Transcription Factors Control the Virulence-Associated Metabolic Enzymes and Membrane Proteins in Response to Nutrient Bioavailability in Cryptococcal Infection

1. Introduction

Cryptococcus neoformans is an opportunistic environmental yeast found within the shrub trees and humous soil contaminated with Aves dropping [1,2,3]. As an encapsulated and obligate aerobe, it becomes pathogenic and infects, invades, and colonises various animal and human organ systems [4]. Being an environmental saprophytic fungus, the internalisation of extracellular nutrients is necessary to survive. Following extracellular digestion, numerous membrane-associated proteins (permeases, transporters, and pumps) help with nutrient internalisation.

The assimilation of extracellularly digested nutrients and micro-nutrients is a complex metabolic system that requires enzyme sequestrations. In C. neoformans, membrane-bound proteins obtain ions such as Cu²⁺, Fe²⁺, and Zn²⁺ for membrane potentials. Pumps, such as Na⁺/K⁺-ATPase and P-type Na⁺-ATPase (Ena1), are used to maintain membrane polarisation, while ABC (Afr1) and ABD-type multidrug homologue pumps (Pdr5, Yor1, and Snq1) are deployed for drug resistance. Several transporters and permeases like Ca²⁺ transporters (Cch1, Eca1, and Vcx1), Na⁺/H⁺ antiporter (Nha1), aquaporin (Aqp1), phosphate affinity permeases (Pho) are implemented for cellular homeostasis, tolerance, resistance, antioxidation, and environmental stress response (oxidative and osmotic stress) [5,6].

One of the attributes of cryptococcal cells is the ability to form a biofilm, which is called cryptococcomas [7]. This is an essential phenotype of invasive pathogenic fungi and bacteria that involves a consortium of microorganisms adhering to one another in a polyfilm extracellular matrix of complex polymeric substances. Cells in this form come together for colonisation, extracellular matrix modelling, quorum sensing, resistance, protection, adhesion, survival, adaptation, communication, sharing, maturation, filamentation, and invasion [8,9,10,11,12,13]. Biofilm formation in C. neoformans is metabolically regulated, induced by unfavourable conditions and capsular components [14]. In vivo or in vitro, C. neoformans and C. gattii can form a biofilm, even on surfaces and invasive clinical instruments [14,15,16,17,18,19]. Redox process, anaerobic metabolism, substrate-level phosphorylation, proteolysis, and anti-osmotic processes are upregulated during biofilm formation, but general metabolic processes, replication, transcription, translation, transportation, permeases, and pump systems are generally repressed [20]. Further work showed that cryptococcal cells are more resistant to antifungal and more virulent in the biofilm state due to elevated expression of Cap59, Lac1, and Ure1 [15], Figure 1.

Environmental abiotic factors (like pH, temperature, light, and CO₂) and predatory biotic interaction with the soil amoeba are challenges to C. neoformans that require adequate cellular taxis. Amoeboid protozoans can “catch” and “kill” internalised cryptococcal cells in a similar way to mammalian macrophages that phagocytose invading pathogens [22]. Surprisingly, cryptococcal cells can be engulfed yet can survive, replicate, and become more virulent intracellularly within the soil amoeba, an unconducive condition that may kill amoeba [23,24]. Morphologically, the engulfed C. neoformans usually display a pleiotropic phenotype forming pseudohyphal or yeast and lacking capsule or melanin with a high-impact mutation in genes like Opt1, Pkr1, and Cpk2 [25,26]. In immunocompromised patients, the immune response against cryptococcal infection is weak and may lead to a life-threatening condition, meningoencephalitis.

Based on their immediate interaction, cryptococcal cells could be environmental or clinical. Both showed similar pathogenicity in animal models [27] because of the similarity in the virulence factors – capsule, melanin, extracellular secretory hydrolytic enzymes (like urease, phosphatase, DNase, lipase, protease, glycosidase), and membrane-associated enzymes (like laccase, IPC synthase, chitin synthase, catalase, and superoxide dismutase) [28]. Via the nutrient sensors like cAMP/Pka, Gpr1, Gpr4, Aap, Gpp2, and Hxt1, C. neoformans reprogram gene expressions to survive inadequate nutrient bioavailability and nutrient adaptation [29]. Nutrient shortages enhance membrane transporter and enzyme upregulations that ensure carbon intermediates shuttling from alternative pathways such as β-oxidation (mitochondria function) and glyoxylate cycle (peroxisome function) when phagocytosed by amoeba/macrophage. These two pathways fetch cryptococcal cells acetyl-CoA [30], which is converted via gluconeogenesis into various sugars needed for the cell wall components [31]. In addition, C. neoformans resorts to anabolic pathways to generate resourceful intermediates to synthesise melanin and capsule. This is observed with increased activity of fructose-2,6-bisphosphatase (encoded by Fbp1) when phagocytosed by amoeba and increased phosphoenolpyruvate carboxykinase activity (encoded by Pck1) when phagocytosed by macrophages [30,32].

Besides, several other upregulated transcription genes during phagocytosis have been reported. These include genes for nutrient absorption (amino acids, copper, and iron), antioxidative stress genes, and genes involved in autophagy, apoptosis, and mating [33]. In extreme cases, the redox process, substrate-level phosphorylation, and anaerobic decarboxylation can be employed to generate energy for cellular function and growth [20]; however, the lack of glucose or other utilisable sugars generally impairs cryptococcal proliferation, virulence, persistence, and tissue invasion as displayed by Δpyk1, Δpyk1Δmig1, Δmig1, Δpck1, Δhxk1, Δhxk2, and Δhxk1Δhxk2 mutants in the presence of glucose, glycerol, or lactate [34]. The Δacl1 mutants showed delayed growth in media with glucose with attenuated virulence and increased antifungal and macrophage susceptibility [35]. In addition, Δpck1 mutants failed to carry out gluconeogenesis and attenuated for virulence in the murine inhalation model of cryptococcosis; however, Δpyk1, Δhxk1, and Δhxk2 failed to utilise glucose and severely attenuated for virulence and CNS persistence [34].

Though similarities occur in the responses of C. neoformans to soil amoeba and mammalian macrophages based on their intracellular attack but the ecological niches surrounding the amoeba are quite different from the mammalian cells. Interaction of C. neoformans with the soil amoeba is usually at the ambient temperature (28 – 30^oC) in a slightly acidic environment, while with the macrophage is at the physiological temperature of 37^oC and a near-neutral pH range (7.2 – 7.4). By equilibrating the natural environment of Acanthamoeba castellanii and murine macrophage to C. neoformans, Derengowski et al. showed massive similarities in the modulation of several enzymes and membrane transporters involved in all significant metabolic systems with at least 2-fold increase as compared to the non-phagocytosed C. neoformans; though only a few numbers of these proteins were differentially modulated [30].

So, deploying several membrane transporters, permeases, and extracellular proteins for cellular nutrient balance and pathogenesis of C. neoformans is critical. We review several of these proteins and discuss their functional relevance to cryptococcal survival and adaptation in nutrient repletion or depletion and as weapons in human infections. Careful consideration of their consequences to mammalian cells is, however, needed if any of these proteins is considered a target for antifungal formulation against invasive cryptococcal infections in humans.

2. Environmental Nutrient Sensors Determine Metabolisms in Cryptococcus with Appropriate Release of Transcription Factors

The environmental transition of C. neoformans enables this fungus to cause infection in a susceptible host. During systemic infection, macrophage engulfs cryptococcal cells and restricts access to nutrients in the phagosomes. For these fungi to survive, alternative routes of nutrient acquisition are needed for adaptation, immune evasion, and virulence. A futile cycle is avoided in C. neoformans by switching on the essential and inevitable transcription factors, while some, considered redundant, are temporarily switched off. When phagocytosed, gene expression for membrane transporters, secretomes, cell wall formation, anti-oxidative and anti-nitrosative stress, anti-autophagy and peroxisome functions, intracellular metabolism, mating, and genomic repair are highly expressed. In contrast, the ribosomal metabolism and translational process are repressed even as the infection progresses [33]. Thus, C. neoformans possess a highly classical environmental sensor, robust enough to help decipher the micro-conditional changes and subsequently activate the fort of transcription factors for nutrient acquisition that translate into cellular morphology for infections and immune evasion.

Numerous environmental sensors are part of the membrane and cell wall integrity of C. neoformans. These sensors can associate directly or get involved in the signalling event induced by nutrient or environmental factors. With Rim21-Rim8 complex, C. neoformans responds to ΔpH; Cam1 senses the ΔpH and temperature; Tco1, Tco2, Ssk2, Ras1, phosphatidylinositol, Pdk1, and Rho-factors are various stress sensors; Can2 senses change in CO₂, and Gpr4 can be induced by methionine for Gpa1-Cac1 activation that produces cAMP/Pka [29].

2.1. Metabolism in C. neoformans is Influenced by cAMP/Pka

C. neoformans possess arrays of metabolic enzymes that re-wire carbon metabolites to favour the sugar-conjugate products and metabolic intermediates such as acetyl-CoA used for capsule and melanin biosynthesis. In addition, the in vitro and in vivo analysis showed numerous membrane transporters specific for each nutrient within their environment [36]. C. neoformans secrete hydrolytic enzymes such as peptidases, esterase lipases, and glycosidases to release amino acids, fatty acids, and sugars, respectively. The plethora of enzymes involved in these metabolic activities is influenced by cAMP signalling pathways. The cAMP-deficient mutants are poor in sensing glucose (via Hxtp – a sugar/hexose transporter and G-protein coupled receptor, like Gpr1p) and amino acids (via the Gpr4p) because these metabolites provide precursors needed for growth, mating, and virulence which are constantly under the regulation of cAMP/Pka signalling factors [37,38]. The Δgpr4 mutants are very poor in sensing certain amino acids, such as Met, which triggers receptor internalisation of Gpr4p and subsequent activation of the cAMP/Pka pathway [29]. However, Δgpr4 mutants showed a normal response to glucose, unlike a poor glucose response from Δhxt mutants. Despite a good response to glucose, the Δgpr4 mutants still displayed impaired capsule formation and mating defect, and both conditions are recoverable when cAMP is supplied exogenously [37].

The significant role of cAMP as a secondary messenger in metabolic regulation is invaluable, coupling environmental sensing to metabolism, virulence, cell differentiation, mating, and growth. Metabolisms generally begin with activating corresponding transporters, permeases, carriers, couplers, and pumps. This enables the endocytic movement of nutrients into the cell for metabolism. The transcriptome of Δpka1 and Δpkr1 mutants have been characterised by higher-level gene transcripts involved in different metabolic pathways [39] (Figure 2). Many of these pathways are activated via the cAMP/Pka pathways and regulated by Pkr1 expression.

C. neoformans is sensitive to glucose limitation, and phagolysosome does this by constantly maintaining acidic pH to minimise glucose availability to pathogens. To circumvent this, gluconeogenesis is promoted in C. neoformans to supply metabolic sugars needed for titanisation and capsule formation. The interdependence of peroxisome, glyoxysome and cytoplasm to break down assimilated fatty acids (β-oxidation) to generate acetyl-CoA is essential in driving the glyoxylate cycle that generates oxaloacetate used in gluconeogenesis.

Contrary to C. albicans, which showed up-regulated genes for key enzymes of gluconeogenesis [40], phagocytosed C. neoformans showed no upregulation of such enzymes [33]. However, transcriptional response and analysis of C. neoformans ingested by macrophages and amoeba showed that key enzymes like phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase were respectively upregulated [30]. The Icl1 (encoding isocitrate lyase), Mls1 (encoding malate synthase in glyoxylate cycle), and genes for β-oxidation enzymes are upregulated in C. neoformans recovered from murine macrophage [33], but Mdh2 (encoding malate dehydrogenase) and genes for ergosterol biosynthesis were downregulated in the phagocytosed C. neoformans [30]. The findings of Rude et al. showed that though the Icl1p and the glyoxylate shunt are essential for ATP production during fungal infection, still Δicl1 mutants failed to display any apparent virulent defect in animal studies and produced a wt level of capsule and melanin without any weakness in macrophage survival or phagocytic index [41].

2.2. Involvement of NADPH in Cryptococcus Metabolism

In most biological reactions, reducing equivalents, such as NADPH, are highly required for redox processes. Hypervirulence-associated protein 1 (encoded by Hva1) has been identified to be associated with NADPH to regulate C. neoformans metabolisms [42]. An exogenous supply of NADPH alone promotes an animal tissue burden of Δhva1 mutant in the spleen and brain than in the lungs [42]; Derengowski et al. discovered a multiple-fold increase in transcript level of Hva1 in amoeba-phagocytosed cryptococcal cells [30]. The absence of Hva1p unexpectedly increased the level of phosphoenolpyruvate kinase but impaired the TCA due to reduced activity of 2-ketoglutarate dehydrogenase complex activity. This condition may attenuate mitochondria oxidative reaction but favours sudden energy increased from preponderant cytoplasmic NADPH via alternative pathways to ATP production. This metabolic energy production shift favours cell proliferation and growth, hence the hypervirulence trait of Δhva1 mutants in the murine model but not in the moths or worms [42], making the Δhva1 virulence traits temperature dependent. Comparing Δhva1 to Δhva1+Hva1 reconstituted mutants, there was no significant difference in the animal, moth, and worm virulence models, as well as in capsule size and structure, melanin production, GXM content, phospholipase and urease activity, growth and doubling time under stressful conditions, survival and fungal burden in macrophage [42]. Therefore, Hva1p plays essential metabolic roles that may support tissue invasion; however, Hva1 is dispensable for cryptococcal virulence and infection.

2.3. Iron Regulators Induced Iron-Dependent Metabolic Enzymes

The exocyst complex vesicles (guided by Sec genes), enzymes (such as glutamate dehydrogenase, p-aminobutyrate aminotransferase, proteasomes, ubiquitin ligase, RNA-processing enzymes, and mRNA splicing proteins), and transporters/permeases (for allantoate, urea, ammonia, and purine-cytosine, involving in nitrogen and amino acid metabolism) have been observed to be elevated under the iron-enriched media [43]. Not only these, but iron also influences several of the amino acid metabolism (anabolism and catabolism).

C. neoformans is among the pathogenic fungi that utilise other carbon sources, including alcohol. Inexpediently, mutants lacking Hap3 and Hap5 showed weaker growth in sucrose, acetate, and ethanol and are more sensitive to 0.01% SDS [44]. In contrast, the ΔhapX mutant and the wt showed similar growth in the glucose, sucrose, acetate, and ethanol, even in 0.01% SDS [44]. Although Hap genes are iron-dependent regulatory genes, surprisingly, supplementing the media with hemin failed to improve the weak growth in hap mutants [44]. Because of this, iron-enrich media specifically improved gene expression encoding several iron-dependent enzymes in the glycolytic pathway, TCA cycle, functional oxidative pathways, and mitochondrial redox processes, but gene expression targeting the pentose phosphate pathway was scarcely tagged in the SAGE analysis [43]. Apart from these elevated key enzymes, other accessory proteins such as ubiquinone, prohibitin (a membrane-bound chaperone stabilising the mitochondria proteins), and ATPases are equally pre-eminent [43]. The expression of TCA-targeting enzymes such as aconitase and succinate dehydrogenase further confirmed the importance of the heme and Fe-S redox proteins to the upkeep of C. neoformans metabolic systems. Aconitase identified in C. neoformans appears as paralogous encoding genes that possibly enhance its extracurricular activities, such as mRNA stability and translation – functions known for iron regulatory proteins (Irp1). By speculation, C. neoformans might regulate excessive iron by converting this Irp1p to cytosolic aconitase, which fails to stabilise mRNA but promotes Irp2 ubiquitination and proteasomal degradation to avoid iron toxicity [45].

In S. cerevisiae, iron may orchestrate the binding of α-isopropylmalate to homodimeric DNA-binding protein Leu3 to regulate branched-chain amino acid biosynthesis, amino acid permeases (Aap), nitrogen, and carbohydrate metabolisms [46,47]. The expression of Lys4 for Lys biosynthesis and Leu1 for Leu biosynthesis is iron-dependent because of the presence of conserved aconitase repeating motifs that bind Fe-S [48], and possibly other amino acids biosynthesis proteins such as Met2p, Met3p, and Met6p for Met; Ilv2p for Ile and Val may have aconitase repeating motifs. In C. neoformans, mutants generated from these transcription factors are primarily auxotrophs, avirulent or attenuated for virulence in the murine infection model of cryptococcosis, and highly sensitive to antifungals [49,50,51]. This iron-coordinated virulence is centred on the interplay of Hap3 and HapX proteins.

Further into the regulatory roles of Hap3 and HapX, these two transcription factors negatively modulate the expression of the orthologue Lys4 (encoding homoaconitase that converts homocitrate to homoisocitrate in lysine biosynthesis) in the LIM. This was confirmed by low detection of Lys4 transcripts in ΔhapX and Δhap3 mutants cultured with 100 μM FeCl₃, but in the absence of FeCl₃, the expression of Lys4 is well induced in Δhap3 and ΔhapX mutants [52]. Relative to the wt, the expression of Lys4 remained unchanged in Δcir1 mutants in either iron enrich media (IEM) or LIM. This confirmed the critical importance of the Cryptococcus iron regulator factor (Cir1p) in the cellular regulation and distribution of iron.

Like Lys4, Leu1 (encoding 2-isopropylmalate synthase) expression was similarly regulated by Hap3 and HapX proteins in the LIM or IEM. Low iron supports the expression of Leu1 and Lys4 in Δhap3 and ΔhapX mutants, but only a high level of iron favours the expression of Leu1 in the Δcir1 mutant [49]. This means that Cir1p positively regulates this cytoplasmic located Leu1p in Δhap3 and ΔhapX mutants cultured in LIM but regulates negatively in IEM. There is speculation, however, that Leu1 expression may be more under the regulation of Hap3 than HapX expression because while ΔhapX mutant produced a comparable Leu1 transcript with the wt, Δhap3 mutant had a significantly more transcript of Leu1 under the same IEM conditions [49]. Like Lys4 expression, Leu1, with a putative ―CCAAT―HAP binding element at the promoter site for repression during the iron-limiting condition, is also very important in Leu biosynthesis; however, the uptake of these amino acids is generally under the influence of nitrogen catabolite repression (NCR).

3. Essential Enzymes of Metabolism and Membrane Transporters/Permeases are Controlled by Transcription Factors in cryptococcal cells for Virulence, Survival, Resistance, and Adaptation

3.1. Deleting Phosphoglucose Isomerase Impairs the Hog1 Pathway, Capsule Production, Membrane Integrity, and Alternative Pathways

To further appreciate the involvement of metabolic systems and their regulatory enzymes in the virulence, cell wall integrity, and stress-resistance phenotypic traits in C. neoformans, the lack of Pgi1 expression (encoding phosphoglucose isomerase/glucose-6-phosphate isomerase), though showed no observable difference in the cell morphology but resulted in reduced capsule biosynthesis, impaired cell wall integrity, fragile cell membrane, osmotic stress hypersensitivity (due to impaired Hog1 pathway), and failure to utilise mannose and fructose [53]. Insertional T-DNA mutation at the promoter site of Pgi1 reduces the activity of Pgip. In the presence of a 2% glucose supplement, the Δpgi1 mutant could produce melanin due to the derepression of Lac1. Further work showed that an exogenous supply of cAMP could restore capsule deficiency in the Δpgi1, but this repressed the Lac1 expression [53] (Figure 2). Even in the presence of glucose, mutation of Pgi1 expression may have limited glucose catabolism and subsequently altered the ATP homeostasis. Conversely, this condition favours Snf1p activation leading to the deactivation of Mig1p and subsequently derepresses Lac1 for melanin production, Figure 2. Therefore, the involvement of the Snf1-Mig1 regulatory pathway through nutrient homeostasis cannot be ruled out in the pathogenesis and virulence of C. neoformans; however, this is tightly regulated by the Pgi activity under a considerable basal glucose level.

3.2. Low Glucose Level Activates Serine/Threonine Protein Kinase 1 Complex to Induce genes with SRE-Promoter Sequence for Stress Response

The mammalian homologue of AMPK protein is Snf1p in the yeast, which is critical to cellular energy homeostasis through oxidative phosphorylation by facilitating sugar and fatty acid uptake. In a normal state, Snf1p (encoding serine/threonine protein kinase 1 complex) is turned off by Reg1-Glc7 type I protein phosphatase 1 (PP1) by dephosphorylation but activated by phosphorylation as the glucose level decreases [54,55,56,57,58]. Mutation in Reg1p or absence of Gpr1 sugar sensor caused increased activation of Snf1p with poor growth in the yeast; however, any alteration in the negative regulatory sub-units of cAMP/Pka (Ira1, Ira2, and Bcy1) will suppress Snf1 pathway activation [54], Figure 2.

High glucose level represses the Snf1 pathway in the yeast [54]. By speculation, the Snf1 pathway in C. neoformans may have been regulated similarly. Increased glucose level activates the Cac1→cAMP→Pka pathway to repress the C₂H₂ zinc-finger nuclear-translocated proteins, Msn2p and Msn4p, Figure 2. The Msn2 and Msn4 are programmable proteins that induce genes containing SRE-sequence promoters, such as Hxt1, Hog1, Ras1 and other genes belonging to NSR, OSR, OMSR, ESR, GSR, HMSR, or CSR (see Supplementary file for definition), which Snf1 activation promotes under low glucose level, Figure 2. In addition, the cAMP/Pka inhibits the redundant upstream Snf1p-activation kinases, Sak1, Tos3, and Elm1, in the yeast to put Snf1 under repression at a high glucose level [54].

Relevant to C. neoformans, the high-glucose-deactivating Snf1 via the Pka leads to the activation of Mig1p by dephosphorylation to enhance the association of Mig1p with Suc2p (Figure 2). The Mig1p is a universal gene repressor in the yeasts [59], which binds the promoters of target genes, including the Lac1 gene, to repress melanin biosynthesis as a response to glucose repression (Figure 2). In low glucose levels, however, the Cac1 activity is reduced to lower the cAMP level, which invariably elevates the Sak1 activity to activate Snf1p [60]. The activated Snf1p further inhibits the Cac1 to promote nuclear translocation of Msn2p to induce Hxt7 and other similar stress response genes, including the Lac1 gene, Figure 2. Unprecedentedly, the involvement of Snf1 expression in antifungal, oxidative, and osmotic stress responses is less significant. Though Hu et al. claimed to have observed increasing sensitivity of Δsnf1 mutants to amphotericin B relative to the wt response; however, the Δsnf1 mutants showed no significant growth defect to AmpB and fluconazole but a very slight growth defect to rapamycin [61].

3.3. Sugar/Hexose transporters (Hxt1 and Hxt7) Are Unexclusively Dependent on the Induction of Serine/Threonine Protein Kinase I Complex (Snf1)

Generally, phenotypic defects of Δsnf1 mutants become conspicuous with increasing temperature. For example, melanin formation decreases above 30^oC and becomes a non-melanised mutant at 37^oC [61]. Surprisingly, though the transcript level of Snf1 is practically increased in a glucose-limited or acetate medium, there is no evidence of increased transcript in the cryptococcal cells recovered from the infected lungs [61]. Besides, Δsnf1 mutants in a glucose-limited medium displayed an increased marginal level of Ady2, Acs1, Ato2, and Pck1 transcripts; a marginal decrease in the expression levels of Ctr4 and Cgl1; and a significant reduction in the Hxt1 and Cft1 transcripts [61]. So, the last two transcription factors depend solely on the Snf1 expression, especially in the glucose-limited medium (Figure 2). In the acetate medium, however, deletion of Snf1 showed no effect on the expression of all these genes except the Hxt1, where an elevated transcript is observed [61]. Additionally, the report showed that Δsnf1 mutants displayed a wt Mls1 transcript irrespective of the media used [61]. The activation of Hxt1p in Δsnf1 mutants cultivated in the acetate medium showed the characteristic of glucose-deprived cryptococcal cells and readiness to assimilate sugars as soon as it is available.

3.4. Low Glucose Level Promotes Melanin and Antioxidative Protein Expression via Serine/Threonine Protein Kinase 1 Complex

In addition to Lac1 regulation, Snf1 expression also affects the SOD activity. A drastic reduction of Lac1 in Δsnf1 mutants cultivated in the glucose-limited medium indicates the Lac1 expression being regulated by Snf1p [61]. Similarly, the Sod1 transcript is reduced in Δsnf1 mutants in a glucose-limited medium, even in the acetate medium [61]. Irrespective of the medium, deletion of Snf1 failed to affect the expression of Fhb1, Tps1, Glr1, and Skn7 significantly, but Lac2, Ssa1, and Tsa1 transcripts are increased considerably in this mutant when cultured in 2% acetate media [61]. This means that in the glucose-limiting medium, Snf1p orchestrates the expression of Lac1 (by preventing the Mig1-Suc2 association, Figure 2) and Lac2 (in alternative carbon source) to produce melanin and the expression of Sod1 against oxidative stress in the wt [61].

Strategically, Lac2 expression may be preferable for melanin synthesis under alternative carbon sources coupled with oxidative and nitrosative stress, which might have been addressed by the concomitant expression of Tsa1 under the same nutrient-limiting medium. Though the Δlac1 and Δlac1Δlac2 mutants are albino in nutrient-limiting conditions, alternative carbon sources appeared to elevate Lac2 expression in the Δlac1 mutants background to cause infection in a murine model [61,62]. In contrast, Δlac2 mutants are melanised in catecholamine-enriched media [63]. Therefore, an elevated Lac2 transcript more than Lac1 in the presence of alternative carbon sources such as acetate [61] is a reminiscence of Lac2 possibly involvement in melanin production when unusual carbon sources weaken Lac1 involvement.

3.5. Deleting Acetyl-CoA Forming Enzymes in C. neoformans Impairs Alternative Use of Carbon Sources and Attenuates the Virulence

The roles of Acs1 (acetyl-CoA synthetase) have been investigated in the cryptococcal cells recovered from the lungs of infected animals. Evidence showed an elevated expression of the Acs1 gene in C. neoformans during infection, as well as Kbc1, coupled with concurrent elevated expression of different membrane transporters and stress response genes, indicating a nutrient-limited environment and the route for alternative carbon sources as facilitated by the expression of Snf1 [61]. Including Acl1 (ATP-citrate lyase) and Kbc1 (2-ketobutyryl-CoA/acetoacetyl-CoA synthetase), any singly mutated of these genes impairs phagocytic resistance of C. neoformans, and doubly mutated genes (Δacs1Δkbc1) reduced CNS fungal burden further [64]. Glucose is equally consumed in the Δkbc1, Δacl1, and Δacs1Δkbc1 mutants compared to the wt; however, Δacs1 and Δkbc1Δacl1 mutants showed a slight reduction in glucose consumption. In addition, Δacs1 and Δacs1Δkbc1 mutants failed to utilise acetate and are significantly susceptible to FCZ [64]. Deleting Acs1 yielded moderately attenuated mutants that could not use alternative carbon sources such as acetate, and deleting Acs1 and Acl1 generated sterile mutants. This shows that Acs1 and Kbc1 may be necessary enzymes needed for an alternative carbon source utilisation in C. neoformans.

3.6. Phospholipases B is a Crucial Enzyme to Cellular Tight-Junction Penetration by C. neoformans

The secretion of extracellular phospholipases (PLs) by C. neoformans occurs as a cluster of PLs comprised of PLB, lysoPL hydrolase, and lysoPL transacylase/acyltransferase (all together referred to as PLB). These three oligomeric proteins are borne on the same coding gene, the Plb gene, just like in other yeasts such as Penicillium notatum [65], S. cerevisiae [66], and C. albicans [67]. PLs, best secreted at 37^oC [68], are a highly diverse group of hydrolytic enzymes targeting the ester linkages of glycerophospholipids carbonyl linkages and can also transfer acyl-chain to the lysophospholipid to form diacylglycerophospholipid. Chen et al. showed that PLs are well secreted between 6 – 10 hours of cell culture at 30^oC, with optimal activity between pH 3.5 – 4.5 and stability at pH 3.8. The enzyme activity was not affected by exogenous serine protease inhibitors such as leupeptin, phenylmethylsulphonyl fluoride (PMSF), divalent cations (Ca²⁺, Mg²⁺, and Zn²⁺), and EDTA [69]. The stability of this enzyme at acidic pH reveals one of the C. neoformans tolerance phenotypes in the phagosome micro-acidic environment. In so doing, the PLs may break down the phagosomal membrane leading to tissue dissemination.

PLs degrade membrane phospholipids in response to signal transduction. It is a complex extracellularly released enzyme that facilitates fungi-host cell attachment, tissue invasion, and hydrolysis of the epithelial plasma membrane for penetration. The activity of the PLs at the cell wall promotes survival against temperature, antifungals, oxidative stress, and cell wall destabilisers [68]. Moreover, the involvement of PLC as a component of PLs in the virulence expression, antifungal resistance, homeostasis and IP3 kinase activity, and cytokinesis displays the significant functions of this membrane-associated enzyme [70,71].

Every Δplb1 mutant is usually marked with a drastic reduction in the activity of each enzyme compared to the wt [69,72]. By correlation, PL activity corroborated the mortality rate and mucosal invasion by Candida albicans in animal models [73]. Similarly, C. neoformans may use these hydrolytic enzymes to invade tissue and cross the blood-brain barrier (BBB). The production of PL appeared similar in environmental and clinical strains of C. neoformans var. gattii, but the environmental isolates of C. neoformans var. neoformans produced more PL than the clinical isolates [69]. Various isolates of C. neoformans possess different degrees of PL secretion. This was shown to correlate with the fungal virulence and tissue burden in the lungs and brains of mice inoculated intravenously [69,72].

The Ste12α gene has been implicated in the regulation of extracellular PLs. Deletion of the Ste12α gene significantly reduced the production of PL in the egg yolk agar media [74]. As crucial as PLs are to the virulence of C. neoformans, the absence of these hydrolytic enzymes may not affect the virulence phenotypes, such as growth at 37^oC, urease activity, capsule, and melanin formation. Paradoxically, such a mutant is less virulent in the in vivo mouse inhalation and rabbit meningitis infection models when compared to the wt in so much that the mutant growth is defective in a macrophage-like cell line [72]. The attenuated virulence observed in the Δplb1 mutant must have come from the lower density of the capsule; notwithstanding, the capsule and the cell wall dimensions are relatively retained compared to the wt [68].

The attenuated virulence observed in the Δplb1 mutant is likely comparable to Δure1 and Δlac1. The PLB and the accessory enzymes are generally associated with C. neoformans virulence, yet an infection can still progress in their absence but at a slower rate [68,72]. Increasing the temperature from 30 – 37^oC promotes transmigration of the PLB from the cell membrane to the cell wall with a concomitant increase in the cytoplasmic translation, perhaps due to constitutive secretion from the Golgi apparatus. However, secretion of PLB under this heat stress is highly minimised to maintain cell wall integrity and promote membrane homeostasis [68].

3.7. Deleting Membrane-Associated Antioxidative Enzymes Severely Reduced PLB, Urease, and Laccase Expression in C. neoformans

Efficient antioxidant control has been linked to PLB production. Mutation of plasma membrane-associated Cu/Zn SOD encoded by the Sod1 gene has been characterised by a severe reduction in the phospholipase B, urease, and laccase expressions, leading to attenuated virulence manifested in the low brain colonisation and persistence in a mouse model of cryptococcosis [75]. Though the Δsod1 mutants appeared normal in their growth, capsule production, mating, and sporulation, the mutants are susceptible to menadione (a redox cycling agent) and PMN (neutrophils) killing [76]. The mutant culture was also characterised by high mannitol production that possibly acted as an antioxidant to complement the loss of Cu/Zn SOD activity [76,77].

Similarly, mutation of mitochondrial SOD (Mn-SOD) encoded by Sod2 produced thermosensitive and growth defective mutants in normoxic conditions coupled with the accumulation of reactive oxidants in the presence of stress-induced agents like antimycin A, menadione, high salt contents, and nutrient limitations [78]. Furthermore, the poor growth of the mutant at 37^oC ensued in either avirulent or severely attenuated infectious conditions in mouse models of cryptococcosis. An exogenous supply of antioxidants like ascorbate could restore poor mutant growth [78].

Sequential deletion of Sod1 and Sod2 genes produced summative disadvantages in C. neoformans infections and tissue invasion. Further virulent attenuation, PMN killing, poor growth at 37^oC, osmotic stress, and susceptibility to Paraquat^TM and oxytetracycline but not to menadione are the general features of Δsod1Δsod2 mutants [78].

3.8. Phospholipase C orchestrates the Release of PLB, DAG, and IP₃ during C. neoformans Infection

PLB covalently attaches to the cell wall chitin protruding β-1,3-linked glucan via its β-1,6-linked glucan. This arrangement is anchored by the glycosylphosphatidylinositol (GPI) within the membrane lipid raft proteins. The release of PLB from the GPI-anchor is possibly orchestrated by the phosphatidylinositol-specific phospholipase C (PI-PLC, putatively encoded by Plc1 and Plc2), and the inclusion of β-1,6-linked glucan to this release confirms the cell wall localisation of the PLB [68,70]. This shows that PLB constitutes part of the proteins involved in the cell wall integrity because Δplb1 mutant exhibited morphological cell wall defect, which is sensitive to SDS and Congo red disruptions but not caffeine [68]. The Δplc1 mutant of C. neoformans var. grubii strain H99 failed to produce melanin, refused to grow at 37^oC and exhibited impaired PLB secretion with concomitant accumulation of cytoplasmic PLB [70,71]. Furthermore, this mutant displayed high antifungal sensitivity, poor replication, and a defective cell wall with irregular morphology due to impaired activation of the Pkc/MAPK pathway [38].

Because the Δplc1 mutant had poor growth at 37^oC, its virulence was attenuated in the mice study. Also, even at 25^oC, this mutant failed to kill a significant number of Caenorhabditis elegans compared to the wt or reconstituted mutant [70,71]. The actual killing of the worms at 25^oC by the wt or reconstituted mutant indicates that the virulence involvement of Plc1 may be temperature independent. Contrarily, the Δplc2 mutant failed to exhibit any significant defect and is as virulent as the wt [70].

Activated PLC breaks membrane-associated phosphatidylinositol-4,5-bisphosphate (PIP₂) into diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP₃). The IP₃ produced by Plc1 can bind its ER receptors to release Ca²⁺ intracellularly or serve as a substrate for IP₃ kinase (encoded by Arg1) to produce inositol polyphosphate. Expectedly, the Δplc1 mutant generally accumulates PIP₂ but a reduced level of IP₃, whereas in the Δarg1 mutant, IP₃ was accumulated [71]. Invariably, these two mutants shared similar phenotypic defects, including impaired thermotolerance, defective cell walls, impaired virulence factors, improper cell division, and defective mating and filamentation [70,71]. Further analysis showed that Δarg1 accumulated larger intracellular vacuoles (excessive vacuolar fusion) than the wt and Δplc1 mutant [71]. Therefore, Arg1p inositol polyphosphate anabolism (IP_4-8) is as important as the Plc1p catabolism for C. neoformans virulence, and IP₃ may be acting as a biological relay molecule for functional Plc1p.

Being a eukaryote, C. neoformans compartmentalise and adopt differential regulation of transcription factors to facilitate catabolism (in the mitochondria, glyoxysome, and peroxisome) or anabolism (in the cytoplasm) depending on the inducing factor and the available nutrients. For example, IEM elevates enzymes (including fatty acid synthase and acetyl-CoA carboxylase) in the cytoplasmic lipogenesis and promotes extracellular digestion of lipids (lipolysis) which is usually commenced with the release of phospholipase B (encoded by Plb1) [43,47,79,80]. Analysis showed that C. neoformans possessed genes for lipase (encoded by Tgl2 among at least three encoding genes) and enoyl-CoA hydratase [43,80]. However, there is no specific evidence of the functional extracellular lipase expressed in this fungus due to the lack of a signal peptide in these proteins, unlike the lipase secreted by C. albicans [79,80].

3.9. Glycerol-3-Phosphate Phosphatase Controls S-Containing Amino Acid Synthesis via Cystathionine-γ-lyase and Calcineurin Pathways

In a DNA microarray analysis, nearly 20% of total gene expression is dedicated to carbohydrates and amino acid metabolism [81]. The expression of Gpp2 encoding glycerol-3-phosphate phosphatase controls various metabolic systems as well as osmotic and cold shock responses. Deletion of Gpp2 induced various permeases/transporters (like Aap, Mup1, Mup3, Ena1, Nha1, Plb1, Hxt1, Stl1, Uga4, sulphite, myo-inositol, and pantothenate transporters), major facilitator superfamily genes (MFS) (encoding secondary transporters like uniporter, symporter, and antiporter), anti-oxidative enzymes (like Cat2, Ccp1, and Gst1), and enzymes for sulphur-containing amino acid biosynthesis (such as Cys3, Sul1, Soa1, Bds1, Met17, and Jlp1) [82]. However, various genes involved in the redox process, the glycolytic pathway, and the TCA cycle are downregulated in Δgpp2 mutant [82,83].

Among the sulfur-containing amino acid biosynthesis regulated by Gpp2p are Met and Cys. The deletion of Gpp2 alters the biosynthetic regulation of these amino acids due to the perpetual activation of amino acid permease (Aap), including Aap4 and Aap5, which most likely explains the increased sensitivity of the Δgpp2 mutants to eugenol because the deletion of Aap4 and Aap5 is hitherto shown to allow eugenol resistance in C. neoformans [82]. The functional absence of Gpp2 has not been shown to be restored by exogenous glycerol or other types of amino acids [82]. Comparatively, Δhog1 mutants are also sensitive to eugenol because Aaps are induced in Δhog1 mutants [84]. Thus, the osmotic-related stress response in C. neoformans is controlled by the release of glycerol in this pathogenic yeast. In the absence of glycerol-coordinated osmotic resistance (Δgpp2 or Δhog1), a reductive glutathione cycle is induced in C. neoformans, such that Cys feeds the glutathione cycle with -SH (sulfhydryl) to regenerate GSH (reduced glutathione), which is channelled against osmotic stress and reactive oxidants [82].

Just as in A. nidulans and N. crasssa, deletion of Cys3 (encoding cystathionine-γ-lyase) in C. neoformans has been shown to reduce the expression of Sul1, and in the synthetic dextrose (SD) media, the two genes are grossly repressed in the presence of S-containing amino acids (Met or Cys) more than in the YPD [83]. Again, Cys appears better as a sulphur source than Met because methionine could be formed from cysteine in a transulphurylation reaction [83]. Further investigation on Δcys3 mutants showed a wt level of urease activity and capsule size yet attenuated for virulence in Galleria mellonella [83]. Bad still, the experimental conditioning with Cys or Met in the culture media failed to support the melanin and phospholipase assay of this mutant [83]. This means the full virulence of C. neoformans is not just an individual and independent involvement of transcription factors but a concerted mechanism of many functional factors.

The proteomic analysis that showed a concurrent induction of Met3, Tef1, Gpd1, Ccp1, Rps0, Rps1, Tif1, Sub2, Tm8, Gpp2, Cna1, and Cnb1 established a metabolic relationship among the S-containing amino acid biosynthesis, glycerol phosphatase, and calcineurin pathway (Cna1/Cnb1) [82,83]. Under the influence of Ca²⁺, the expression of Cna1/Cnb1 induces and maintains Cys3p protein levels for nuclear localisation and subsequently increases the Cys3p-target genes such as Sul1, Met2, Met3, Met10, Str1, and Sam1 to promote sulphur, purine, Gly, Ser, Thr, Asp, Asn, Cys, and Met metabolism. However, Gpp2p orchestrates the inactivation of Cys3p by proteolytic degradation to maintain amino acid homeostasis. A proposed mechanism is that inorganic sulphur induces Cna1/Cnb1 activation to deactivate Gpp2p. The Gpp2p deactivation promotes Cys3p and activates downstream genes involved in S-containing amino acid biosynthesis [83]. However, organic sulphur in Met and Cys deactivates Cna1/Cnb1, which activates Gpp2p to deactivate Cys3p and repress the downstream genes involved in S-containing amino acid biosynthesis [83], Figure 3.

Among the genes induced in Δcys3 mutant are putative Aro3 and Aro4 involved in aromatic amino acids biosynthesis; Jlp1 in sulfonate metabolism to provide SO₄^2- for Met5/Met10p; sulphiredoxin in oxidative stress response; and Clr6 that encodes class I histone deacetylase [85]. However, it is interesting that repressing Clr6 in the C. neoformans wt promotes capsule formation and biosynthesis of various amino acids [86] (Figure 3).

Figure 3. Gpp2, Cys3, and Cna1/Cnb1 interplay control C. neoformans response to inorganic or organic sulphur (Level 1). At (Level 2), Cys3p is activated in the presence of inorganic sulphur via Cna1/Cnb1 cascade to induce various enzymes involved in synthesising S-containing amino acids. Met or Cys represses these pathways but promotes Gpp2 activation that inhibits Cys3. Numerous metabolic-involving enzymes are induced in Gpp2 and Cys3 mutants (Δgpp2 and Δcys3) (Level 3), and these include amino acid permeases, ionic transporters, PLB, antioxidant enzymes, and hexose transporters in Δgpp2 while Δcys3 induced aromatic amino acid metabolic enzymes. Jlp1p is an important sulphonate enzyme induced in Δgpp2 and Δcys3 and channels sulphonate for Met synthesising enzymes. Certain clusters of genes (Level 4) could also affect the general metabolisms at different levels, as shown in Level 5. From the mapping work of Maier et al. [86] on gene clusters and regulation of virulence in C. neoformans, Clr6, along with Hog1, Mbs1, Ste12α, and Tup1, is classified as a cluster of genes induced against reactive oxidants but repressor of mitochondrial activities and also a repressor of sugar, amino acid, and ionic transporters that are needed for growth. The cluster of Ada2, Clr3, Clr4, Clr5, Ecm2201, Fkh101, Gat201, Hap3, and Rim101 induced ionic transporters but repressed chitin synthesis. The cluster of Cac1, Cep3, Cir1, Clr2, Fap1, Fhl1, Fkh2, Gat1, Mcm1, Pdr802, Sp1, Swi6, and Usv101 are involved in the mitochondrial respiratory process and the activation of proteins/enzymes involved in gluconeogenesis and ionic transporters for osmoregulation; however, cAMP signalling is repressed, and the cluster of Clr1, Hap5, Nrg1, Pkr1, and Ssn801 enhanced ribosomal biogenesis, response to oxidative stress, amino acid biosynthesis, cAMP signalling but repressed mitochondrial activities, ionic transporters, gluconeogenesis, and osmoregulatory process. Induced/activated (⁺); Repressed/inhibited (^×).

Figure 3. Gpp2, Cys3, and Cna1/Cnb1 interplay control C. neoformans response to inorganic or organic sulphur (Level 1). At (Level 2), Cys3p is activated in the presence of inorganic sulphur via Cna1/Cnb1 cascade to induce various enzymes involved in synthesising S-containing amino acids. Met or Cys represses these pathways but promotes Gpp2 activation that inhibits Cys3. Numerous metabolic-involving enzymes are induced in Gpp2 and Cys3 mutants (Δgpp2 and Δcys3) (Level 3), and these include amino acid permeases, ionic transporters, PLB, antioxidant enzymes, and hexose transporters in Δgpp2 while Δcys3 induced aromatic amino acid metabolic enzymes. Jlp1p is an important sulphonate enzyme induced in Δgpp2 and Δcys3 and channels sulphonate for Met synthesising enzymes. Certain clusters of genes (Level 4) could also affect the general metabolisms at different levels, as shown in Level 5. From the mapping work of Maier et al. [86] on gene clusters and regulation of virulence in C. neoformans, Clr6, along with Hog1, Mbs1, Ste12α, and Tup1, is classified as a cluster of genes induced against reactive oxidants but repressor of mitochondrial activities and also a repressor of sugar, amino acid, and ionic transporters that are needed for growth. The cluster of Ada2, Clr3, Clr4, Clr5, Ecm2201, Fkh101, Gat201, Hap3, and Rim101 induced ionic transporters but repressed chitin synthesis. The cluster of Cac1, Cep3, Cir1, Clr2, Fap1, Fhl1, Fkh2, Gat1, Mcm1, Pdr802, Sp1, Swi6, and Usv101 are involved in the mitochondrial respiratory process and the activation of proteins/enzymes involved in gluconeogenesis and ionic transporters for osmoregulation; however, cAMP signalling is repressed, and the cluster of Clr1, Hap5, Nrg1, Pkr1, and Ssn801 enhanced ribosomal biogenesis, response to oxidative stress, amino acid biosynthesis, cAMP signalling but repressed mitochondrial activities, ionic transporters, gluconeogenesis, and osmoregulatory process. Induced/activated (⁺); Repressed/inhibited (^×).

3.10. Transcriptional Activation Factor Controls the Lipid and Sterol Biosynthesis in Cryptococcal Cells for Membrane Integrity and Thermotolerance

Lipid biosynthesis is an essential biochemical pathway leading to cell membrane integrity. The Mga2 is an orthologue transcription activation factor for the component of fatty acid synthesis and regulates in vitro normal growth at a range of temperatures [87]. Therefore, mutation of Mga2 makes C. neoformans thermosensitive and hypersensitive to FCZ with a concomitant reduction in the expression of Fas1, Acc1, Rpl11, Rps7, Leu1, Pho, Pdh, eEF3, and other orthologue proteins. However, expression of Lys2, Hxt, Cat, Pak1, Chs, Mdr1, ABC-family, Vps, Hsp78, Hsp104, calnexin, Rho-GAP domain protein, exo-1,3-β-glucanase, and other relevant proteins are induced in this mutant [87]. Ambiguously, it is unclear how the presence of Mga2 regulates polarisation and filamentation growth in C. neoformans from the work of Kraus et al. because Δmga2 mutants are characterised with elevated expression of the homologues of small GTPase effectors, which control the cell polarisation, and the Pak1 that controls the filamentation [87]. Notwithstanding, it is possible that during this morphogenesis, Mga2 expression is repressed at ≤30^oC for GTPases and Pak1 expressions that will promote polarisation during growth, but this, however, will contradict the involvement of Mga2 in thermotolerance and morphogenesis as reported by Kraus et al. [87]. Besides, the dependency of Fas1 (fatty acid synthase β-subunit) and Acc1 (acetyl-CoA decarboxylase) expressions on Mga1 for lipid and sterol biosynthesis through the elevated acyl-CoA desaturase I, Ole1 (an ER membrane enzyme for the production of unsaturated fatty acids), will promote polarisation growth, mating, and haploid fruiting [88,89]. Thus, the background expression of Mga1 may have promoted polarisation and cell growth in Δmga2 mutant.

3.11. Cryptococcus Virulence Is under Nitrogen Catabolite Repression Regulated by GATA-Sequence Binding Factors

With NCR, certain nitrogen sources are not assimilated, perhaps because of the energy cost of catabolism. Deletion of Gat1, among other GATA transcription factors, promotes poor to no growth of C. neoformans in the majority of different nitrogen sources except in the presence of Pro and, to some extent, Arg, while single deletion of other GATA family genes (Δgat201, Δgat204, Δbwc2 or Δcir1) failed to shut down totally the fungal growth in all the popular nitrogen sources [90]. This means that various amino acid permeases (Aaps) may still be activated to assimilate other nitrogen sources under later conditions, contrary to Δgat1 mutants where ammonium transporter (Amt) and most Aap gene expressions are impaired. However, Pro and Arg may not be under NCR control.

To affirm this, the Pro medium induces Put1 gene expression for proline oxidase in the wt under the influence of NCR as mediated by Gat1 expression in the presence of NH₄⁺ [90]. In addition, the expression of Put5 (a paralogue of Put1) and Put2 genes, which are involved in the downstream catabolism of Pro to Glu, remained unchanged in Δgat1 mutants and wt [90]. This means that though Gat1 mediates NCR, Pro metabolism is Gat1-independent. The ability to form Glu from Arg may also explain the moderate growth of Δgat1 mutants in Arg-containing media. Gln and His can also form Glu directly. However, because the cytoplasmic content of Gln is always high as a form of nitrogen donor in most metabolic reactions and His metabolism is also very complex, this may explain why Δgat1 mutants had poor growth in a medium with each of these two amino acids as nitrogen sources.

The growth defect of Δgat1 strain H99 mutant in urea, urate, or creatinine-containing media with reduced expression of genes involved in nitrogen metabolism (such as Gdh1, Amt1, and Amt2) is a serendipitous observation that contradicts the ecological niche of C. neoformans. A similar survival growth observed with the wt and Δgat1 mutants cultivated in pigeon guano media is an indication that though this ecological niche is rich in urea, urate, and creatine, which may probably be selective by the NCR, at the same time, the survival of the Δgat1 mutant in the same environment poses an investigation into the unique nitrogen sources suitable for such a mutant. Perhaps, such media are rich in Pro or Arg, as inferred from Lee et al. observations [90]. Not only this, but Kmetzsch et al. also observed the growth of Δgat1 mutant in Pro, Arg, urea, and NH₄⁺-containing media [6,91]. Notwithstanding, limited nitrogen sources in the environmental pigeon guano may be suspected to have repressed the Gat1 mutant and prevented the mating process that promotes propagule/basidiospore formation in the filamentous stage of this opportunistic human infectious fungus. Lee et al., instead, discovered that mating is greatly enhanced through the robust filament and basidia formation in the unilateral or bilateral crossing of Δgat1 mutants [90].

In the same way, deletion of Gat1 drastically reduces capsule formation, enhances melanin formation at physiological temperature, and promotes extreme thermotolerance but shows no significant effect on urease activity. Unexpectedly, these conditions seem to reduce the pathogenic efficiency of Δgat1 mutants moderately compared to the wt [90]. This shows that the critical virulence factor of C. neoformans is firmly under the influence of NCR [91].

Keeping NCR under repression by Gat1p is necessary to facilitate the utilisation of other nitrogen sources. Lee et al. discovered that capsule formation increased considerably in the strain H99 when cultivated in a medium containing Asn, urea, urate, or creatinine but not Gln, Pro or Ala-containing medium. This capsule formation is prosaically affected by the presence of NH₄⁺ in each media [90]. This shows that NH₄⁺ represses capsule formation by activating NCR even in capsule-induced nitrogen sources, but, on the contrary, the presence of NH₄⁺ induces melanin formation. To buttress this, Lee et al. observed melanin formation in each media mentioned above when prepared with NH₄⁺ except in the Pro medium, where a slight melanisation was observed even when NH₄⁺ was present [90]. Without NH₄⁺, creatinine and urate failed to support melanin formation, and only minimal melanisation was observed in the Ala medium. However, melanin was significantly induced in urea, Asn, or Gln-containing media [90]. This shows that NCR differentially regulates virulence accessory factors and that certain amino acids can override the NCR effect in the dire need for virulence. So, while NCR induces NH₄⁺ to repress capsule formation in the presence of different nitrogen sources, melanin is significantly induced.

3.12. A Limited Number of Amino Acid Permeases/Transporters Are Crucial for Cryptococcus Virulence

It was shown that among the numerous amino acid permeases, Aap2, Aap4, Aap5, Aap6, and Aap8 are more differentially expressed in the amino acid-fortified synthetic dextrose (SD) minimal media than the rich media (YPD) [92,93], which means that NCR is highly induced in YPD compared to the SD. Without NH₄⁺ (a preferred nitrogen source), global amino acid control is triggered by nitrogen starvation leading to preponderant production of t-RNA for amino acid biosynthesis in the yeast. For example, nitrogen starvation induces Trp biosynthetic enzymes encoded by Trp2, Trp3, Trp4, and Trp5 [92]; also, under repressive NCR, Aap2 and Aap3 are induced in the presence of Lys-containing media [52]. However, amino acid supplements in SD do repress the general amino acid control (GAAC) but promote the APC-superfamily membrane transporters/permeases, such as APC, AAAP, AGCS, CCC, HAAAP, BCCT, SSS, NSS, NCS1, NCS2, and SulP families [94,95,96].

The transcription analysis further showed that NCR could repress Aap2, Aap5, and Aap8 in the presence of NH₄⁺ but not Aap4, which means Aap4 is sensitive to any nitrogen sources; however, Aap1 and Aap7 remained minimally expressed [92]. Though the NCR selectively represses Aap, carbon catabolite repression (CCR) appears to repress all the Aap expression in the presence of glucose (preferred carbon source) compared to galactose (alternative carbon source) [92].

The Δaap1Δaap2 mutants are thermosensitive (at 37^oC in synthetic media fortified with amino acids), hypocapsulated, and hypovirulent in the G. mellonella infection model [97]. Mutants such as Δaap1, Δaap2, Δaap6, Δaap8, and Δaap1Δaap2 failed to display any significant difference in growth when compared to the wt at 30 or 37^oC in SD fortified with amino acids/NH₄⁺ or in YPD; however, growth reduction was observed in liquid culture of Δaap1 containing Met or Pro at 30 or 37^oC [97]. Also, a liquid culture of Δaap1Δaap2 mutant containing Gln or Arg displayed growth reduction at 30^oC, and Δaap8 mutant showed growth reduction in media containing Met, Glu, and Trp [97].

Generally, Δaap1Δaap2 mutants appeared to show defective growth in 60% of all culturable amino acid media (AAM), which means these amino acid permeases are essential for growth at 37^oC on fortified SD media, just like Aap4 and Aap5. However, Aap1, Aap2, Aap6, and Aap8 appeared non-essential for virulence because mutants of these genes, including Δaap1Δaap2 mutant, failed to show any significant difference in the mating, filamentation, melanin expression, PLB and urease activities when compared to the wt [97]. Apart from being non-essential for virulence, none of the mutants seemed affected by oxidative stress agents, alkaline conditions, osmotic and salt stress agents, and cell wall stress agents at 30 or 37^oC. Surprisingly, unlike the single mutants (Δaap1, Δaap2, Δaap4, Δaap5, Δmup1, and Δmup3), the double mutants Δaap1Δaap2, Δaap4Δaap5, and Δmup1Δmup3 displayed reduced capsule size at 37^oC when compared to the wt. However, the capsules are similar at 30^oC, and the virulence of Δaap1Δaap2 and Δaap8 mutants only is attenuated in G. mellonella [97].

From Martho et al., Δaap2, Δaap4, Δaap5, Δmup1, Δmup3, and Δmup1Δmup3 showed no sign of attenuated virulence in G. mellonella, but Δaap4Δaap5 mutants are hypovirulent at 30 or 37^oC [98]. Furthermore, virulence investigation in the murine model showed that mice inoculated with the wt, Δaap4, and Δaap5 died within a month, while the mice inoculated with Δaap4Δaap5 mutants and PBS-treated strain survived with viable recovery colonies from the lungs and livers [98]. In addition, Δaap2, Δaap4, Δaap5, and Δaap4Δaap5 mutants displayed wt phenotypic sensitivity to AmpB but are hypersensitive to FCZ, and each of the mutants is hyper-resistant to eugenol due to Aap inactivation. This shows that Aap4 and Aap5 are important permeases for thermotolerance, antifungal resistance, and oxidative stress response for tissue invasion, survival, and virulence in the animal model. By speculation, they are a potential target for therapeutic application [99]; notwithstanding, the compensatory essence of permeases/transporters cannot be ruled out. By harmonising several works on amino acid permeases (Aap, Mup, and Dao), all mutants of Aap and Mup:

❖

are not affected in rich media (YPD) at high temperatures except Δaap4Δaap5,

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are not affected in AAM at 37^oC and in capsule formation except Δaap1Δaap2, Δaap4Δaap5, and Δmup1Δmup3,

❖

are not sensitive to any stress agent except Δaap4Δaap5,

❖

are virulent in G. mellonella except for Δaap1Δaap2 and Δaap8 (hypovirulent), Δaap4Δaap5 (avirulent as well as in murine model),

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shared appreciable level of sequence homology (Aap1 vs Aap2, 80.9%; Aap1 vs Aap2 vs Aap3, 49%; Aap4 vs Aap5, 89.5%; and Aap6 vs Aap7, 41.4%), and each of these Aaps in the doubly mutated yeast appeared redundant in their functions,

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the Aap3 and Aap7 expressions are below detection in YPD or SD; however, Lys-containing medium induced Aap2 and Aap3 expressions under alleviated NCR,

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irrespective of the nitrogen sources, the expression of Aap6 remained relatively unchanged,

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the Aap8 responds to amino acid-supplemented media only, but the highest expression is usually found in Aap2, Aap4, and Aap5,

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Mup1 and Mup3 expressions are under the NCR regulation as Aap2 and Aap5 but can be induced by His, Trp, and Met when NRC is shut down,

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only Mup1 can be induced by S-containing amino acids,

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galactose induces the expression of all the Aap and Mup genes,

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expression of Aap6 and Aap8 is temperature-independent,

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Aap4, Aap5, and Mup1 induction increased from 30 to 37^oC in SD medium,

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Aap2, Aap4, Aap5, and Mup3 expressions are further repressed from 30 to 37^oC except for Mup1, which is further induced,

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irrespective of the growing media, there is no significant change in the growth of Δaap2, Δaap4, Δaap5, Δmup1, Δmup3 and Δmup1Δmup3 mutants at 30 or 37^oC,

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the significant growth defect of Δaap4Δaap5 mutants at especially 37^oC in YPD or SD showed that the two permeases (or at least one of them) are important for thermotolerance,

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the use of amino acids as nitrogen sources impaired the growth in Δaap4Δaap5 mutants,

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relative to NH₄⁺ at 30^oC, Val, Ile, and Met-containing SD media poorly support the growth of C. neoformans, but Leu, Ser, Lys, and Phe are better nitrogen sources,

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Gly, Asp, Asn, Glu, Gln, Arg, Trp, and Pro are highly competitive with NH₄⁺ in culturable AAM,

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at 37^oC, Val and Met poorly support the growth of C. neoformans in SD media, but Gly, Leu, Ile, Ser, Trp, and Phe are good nitrogen sources, while Asp, Asn, Glu, Gln, Arg, Lys, and Pro are better nitrogen sources,

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stereospecifically, var. gattii metabolise D-amino acids because of the more active expression of Dao1, Dao2, and Dao3 genes (encoding D-amino acid oxidase) but less metabolisable for var. neoformans, which prefers L-amino acids as nitrogen sources due to the inefficient evolutionary expression of Dao genes,

❖

in all, growth is denser in L-amino acids containing media than the corresponding D-amino acids media,

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pathologically, Δdao mutants of C. neoformans are virulent, but Δdao mutants of C. gattii are attenuated,

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positive correlation of Gat1 expression that represses NCR is confirmed to aid the expression of Aap when the preferred nitrogen source is absent/limited but uncertain with Dao expression,

❖

L-Tyr failed to dissolve at permissive pH for culturing C. neoformans, hence not suitable to be tested,

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Aap2, Aap3, Mup1, and Mup3 may be global amino acid permeases/transporters because of the most significant growth defect of their corresponding mutants, especially in their double mutant states, from 30 to 37^oC and their ability to be induced by various amino acids,

❖

in addition to being a global permease and redundant transcription factors, Aap4 and Aap5 promote thermotolerance and response to oxidative stress, and the growth of the double mutant is significantly impacted from 30 to 37^oC in a single amino acid medium or the presence of ≥5 mM H₂O₂,

❖

unlike the Δaap4 and Δaap5 mutants, the growth defect of Δaap4Δaap5 mutants at 37^oC appeared to be restored as pH increased gradually into the alkaline state or when supplemented with 0.75 M NaCl (this condition generates H⁺ via Na⁺/H⁺ antiporter that drives other amino acid permeases to compensate for the deletion of Aap4 and Aap5),

❖

functionally, Aap2, Aap3, Mup1, and Mup5 are regarded as minor permeases while Aap4 and Aap5 are major permeases [97,98,100].

3.13. Mutating the Ras1 Gene Attenuates Cryptococcus Thermotolerance, Capsule Formation, and a More Significant Proportion of Amino Acid Permeases/Transporters

The thermotolerance in cryptococcal cells is majorly controlled by Ras1. Surprisingly, it has been shown that the Ras1 pathway also controls amino acid permeases leading to thermotolerance [97]. The Δras1 mutants had shown growth reduction compared to the wt in various amino acid-fortified cultures at 30 or 37^oC except for media with Ala, Cys, His, Tyr, and Thr that failed to support the growth of Δras1 mutants [97]. The involvement of Ras1 in the assimilation of amino acids is a unique feature of this transcription factor to facilitate thermotolerance [101,102,103]; however, Aap4, Aap5, and Mup1 are among the few selected expressible permeases in this non-permissive temperature. Nutritionally, all Aap are generally more induced in fortified SD media (without NH₄⁺) than YPD at 30^oC [104].

The controlling effect of Ras1p on the expression of Aap was further investigated by quantifying the expression level of each Aap transcript in Δras1 mutants. All Aap and Mup transcripts were repressed in Δras1 mutants in SD fortified with Trp, His, and Met at 30 and 37^oC except Aap5, which showed a wt transcript level at 37^oC [97]. The exception of Aap5 may suggest a different regulatory mechanism in addition to or different from Ras1 expression. This shows the importance of the Ras1/MAPK signalling pathway in the nutritional balance of C. neoformans against thermal stress, alternative nutrient source, morphogenesis, and virulence. Just as Aap4 and Aap5 were identified as potential drug targets against cryptococcal infection, the unique region of sequence homology within the regulatory domain of Ras1 protein across a few numbers of pathogenic yeast has also been speculated as a drug target [105].

4. Transcriptional Factors Regulate Virulence-Associated Membrane-Anchored Proteins, Extracellular Enzymes, and Vesicular Secretions in Cryptococcal Cells for Morphology, Growth, and Virulence

Several extracellular hydrolytic enzymes from C. neoformans have been studied with diverse functions. They are freely secreted under the regulatory N-terminal signal peptide or cleavage from the cell wall located GPI-anchored proteins or remain covalently attached to the cell wall glycans. Though the specific functional proteins, such as those designated for antioxidative functions, may lack N-terminal signal peptide and GPI-anchor [106], most of the proteins involved in adhesion may require GPI-anchorage.

Cryptococcal secretomes are controlled by several Sec expressions, which encode factors that orchestrate the secretory pathways of extracellular proteins, especially the glycosylated proteins. S. cerevisiae has been well-studied regarding the mechanism of exocytic secretion of proteins. Several haploid double Δsec mutants of S. cerevisiae on invertase secretion showed that extracellular secretion by yeast required several forms of Sec factors, ATP, temperature, and a moderate level of glucose (<1%) [107]. The endoplasmic reticulum-Golgi Body migration of secretory protein required energy for posttranslational modification. The final vesicular packaging and plasma membrane fusion for budding also required energy, permissive temperature, and several other Sec factors.

Secretions are generally poor at non-permissive temperatures but are reversible when transferred to permissive temperature in the presence of glucose, as found in intracellular accumulation of invertase by Δsec7 and Δsec16 mutants [107]. Mutation of Sec7 impairs secretome activity, followed by intracellular accumulation of different organelles involved in the secretion. Furthermore, Sec expression has a way of regulating each other. For example, Sec18 was epistatic to Sec1 and Sec17, which was shown by intracellular accumulation of invertase in Δsec18, Δsec1Δsec18, and Δsec7Δsec18 mutants but not in Δsec1 and Δsec7 [107].

Extracellularly released proteins could be inducible/constitutive depending on the signalling factors. Proteases are released to degrade proteins, lipases degrade triglycerides, phospholipases degrade phospholipids and sphingolipids, urease degrades urea to cytotoxic ammonia and CO₂, laccase is involved in pigmentation, and chitin synthase is involved in cell wall chitin formation. Each of these enzymes is examined further as follows:

4.1. Proteases (Proteinases/Peptidases)

Various hydrolytic enzymes, including endoproteinases and exoproteinases, have been implicated in the cryptococcal invasion and colonisation of the CNS (neurotropism). Vu et al. discovered that extracellular secreted metalloproteinase, which is an M36 protease class of fungalysins, is highly required for fungal colonisation of the CNS and brain endothelium but not dissemination and the deletion of the Mpr1 gene incapacitated the fungal penetration of the BBB endothelial layer to establish infection [108]. This prolongs the survival of the infected host due to reduced brain pathology.

The nature of the media determines the type of secreted protease. Animal tissue culture induces metalloproteinase and serine endoprotease as major peptidases, but aspartyl endoprotease activity dominates in minimal media. Remarkably, the deletion of Qsp1 reduced aspartyl endoprotease in the minimal media but elevated the secretion of metalloproteinase and serine endoprotease in animal tissue culture more than the wt [109]. Likewise, Brueske discovered that media supplemented with NH₄⁺ and glucose could inhibit the total secreted proteolytic enzymes of C. neoformans, while growth in essential salts medium supplemented with bovine serum albumin only enhanced the extracellular protease profile [110].

The involvement of C. neoformans extracellular proteinases in the degradation of host immunological proteins has been demonstrated, and this is responsible for fungal invasion and dissemination. Such proteinases could degrade β-casein, murine IgG₁, bovine IgG, human C₅, haemoglobin, and γ-globulin in media containing carbon and nitrogen sources [111,112]. Regarding the potential to cause infection, a relatively low proteinase profile of C. neoformans var. gattii may have reduced tissue invasion, dissemination, and systemic spread of this serotype compared to the C. neoformans var. neoformans. However, there is no difference in the total proteolytic activities of the environmental isolates compared to the clinical isolates of C. neoformans [113]. Proteases are released at permissive and non-permissive temperatures, irrespective of their sources. A positive correlation exists between the capsule size and the protease activity in the clinical isolates, which was not found in the environmental/bird-dropping isolates [114]. The majority of these proteinases are serine proteases having optimal activities between pH 7 – 8 at 37^oC [111]

In another related study, aspartyl endopeptidase (putatively encoded by May1) and carboxypeptidase D1 (putatively encoded by Cxd1, Cxd2 or Cxd3) have been identified as the major extracellular endoprotease and carboxypeptidase, respectively, in C. neoformans that help cope with the acidic environment and significantly enhance virulence [115]. Specifically, the total proteolytic activity in the mutants lacking metalloendopeptidase or metalloproteinase (encoded by Mpr1), serine carboxypeptidase (encoded by Scx1), carboxypeptidase C (encoded by Prc1), serine endopeptidase (encoded by Prb1), carboxypeptidase D (encoded by Cxd2, Cxd3), and aspartyl endopeptidase (encoded by Pep4) was not significantly different from the wt in an enriched medium. However, Δmay1 and Δcxd1 mutants had significantly reduced activity of the extracellular proteases because they encoded major secreted protease tagged major pepsin-like aspartyl endopeptidase with optimal expression in an acidic medium.

This aspartyl endopeptidase, by mechanisms, can be inhibited by pepstatin A and antiviral protease inhibitors [115,116,117]. Mutation of these extracellular protease-encoding genes might not affect melanin production. Still, since melanin synthesis is also a cell wall localised process enriched with mixed GPI-anchored proteins, chitin, chitosan, and glycan, the deletion of Prb1 has shown hypomelanisation in such a mutant [115].

Targeting the proteolytic activities of pathogens is a significant way of therapeutic approach against infections. A few works have shown that antiretroviral drugs in AIDS patients could antagonise some key extracellularly released hydrolytic enzymes and virulence factors in pathogenic fungi such as C. albicans and C. neoformans, and this invariably inhibits fungal growth. Unlike Saquinavir, Nelfinavir, Lopinavir, Darunavir, Tipranavir, Atazanavir, Amprenavir, Brecanavir, Darunavir, Indinavir, Ritonavir, and Saquinavir as well as macrocycles have been shown to inhibit fungal growth, attenuate capsule production, and inhibit urease and proteases but not phospholipase and melanin production in C. neoformans [115,116,117].

The use of specific and effective antiretroviral drugs in a co-morbidity patient with cryptococcosis may be responsible for the higher clearance of the cryptococcal cells because of the increased susceptibility of the weakened cryptococcal cells to the killing effect of innate effector cells, such as monocytes, neutrophils, and macrophages [116]. Unlike C. albicans, which are easily cleared by antiretroviral drugs, C. neoformans may be relatively difficult. C. neoformans aspartyl protease is one of the proteases not inhibited by highly active antiretroviral therapy, such as Indinavir, Lopinavir, and Ritonavir. Apparently, the transcriptional level of aspartyl protease is strategically independent of the media used [118].

4.2. Urease

Urease (encoded by Ure1) is another hydrolytic metalloenzyme that hydrolyses the urea to ammonia and carbamate. Apart from Ure1, other transcription factors like Ure4, Ure6, and Ure7 have been associated with the production of accessory proteins required for the nickel-dependent functional protein complex [119]. Via the nickel/cobalt transporter (encoded by Nic1), Ni²⁺ is shuttled into the cytoplasm to combine with the apo-urease (encoded by Ure1) via Ure7p to form an active metallocentric urease. The urease reaction may increase the media pH to create an alkali environment. While this may be deleterious to the infected host cells, C. neoformans deploy the pH-sensing signalling factors, such as Rim21-Rim8-Rim20-Rim13-complex, to counter the pH surge via the activation of Rim101 signalling pathway [29].

The Δure1 mutants displayed comparable phenotypic and virulence features with the wt; however, evidence existed that deleting this gene may attenuate the progression of infection caused by C. neoformans. This is corroborated by the outcome of Cox et al. that murine infected with Δure1 mutant, whether intranasally/intravenously, failed to show pulmonary distress, displayed delayed hydrocephalus, and wasting, and significantly lived longer than the wt-infected mice [120]. Hydrocephalus and wasting are delayed manifestations of meningoencephalitis caused by Δure1 mutants after dissemination into the brain, but the infection caused by urease-positive strains proceeds rapidly in the lungs to result in acute pneumonitis capable of killing the mice before the brain encephalitis sets in [120]. Notwithstanding, wholistic urease activation is essential for the microcapillary sequestration, dissemination, and brain invasion of C. neoformans because Δure1, Δure7, and Δnic1 mutants, which lacked urease activities, significantly displayed defective growth in the presence of urea with poor blood-to-brain invasion in an animal model [119,121]. Though urease seems to be essential to the dissemination, invasion, and survival of C. neoformans in vivo yet, clinically infectious urease-negative isolates capable of dissemination and invasion have been reported in AIDs patients [122,123], which means this enzyme may be playing second fiddle in the pathogenesis of C. neoformans [124].

4.3. Laccase

Laccase is a cell-wall-associated pigment-forming enzyme in C. neoformans. This iron/copper-dependent metalloenzyme is encoded by Lac1 (major) and Lac2 (minor) genes [62,125]. Four major transcription factors, Bzp4, Usv101, Mbs1, and Hob1, are involved in the induction and regulation of the Lac1 gene [126]. The enzyme can act on several neurotransmitters and other amino compounds to synthesise melanin that confers camouflaging [127,128], resistance (against fungicides, photolysis, thermolysis, acidolysis, oxidants, denaturants) [129,130,131,132,133],[134,135], and elongating propagule lifespan [136] in C. neoformans. Several mutants of cryptococcal cells displayed impaired melanin formation. Mutating the Snf7 gene in cryptococcal cells impaired polysaccharide secretion, capsule, and melanin formation, especially at physiological temperature, with a complete loss of virulence in an intranasal model of murine cryptococcosis [137].

Low glucose level facilitates the nuclear translocation of Bzp4 and Usv101; and, together with the residential nuclear transcription factor ( Mbs1), initiates the transcription of the Lac1 gene [126]. Conversely, Hog1 provides a repressive regulation of Bzp4 and Lac1 to coordinate melanin production. The Lac1 expression was particularly shown to be upregulated in Δssk1, Δhog1, and Δskn7 mutants [84]. In the early investigation, Snf5 and Mbf1 have been implicated in regulating laccase transcription, poor growth observed in non-glucose media, bilateral sterility, and mating defect in C. neoformans [138]. Also, because melanin production is anchored by the cell wall chitin and governed by the cytoplasmic ionic homeostasis, Chs3, Ccc2, Atx1, Cuf1, and Sit1 have been further identified as important transcription factors for melanisation [138].

Two oppositely regulated signalling pathways govern melanin production – cAMP/Pka and Hog1 [139,140] (Figure 4). Low glucose concentration activates cAMP/Pka-repressing pathways while repressing the Hog1 signalling pathways. This condition induces the Lac1 gene for melanin production. Hog1 expression could hinder the Pka downstream activity leading to melanin production in serotype A, Hog1 deletion effectively restored the melanin production in hitherto non-melanized Δgpa1, Δcac1, or Δpka1 mutant cultured in 0.1% glucose media (Figure 4); however, Hog1 mutation failed to restore melanin production in serotype D Δpka2 mutant [140]. From the two-component system of the Hog1 pathway, deletion of Tco genes did not contribute significantly to melanisation, but Δtco1 or Δtco1Δtoc2 mutants showed enhanced melanisation at 37^oC even in up to 2% glucose media, and this melanin content was considerably higher than the Δhog1 mutant [141]. This shows that Tco1 kinase is a key repressor of Lac1 expression, just like Ssk1, Pbs2, Hog1, and Skn7 proteins. However, complementing the Δtco1 mutant (Δtco1::Tco1) reduced the melanin to the wt level.

The Ypd1 is also a component of the Hog1 pathway, which regulates melanin and capsule formation. At 0.1% glucose, Δypd1Δhog1 mutant produced a highly significant level of melanin just like Δhog1, Δssk1 and Δskn7 mutants at 30^oC, and at 37^oC, only the Δypd1Δhog1 and Δskn7 could retain the melanin [142]. Similarly, Bahn et al. reported comparable melanin formation in the wt, Δhog1, Δpbs2, Δssk1, and Δskn7 mutants at 37^oC [141]. In 1.0% glucose medium, Δssk1, Δhog1, and Δskn7 mutants melanised as wt, but all mutants, including the wt, failed to retain this melanin at 37^oC. This shows that the efficiency of melanin formation is more negatively affected by increasing glucose levels than by increasing temperature.

Surprisingly, the deletion of Ypd1 represses melanin formation in the Δhog1 background mutant irrespective of the temperature [142] (Figure 4). Contrary to this, Δhog1, Δpbs2, Δssk1, and Δskn7 mutants still retained pigmentation in 1.0% glucose media at 37^oC while melanin formation had already been lost in the wt and reconstituted Δssk1. Yet, in 2% glucose media, pigmentations are still found in the same set of mutants with significant melanisation in the Δskn7 than the rest of the mutants [141]. Because of the regulatory effect of Hog1p on the expression of Mbs1 (encoding DNA-binding basic helix-loop-helix protein 1), then Song et al. showed significant melanin and capsule defect in Δcac1 compared to Δmbs1 mutants at 37^oC, notwithstanding Δmbs1 mutant still showed a reduced virulence with low degree tissue fungal burden and titanisation compared to the wt or complemented Δmbs1 mutant (Δmbs1::Mbs1) [143].

While it is reasonable to summate that increasing cAMP production would elevate melanin synthesis, D'Souza et al. showed that high levels of cAMP led to overexpression of Pka1 and the repression of melanin production as compared to the wt [144] (Figure 4). However, capsule production appeared highly induced [144]. This means that capsule production may be more critical to virulence than melanin. Though the same upstream regulatory factors may control melanin and capsule productions, different transcription factors are involved in the terminal responses. Evidence exists that melanin is produced in the infected tissue isolates, but not as much is produced in vitro. Again, the fact that larger capsules have been produced in the infected tissue isolates than culture media means that capsules are more involved in the C. neoformans in vivo virulence and pathogenesis than melanin. Notwithstanding, an appreciable level of melanin is necessary for effective virulence, tissue invasion, antifungal resistance, and macrophage survival within the infected tissues [145,146,147,148].

Ironically, the presence of melanin in the infected tissues has been proposed with uncertainty. Liu et al. discovered that laccase-dependent catecholamine oxidative products such as pyrrole-2,3,5-tricarboxylic and pyrrole-2,3-dicarboxylic acids rather than melanin might be produced in the mouse brain during infection to induce oxidative cytotoxic effects within the infected tissue [149]. The accrued evidence came from the work of Ito et al. that dopamine-o-quinone is one of the reactive catecholamine oxidative intermediates, which are formed during melanin synthesis, and this intermediate strongly attacks the sulfhydryl groups of protein cysteine [150]. This can be further oxidised to pyrrole acids in alkaline peroxide [151,152]. Therefore, this contrary discovery means that if melanin is actually produced during infection, then it may be stringently controlled for moderate pigmentation against oxidative damage caused by heterologous quinone-like derivatives, which are cytotoxic catecholamine oxidative intermediate products channelled to destroy the host cells.

Mutation of the Pka1 gene in C. neoformans usually leads to sterility and avirulent because the strain failed to produce melanin and capsules; however, overexpression of Ste12α in the Δpka1 mutant restored the mating but was unable to restore the virulence of Δpka1 mutant [144]. Contrarily, the mutation of the Pkr1 gene, encoding the regulatory Pka subunit protein, failed significantly to affect melanin production in serotype A MATα, unlike the mutation of Gpa1. Interestingly, it was shown that the Δpkr1Δgpa1 double mutant produced a comparable level of melanin to the wt better than the Δgpa1 mutant [144]. This indicates that melanin and capsule production is under the multifactorial transcription factors downstream of the Pka protein. Furthermore, mutation of the Crg1 gene has been shown to enhance melanin production in the MATα strain, which depends on the Ste12α expression. This mutation directly increased the virulence in the animal study in a Cpk1-independent manner [153].

Deleting the Sit1 gene (encoding siderochrome-iron uptake transporter) in the serotype D background enhances melanisation better than the serotype A Δsit1 mutant. The melanin production seemed better with increasing DOPA concentration in the medium—however, 1.0% glucose repressed melanin formation in the Δsit1 mutants of the two serotypes. Additionally, the Sit1 expression appears to control copper availability to Cu²⁺-binding pocket of laccase. So, loss of Sit1 gene apparently attenuates ionic homeostasis and increases laccase activity more significantly in the mutants than the wt. This means laccase in the mutants has more access to copper because of the absence of Sit1p, which ordinarily could have distributed the copper among other copper-dependent proteins. In this case, Tangen et al. exogenously supplied copper and discovered an elevated laccase activity in the wt and reconstituted Δsit1 strain, comparable to the Δsit1 mutant [125]. Notwithstanding, an exogenous supply of copper or iron in the absence of glucose generally seemed to favour melanisation in serotype D ΔSit1 mutant.

4.4. Inositol-Phosphorylceramide Synthase

Luberto et al. exposed the vital role of the Ipc1/Aur1 gene encoding inositol-phosphorylceramide synthase 1, which catalyses the formation of membrane-associated sphingolipids complex called inositol-phosphorylceramide (IPC) and diacylglycerol (DAG) from phytoceramides and phosphatidylinositol (PI) in fungi [154]. The DAG is a second messenger of mitogen and protein kinase c (Pkc) activation. Based on this function, inositol-phosphorylceramide synthase 1 is critical in membrane integrity during growth.

Suppressing the Ipc1 expression under the P_gal7-regulated inducible promoter, it was shown that while the glucose-repressing condition reduced melanin production by 60%, the galactose-inducing condition increased melanin production by 80% [154]. Furthermore, this repressive condition impaired the virulence, survival rate, in vivo growth, replication, and cellular diffusion of infectious strain H99 in immunocompromised animal and macrophage-like cell line models [154]. Again, because of the increased melanin production in the Hog1 deleted background mutant, Ko et al. discovered that upregulation of Ipc1 in the Δssk1 and Δhog1 mutants might synergistically enhance melanin production in C. neoformans [84]. This condition may be adopted by cryptococcal cells in vivo during infection to enhance resistance, stability, adaptation, growth, and development.

4.5. Chitin Synthase

Though the presence of chitin and other unique cell wall components predisposes the cryptococcal cells to the host PAMPs-PRRs (pathogen-associated molecular patterns-pattern recognition receptors) recognition to induce anti-cryptococcal defence strategies [155] but with the capsule formation and vesicular secretion of capsular components, C. neoformans could shield the chitin and circumvent the host’s PAMPs effect [28,156]. Not only this, but Cryptococcus also has intrinsic chitin deacetylase encoded by seemingly redundant Cda1, Cda2, and Cda3 functional genes (the fourth, Fdp1, remained undetermined), which can convert cell wall chitin produced by chitin synthase (encoded by Chs3), in the presence of its regulator (encoded by Csr2), to chitosan (a non-rigid soluble polymer) [157,158]. Interestingly, C. neoformans prefers the Cda1 gene, which is highly upregulated in the recovery strain from mammalian lungs and primarily selected for fungal proliferation [159].

Eight genes encode chitin synthase, viz Chs1 to Chs8, but only three chitin synthase regulators, Csr1 – Csr3, are functionally active. Unexplainably, as many of these encoding genes are in cryptococcal cells, a compensatory expression for each mutant rarely occurs except for Δchs3, where a slight increase in Chs5 and Chs7 was detected [157]. Furthermore, the Chs3-Csr2-Cda1 complex is essential for chitosan production for the cryptococcal cells to grow, disseminate, and invade the host at physiological conditions [160,161]. Under vegetative growth, Chs7 expression is entirely repressed, while Chs6 is remarkably and minimally detected in contrast to Chs1 and Chs4 expressions. However, Chs2, Chs3, Chs5, and Chs8 are highly induced during vegetative growth [157]. Surprisingly, none of these chitin-associated genes or regulators is essential for cryptococcal viability at 30^oC, but Δchs3 and Δcsr2 mutants are particularly hypersensitive to non-permissive temperatures and cell wall stressors such as Calcofluor White (CFW), Congo red, SDS, and caffeine.

4.6. Dnases

Cryptococcal cells can assimilate nitrogenous bases and nucleosides to produce endogenous nucleotides via the salvage pathway. By producing extracellular deoxyribonucleases (DNases), most infectious and parasitic fungi degrade the host and surrounding nucleic acids to salvage intermediates molecules needed to build nucleotides. The release of DNases is generally correlated with the increased activity of ureases that degrade excessive nitrogenous compounds in most fungi except in a few isolates of Endomycopsis fibuligera and Lipomyces starkeyi [162]. Though not too many studies are available on the cryptococcal extracellular nucleases [162]; however, available few studies have predicted the inclusion of secreted nucleases as virulence factors [163,164] because it may play a role in innate immune evasion as found with Group A Streptococcus infection characterised with polymorphonuclear neutrophil infiltration at the inflammatory region caused by bacterial infection [165,166]. A wide range of cryptococcal strain assessments for the production of DNase showed that neoformans and gattii species could secrete DNase. Based on their natural environment, the clinical isolates usually produce higher levels of DNase than environmental isolates [163,164].

4.7. Phosphatase

Ectophosphatase is a membrane-surface hydrolytic enzyme that often releases phosphate preferentially from phosphothreonine, phosphotyrosine, and phosphoserine substrates and is liable to be inhibited irreversibly by sodium orthovanadate (Na₃VO₄) [167]. Irreversible inhibition of this enzyme in C. neoformans has been characterised by reduced adhesion to animal epithelial cells – an important step towards fungal pathogenesis and tissue invasion [167].

The removal of phosphate groups (dephosphorylation) by phosphatase from transcription factors that are usually activated/deactivated by serine/threonine/tyrosine phosphorylation catalysed by protein kinases is one of the core regulatory pathways of MAPK. This controls the on/off regulation of signalling pathways orchestrated by transcription factors such as Hog1 MAPK, Ste20/Pak kinase, Pkc1 MAPK, and cAMP/Pka. Such phosphatase could operate optimally in an acidic medium (acid phosphatase) or alkali medium (alkaline phosphatase). For example, C. neoformans can release extracellular acid phosphatase encoded by Aph1 [168], which facilitates fungal adhesion to monocytes, but deletion of this gene attenuates the fungal virulence [167,169].

In a relevant study, an ecto-ATPase has been identified in C. neoformans with totally different activities compared to phosphatase and other types of ATPases. Cryptococcal ecto-ATPase, classified as E-type ATPase [170], functioned optimally in the presence of Mg²⁺, Mn²⁺, Zn²⁺, and Ca²⁺ in that others with a variety of nucleoside triphosphates as substrates (preferentially from ATP>GTP>UTP>ITP>CTP) [171]. Surprisingly, the secretion of this enzyme induced moderate antifungal resistance to FCZ in the presence of ATP [171].

4.8. Multifunctional Hydrolytic Enzymes in C. neoformans

The lack of trypsin, chymotrypsin, β-galactosidase, β-glucuronidase, and α-mannosidase in the species Cryptococcus showed that certain proteins and β-D-sugars might not be suitable for culturing this fungus [79]. However, many species of cryptococcal cells do possess esterase lipase (active against triacylglycerol with C4 – C8 acyl chain), α-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-fucosidase, galactokinase, D-lactate dehydrogenase [43,79], which means that various α-D-sugars, amino sugars, and disaccharides are suitable carbon sources for cryptococcal cells. Special attention is needed on some extracellular lipases capable of inducing antibodies in infected animal studies. Chen et al. discovered that 92% of the tested cryptococcal strains secreted butyrate and caprylate esterase lipases [172]. At the same time, some also produced acid phosphatase, naphthol-AS-BI-phosphohydrolase, and β-glucosidase, and the sera of infected rodents could precipitate several of these secreted proteins [172]. This shows that extracellular vesicles from C. neoformans contain proteins of immunological functions, which can be modified as vaccine candidates against systemic cryptococcosis.

4.9. Extracellular Vesicles

Extracellular vesicles (EVs) described as “virulence bags” are exosomal secretory vesicles containing capsular components (GXM, GalXM, and MP88) and enzymes like laccase (Lac1), urease, chitin deacetylase (Cda1, Cda2, and Cda3), glyoxal oxidase (Gox1, Gox2, and Gox3), multicopper oxidase (Cfo1 and Cfo2), ricin-type lectin-domain containing proteins (Ril1, Ril2, and Ril3), Sul7/PalI family motif protein (Tsh1, Tsh2, and Tsh3), Pr4/Barwin domain protein family (Blp2 and Blp4), and phosphatase [28,173,174,175,176]. Studies showed that the key components of EVs are virulent and antigenic, capable of activating macrophages to produce pro-inflammatory cytokines and inducing protection in animal studies [174,177,178,179]. As a standalone component, it could enhance cryptococcal cell infectivity and BBB penetration [180]. The production, packaging, and secretion of these vesicles are controlled by Sec genes, such as Sec6 [181].

The proteomic analysis of the C. neoformans secretory vesicles showed diverse functional proteins and enzymes classified as antioxidative and heat-shock proteins (Hsp70 and Hsp90), signal transduction and nucleotide salvage enzymes, ribosomal proteins, metabolic enzymes (for sugar, lipid, protein, and amino acid), nucleoproteins, membrane proteins (transporters, carriers, pumps, channels, adhesins, inositol-3-phosphate synthase, Rab proteins), cytoskeleton proteins (actin and actin-binding proteins, tubulin, and annexin), and a few mitochondria membrane proteins [28]. In addition, the presence of UDP-glucose dehydrogenase (encoded by Ugd1) and UDP-glucuronate decarboxylase (encoded by Uxs1) in the extracellular vesicles of C. neoformans showed the presence of functional enzymes that not only promote capsule formation but also enhance cell wall integrity and thermotolerance [182]. Cryptococcal EVs witnessed a considerably wide range of studies recently and have opened a new way of presenting the component of these vesicles as potential vaccine candidates against cryptococcal infections [183]. Many more studies will be needed to characterise the strain specificity in the antigenic properties of these vesicles.

5. Conclusions and Perspectives

The significant involvement of different hydrolytic enzymes, secreted proteins, and membrane-associated proteins like pumps, permeases, transporters, laccase, redox proteins, and ion-affinity proteins related to the pathogenesis of C. neoformans was discussed in this article. While each of these proteins plays a vital role in the survival, adaptation, and pathogenesis of C. neoformans in animals and humans, some are redundant with barely known functions. These attributes are common in other invasive fungi that cause candidiasis and aspergillosis. The multiplicity and redundancy of these proteins may be critical in catabolite repression to rescue the fungi when in an environment with alternative carbon sources, low oxygen, fluctuating pH, high salt contents, high oxidant level, low phosphate content, low ammonium content, and low micronutrient elements. The ability to secrete extracellular enzymes and virulence proteins is an alternative way of inducing cellular morphology, biofilm formation, cellular communication, filamentation, fruiting, mating, and nutrient assimilation. We described these proteins as weapons that facilitate tissue invasion, persistence, and infection in animal models of cryptococcosis. With the increasingly unfriendly environment and incessant use of antibiotics, pathogenic fungi are developing resistance to classical antifungals coupled with the emergence of sporadic strains that pose a threat to humans. Therefore, studies that target virulence-induced proteins and the membrane proteins necessary for fungi nutrient assimilation and survival should be targeted for new antifungal drug development; however, a careful consideration that will preclude the consequences in humans is highly recommended.