/S001301/1 and BBSRC grant -BB/S016899/1. D.L.M. and S.S. are further supported by Unilever and Sanofi.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

### **References**

