Minireviews

Virulence-Associated Enzymes of Cryptococcus neoformans

Fausto Almeida, Julie M. Wolf, Arturo Casadevall
DOI: 10.1128/EC.00103-15

ABSTRACT

Enzymes play key roles in fungal pathogenesis. Manipulation of enzyme expression or activity can significantly alter the infection process, and enzyme expression profiles can be a hallmark of disease. Hence, enzymes are worthy targets for better understanding pathogenesis and identifying new options for combatting fungal infections. Advances in genomics, proteomics, transcriptomics, and mass spectrometry have enabled the identification and characterization of new fungal enzymes. This review focuses on recent developments in the virulence-associated enzymes from Cryptococcus neoformans. The enzymatic suite of C. neoformans has evolved for environmental survival, but several of these enzymes play a dual role in colonizing the mammalian host. We also discuss new therapeutic and diagnostic strategies that could be based on the underlying enzymology.

INTRODUCTION

The facultative intracellular fungal pathogen Cryptococcus neoformans is the causative agent of cryptococcosis, a disease that primarily affects individuals with impaired immunity, such as those with advanced HIV infection (1, 2). C. neoformans is a ubiquitous environmental fungus associated with both pigeon guano and eucalyptus trees, and its environmental niche ranges from the tropical to the temperate (3). C. neoformans infection is acquired from the environment via inhalation, after which it forms a local infection in the lungs. This infection may be cleared, may be contained as a granuloma, or may disseminate from this initial site, leading to pneumonia and/or meningoencephalitis, the latter being uniformly fatal if untreated. Despite the availability of antifungal therapy, more than 650,000 people die each year from C. neoformans infection (1, 2, 4). The principal virulence factors of C. neoformans are a polysaccharide capsule, melanin production (5, 6), the ability to grow at body temperature (7), and the secretion of extracellular enzymes (7). These virulence factors confer a selective advantage to C. neoformans for both residing in the environment and in a mammalian host. Tightly controlled regulation leads to expression of enzymes required for fungal survival and host damage once inside its mammalian host (8).

Many enzymes contribute to the composite cryptococcal virulence phenotype. Dissection of the pathogenic role of these enzymes will enhance our understanding of cryptococcal pathogenic mechanisms and facilitate directed inhibitor development and/or vaccine discovery. We have included a table summarizing basic information regarding global C. neoformans enzymology (Table 1) and a schematic displaying localization of most of the highlighted enzymes discussed (Fig. 1). In this review, we discuss in detail the most important virulence-associated enzymes (Table 2), as well as additional target enzymes with potential for rational antifungal drug design (Table 3). We examine this information in the context of infection and analyze candidate target enzymes for drug inhibition and vaccine discovery.

View this table:
TABLE 1

Described enzymes in Cryptococcus neoformans

FIG 1
FIG 1

Enzymes are crucial for fungal pathogenesis and can alter the infection process. These enzymes are potential targets for new antifungal agents. (A) Some pathogenesis-related enzymes are retained to be active inside the cell body, while others are secreted. Some, like laccase, are both retained and secreted. (B) Of those released, some are secreted using traditional secretion systems, while others are included as cargo in extracellular vesicles.

View this table:
TABLE 2

Enzymes related to the virulence in Cryptococcus neoformans

View this table:
TABLE 3

Possible target enzymes for rational antifungal drug design

POLYSACCHARIDE CAPSULE

C. neoformans is the only fungal pathogen with a polysaccharide capsule, an outermost polysaccharide structure located just outside the cell wall. The two major polysaccharide capsule constituents are glucuronoxylomannan (GXM) and glucuroxylomannogalactan (GXMGal) (9–11). GXM is the major component of C. neoformans, a compound of α-1,3-linked mannose residues with xylosyl and glucuronyl side groups (12), whereas GXMGal is made of α-1,6-linked galactose residues with xylose, mannose, and glucuronic acid (13). The capsule also contains nonpolysaccharide components, such as mannoprotein (MP) (10, 14, 15), although these MP components may represent transient components destined for cellular export.

The role of capsule in environmental growth is unknown, although speculations have been made that the capsule protects the fungus from desiccation or acts as a food source (16). During mammalian infection, the capsule participates in resisting phagocytosis and modulating the immune response (17–21). Not only protective against phagocytosis in both mammalian and lepidopteran hosts (22, 23), the capsule also protects the fungus after ingestion by serving as a free radical sink that can shield the cell from oxidative bursts (24). Hence, while the capsule is not part of the enzymatic microbial arsenal, the machinery responsible for capsule synthesis and assembly does directly contribute to cryptococcal virulence. The primary structures of GXM and GXMGal subunits have been defined, but the mechanisms of subunit assembly into >106-Da branched structures have not (25, 26). The degree of branching and conformation of polysaccharides imply an elaborate assembly and regulatory enzymatic machinery (27).

The subunits of GXM and GXMGal are large glycans that require several glycosyltransferases for synthesis. Both xylosyltransferase and glucuronyltransferase activities are involved in capsular polysaccharide biosynthesis (28–31). A xylosyltransferase, Cxt1, was the first glycosyltransferase identified with a defined role in capsule synthesis (31). It is a large transmembrane protein with β-1,2-xylosyltransferase activity (31), and deletion of the corresponding gene (CXT1) decreased capsular β-1,2-xylose linkages and fungal growth in the lung in a mouse model of infection (30).

Several acapsular mutants were obtained through identification of rough colonies. This type of screen identified four genes required for capsule formation: CAP10, CAP59, CAP60, and CAP64. Although these genes are not essential, their mutation does confer defects in growth and in mouse models of infection (17, 32–35). Cells from these mutant strains lacked or produced extremely reduced capsule, but these mutations did not correlate with enzymatic deficiency in UDP-glucose dehydrogenase, UDP-glucuronate decarboxylase, UDP-glucuronyl:acceptor transferase, UDP-xylosyl:acceptor transferase, or lipid-linked oligosaccharide biosynthetic pathways. CAP10 is a putative xylosyltransferase gene, and cap10Δ mutants show a pleiotropic phenotype, which includes enlarged cell size, smaller extracellular vesicles, and affected expression of some virulence factors (36). CAP10 therefore is required for both capsule formation and other aspects of fungal virulence.

Capsular lactonohydrolase also affects multiple capsule-related phenotypes (37). A strain lacking lactonohydrolase (lhc1Δ) produced capsules with a larger size and altered branching, density, and solvation compared to the parental strain. These capsular structure alterations increased virulence in murine infection (37). Taken together, these results suggest that lactone may be involved in cross-linking of the capsule.

α-1,3-Mannosyltransferase (encoded by CMT1) synthesizes the mannose backbone of GXM and thus plays a crucial role in capsule synthesis. However, α-1,3-mannosyltransferase activity is more involved in in serotype A capsule biosynthesis than in the serotype D C. neoformans (38, 39). Serotypes A and D represent two of the four C. neoformans serotypes: C. neoformans var. neoformans (serotypes A and D) and C. neoformans var. gattii (serotypes B and C), which can be distinguished according to their growth differences on diagnostic media (40). The strain-specific capsule synthesis differences, such as the role of CMT1, show the importance of studying multiple strain backgrounds.

Much remains to be learned about the enzymatic machinery involved in capsule synthesis, including enzyme localization and kinetics. Detailed studies of capsule structure and the enzymatic machinery involved are critical for a better understanding of the function of the capsule production and regulation.

MELANIN SYNTHESIS

Melanin formation protects C. neoformans from oxidative damage as well as from both heat and cold (41, 42). Melanin is synthesized on 2,3- or 3,4-diphenol substrates by a phenoloxidase and accumulates in the C. neoformans cell wall (43, 44). The melanin-synthesizing enzyme has two classical laccase characteristics: a glycosylated copper-containing protein with the ability to oxidize diphenolic substrates and the ability to produce decarboxy dopachrome (45, 46). C. neoformans melanin synthesis occurs only in the presence of exogenous dihydroxyphenols, since no known C. neoformans endogenous substrate exists. Several diphenols can serve as the substrates for pigment synthesis by C. neoformans laccase (47), such as the substrates consisting of para- and ortho-diphenols, monophenols, l-dopa, and esculin, indicating that the enzyme has broad specificity and the ability to generate pigments from different compounds (47–53). Iron increases laccase activity, but hydrogen peroxide has no effect on enzymatic activity, despite the antioxidant properties of melanin (54).

The genes LAC1 and LAC2 encode two laccases, but a single deletion in LAC1 is able to prevent melanin production (55–58). Lac1 localizes in the cell wall, while Lac2 is cytoplasmic, but Lac2 can localize to the cell wall in the absence of Lac1 (55). lac1Δ mutants are easily identified as white colonies when cultivated on catecholamine-containing media (59). The lac1Δ mutant shows decreased virulence in survival studies with rabbit infection (59), corroborating the important role in the fungal virulence (5, 46). In addition to its cell wall localization, laccase is packaged into extracellular vesicles, a nontraditional mechanism of secretion, and can therefore mediate damage away from the laccase-producing fungal cell (Fig. 1).

Melanin is considered a powerful antioxidant, since it may protect cryptococcal cells against oxygen- and nitrogen-derived oxidants of the type made by host effector cells (5, 60–62). In addition to its capacity to absorb free radical fluxes, melanin can also contribute to acquired resistance against to the antifungals amphotericin B and caspofungin, since nonmelanized cryptococcal cells are more susceptible than melanized cells to amphotericin B and caspofugin. Moreover, killing assays demonstrated that addition of melanin particles to amphotericin B or caspofungin significantly reduces their toxicities against C. neoformans (63–65). Thus, melanin and laccase are considered promising targets for drugs against C. neoformans infection.

EXTRACELLULAR ENZYMES

As nature's “recyclers,” environmental fungi secrete a number of degradative enzymes to breakdown macromolecules and obtain nutrients in the environment (7, 66–69). C. neoformans is no exception and releases a number of lipases, proteases, and DNases. However, during the infection process, the same degradative enzymes contribute to virulence by destroying tissues, promoting fungal survival, and interfering with effective immune responses.

Urease is almost universally expressed by C. neoformans isolates. In the environment, C. neoformans is often isolated from avian excreta (70, 71). To survive and grow on this medium, the fungus must metabolize creatinine, xanthines, and uric acid. High urease activity may benefit the fungus under these conditions (72–74), as the enzyme catalyzes the hydrolysis of urea to ammonia and carbamate. Urease is considered a major cryptococcal virulence factor (75). A urease knockout (URE1) strain of C. neoformans was significantly less virulent than the wild-type strain in a mouse model of infection (76). Urease plays a role in fungal escape from the lung to cross the blood-brain barrier but is not required for fungal growth once inside the brain (76). Urease production varies among clinical isolates; however, the vast majority (99.6%) demonstrate some level of urease activity (74, 77, 78). Nevertheless, occasional urease-negative variants have been isolated in clinical isolates (77), suggesting that this enzyme can be dispensable, provided that there are compensatory virulence mechanisms.

Extracellular DNase is produced by C. neoformans in high quantities (79). This DNase may degrade host DNA secreted by neutrophils as part of the innate immune response (80) and additionally may supply C. neoformans with nucleotides. A survey of several yeast species, including C. neoformans, suggests a correlation between urease activity and extracellular DNase production (79). DNase activity is stronger in clinical strains than in environmental strains, further suggesting DNase may play a role as a virulence factor (81).

Superoxide dismutases (SODs) convert superoxide to hydrogen peroxide and oxygen (82). Two SODs have been described in C. neoformans (83–88). SOD contributes to virulence of C. neoformans by facilitating growth within macrophages (89), through a mechanism that is likely to involve protection of the fungus against superoxide generated by host immune response (2). In this regard, melanin and SOD may stimulate complementary defenses for the C. neoformans cells' protection against oxidative damage. SOD production is regulated by temperature, with increases in expression at 37°C compared to 25°C. Thus, increased SOD production at body temperatures may protect the fungus against oxidizing agents produced from host effector cells (90).

Phospholipases degrade cell membrane phospholipids in an enzyme-dependent mechanism. C. neoformans extracellular supernatants contain phospholipase B, phospholipase C, lysophospholipase, and acyltransferase (91–93), and phospholipase activity supports fungal attachment to host cells (94). Phospholipase B promotes fungal invasion of host tissue (95) and hydrolyzes phospholipids in lung surfactant and the plasma membrane (92, 96). Moreover, it contributes to fungal survival by maintaining cell wall integrity (97) and provides nutrients that can be used as sole carbon sources by C. neoformans during the infection (98, 99). As described above, it has also been localized to the cell wall (97), and its transport to the plasma membrane and cell wall is N-glycan dependent (100). Phospholipase C is crucial for several virulence phenotypes (melanin production, growth at 37°C, phospholipase B secretion, and antifungal drug resistance) and is also involved in homeostasis regulation, cell separation following cytokinesis, and cell wall integrity (101, 102).

Phosphatases remove a phosphate group from their substrates and play important roles in regulating protein structure and signaling cascades (103, 104). A secreted acid phosphatase is involved in fungal cell adhesion to host tissues, suggesting an important role in establishing infection (105). Acid phosphatase is encoded by the gene APH1 in C. neoformans. In both wax worm and murine models of cryptococcosis, aph1Δ strain-infected animals survived longer than those in the wild-type-infected model (106), demonstrating the importance of this enzyme during infection.

Proteases break down proteins and are considered important virulence factors, contributing to tissue invasion, colonization, and alteration of the host defense response. Protease activity in C. neoformans cultures has been reported by several investigators (107–111). Proteases play important roles in host cell penetration and virulence of C. neoformans (112). Recently, a metalloprotease was identified by proteomic analyses of the extracellular proteins from C. neoformans and found to be required for invasion of the central nervous system in murine infection of C. neoformans (113). Moreover, the metalloprotease knockout (mpr1Δ) strain was unable to cross the endothelium in an in vitro model of the human blood-brain barrier (113).

DRUG DESIGN AND RESISTANCE

Definition of enzymatic pathways can provide crucial targets for antimicrobial drug design. One way to identify targets is to identify unique metabolic requirements for cryptococcal growth and/or virulence. An example of this is the C. neoformans phosphoribosylaminoimidazole carboxylase gene (ADE2). Mutants with mutations in this gene lack an enzyme required for adenine synthesis and thus have reduced virulence compared to the wild-type strain (114). This observation suggests potential for rational drug design utilizing differences in adenine synthesis pathways between host and pathogen (as first suggested in reference 7). Several candidate enzymes in C. neoformans have been studied regarding fungal amino acid synthesis (e.g., homocitrate synthase, homoisocitrate dehydrogenase, α-aminoadipate reductase, saccharopine reductase, and saccharopine dehydrogenase) (115). However, comparisons between C. neoformans var. neoformans and C. neoformans var. gattii have shown that candidate targets do not necessarily translate across Cryptococcus species. Saccharopine reductase, an enzyme involved in lysine synthesis, was not detected in C. neoformans var. gattii but was detected in C. neoformans var. neoformans. This C. neoformans var. gattii strain was able to grow even in the absence of lysine (115), indicating that further research to identify enzymes essential across all Cryptococcus species is required.

Another essential process for C. neoformans is protein myristoylation. C. neoformans myristoyltransferase catalyzes the transfer of myristate from coenzyme A (CoA) to the amino-terminal glycine residue of a subset of cellular proteins, and this enzyme is essential for C. neoformans viability (116, 117). N-Myristoyl proteins and myristoylation inhibition by the myristic acid analog 4-oxatetradecanoic acid are crucial for this organism (118). Thus, therapies directed at myristoylation may also be a possible target for rational antifungal drug design.

In some cases, an antifungal target is well defined, but multiple enzymes involved in target synthesis provide several inhibitory strategies. Sterols and their synthetic pathways are major antifungal targets in many fungi, but resistance leads to difficulties in patient treatment. Fluconazole-resistant strains require a 10-fold-higher drug concentration to inhibit sterol 14α-demethylation (119), rendering the drug clinically unfeasible. The molecular basis for differential enzyme function has been identified in several clinical C. neoformans strains (120). One documented fluconazole- and amphotericin-resistant C. neoformans patient isolate showed reduced relative sterol content and a defect in δ-8-isomerase, depleted ergosterol, and accumulated aberrant δ-8-double-bonded ergosterol precursors (121, 122), suggesting the ability to form membrane pores due to aggregation and formation of amphotericin-ergosterol complexes. Another study evaluating fluconazole- and amphotericin-resistant isolates observed reduced ergosterol content in the isolates, as well as reduced sensitivity of P450 14α-demethylase to inhibition by fluconazole, and a defect in sterol Δ8-Δ7 isomerase (123). Another C. neoformans strain with defective sterol Δ8-Δ7 isomerase was discovered in an amphotericin B-resistant isolate from an AIDS patient (124). These mutations in sterol synthesis enzymes explain resistance evolution and generate targets to fight it with. This information can also help in rational drug design methodologies.

Identification of key virulence-related enzymes is yet another route toward finding an effective drug target. Glycosphingolipids are essential to regulate survival and/or replication of C. neoformans in the phagolysosome, as well as in the extracellular environment of the host (125–127). Glucosylceramide plays critical role in pathogenicity of C. neoformans, since glucosylceramide synthase (Gcs1) is required for virulence in the murine model of infection (128). gcs1Δ mutants corroborate the crucial role of the glycosphingolipid synthesis in regulation of this considerable aspect of C. neoformans virulence (127). Thus, the glycosphingolipid pathway may also be a reasonable target for antifungal therapies.

Laccase has been considered a drug target in C. neoformans because melanization is critical to virulence. Inhibition of fungal melanization in murine infection using the herbicide glyphosate prolonged average mouse survival. Glyphosate is an inhibitor of both the shikimate acid pathway and l-dopa polymerization (129). Thus, therapies directed at melanization may also be a potential target for antifungal drug design.

Occasionally, a drug proven to work on one microbial pathogen will also be effective against another. This appears to be the case with several viral medications. Drugs such as indinavir and oseltamivir inhibit human immunodeficiency virus (HIV) protease or influenza virus neuraminidase, respectively, and demonstrate the impact an enzymatic inhibitor can have in the clinic (130, 131). The use of protease inhibitors has shown positive effects on C. neoformans and Candida albicans infections, where drug treatment was associated with inhibition of fungal growth and proliferation in vitro (132, 133). These are likely inhibiting the fungal proteases, both cell associated and as part of the fungal secretome.

CONCLUSION

Recent advances in genomics, proteomics, transcriptomics, and mass spectrometry have facilitated the identification and characterization of new fungal enzymes, including those specific to both fungi and C. neoformans. These enzymes are required for many important biological processes, including growth and infection. The importance of the secretome in cryptococcal pathogenesis is apparent from the fact that strain differences in secreted enzymes correlate with their virulence (134). Nonetheless, important questions remain. Future research on cryptococcal enzymology will not only identify new enzymes and their roles during infection but also pinpoint enzymatic targets for the development of antifungal agents.

ADDENDUM IN PROOF

There are, of course, many enzymes involved in signaling cascades, most of which were not discussed in this review. One such enzyme is vital to stress response in C. neoformans and other pathogenic fungi and thus merits a well-deserved mention: the calcium-dependent phosphatase calcineurin (W. J. Steinbach, J. L. Reedy, R. A. Cramer, Jr., J. R. Perfect, J. Heitman, Nat Rev Microbiol 5:418–430, 2008). This enzyme is required for growth in a mammalian host and therefore is necessary to cause disease (A. Odom, S. Muir, E. Lim, D. L. Toffaletti, J. Perfect, J. Heitman, EMBO J 16:2576–2589, 1997). Studies utilizing calcineurin inhibitors for invasive disease in animal models have shown promising results, and this work is now moving into translational stages (D. P. Kontoyiannis, R. E. Lewis, B. D. Alexander, O. Lortholary, F. Dromer, K. L. Gupta, G. T. John, R. del Busto, G. B. Klintmalm, J. Somani, G. M. Lyon, K. Pursell, V. Stosor, P. Munoz, A. P. Limaye, A. C. Kalil, T. L. Pruett, J. Garcia-Diaz, A. Humar, S. Houston, A. A. House, D. Wray, S. Orloff, L. A. Dowdy, R. A. Fisher, J. Heitman, N. D. Albert, M. M. Wagener, N. Singh, Antimicrob Agents Chemother 52:735–738, 2008, http://dx.doi.org/10.1128/AAC.00990-07). Other enzymes involved in stress responses may similarly be identified and targeted in the future.

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