Laccase is an important virulence factor for the human pathogen, Cryptococcus neoformans. In this review, we examine the structural, biological and genetic features of the enzyme and its role in the pathogenesis of cryptococcosis. Laccase is expressed in C. neoformans as a cell wall enzyme that possesses a broad spectrum of activity oxidizing both polyphenolic compounds and iron. Two paralogs, CNLAC1 and CNLAC2, are present in the fungus, of which the first one expresses the dominant enzyme activity under glucose starvation conditions. Regulation of the enzyme is in response to various environmental signals including nutrient starvation, the presence of multivalent cations and temperature stress, and is mediated through multiple signal transduction pathways. Study of the function and regulation of this important virulence factor has led to further understanding of mechanisms of fungal pathogenesis and the regulation of stress response in the host cell environment.
Cryptococcus neoformans has emerged as a major fungal pathogen in immuno-compromised individuals such as patients with AIDS, organ transplant recipients and those receiving high doses of corticosteroid treatment . However, the spectrum of disease ranges beyond those with profound immunosuppression, as cases in immune-competent individuals have also been reported as well as a cryptococcal immune reconstitution syndrome in patients successfully treated for HIV [2–4]. Three subspecies of C. neoformans have been recognized, i.e., var. grubii (serotype A) , var. gattii (serotype B and C) and var. neoformans (serotype D) . In the United States, more than 80% of cryptococcosis in HIV-infected patients is caused by var. grubii, whereas in Europe, approximately half of all infections are caused by serotype D strains [5,7]. Three of the best-known virulence-associated attributes of C. neoformans are: (1) an extensive polysaccharide capsule, (2) the ability to grow at 37 °C and (3) expression of the virulence factor laccase . Laccase is responsible for a brown pigment produced by the organism on Niger seed agar, which has been used for almost 40 years as a selective medium for the isolation and identification of C. neoformans. Laccase-derived pigments require the addition of catecholamine substrates, in contrast to the dihydroxynaphthalene (DHN)-melanins of fungi such as Wangiella dermatitidis and Alternaria alternata which are produced from endogenous substrates . Laccases are shared by a broad family of organisms and provide a diverse array of biological functions including the breakdown of cellulose to provide nutrients to fungi, lignification of plant walls and the production of melanins in the insect midgut as a primitive immune defense against parasites (Fig. 1) . Using the diverse functions of this enzyme, C. neoformans has co-opted laccase into a sinister role as a virulence factor that converts mammalian substrates into reactive intermediates that protect the fungus and allow damage to the mammalian host. Molecular study of this enzyme has yielded insights into the workings of laccase and provides understanding to how a fungal saprophyte becomes a dangerous pathogen.
2Laccase activity and virulence of C. neoformans
Staib first described the formation of pigments produced by C. neoformans on Niger seed agar (Guizotia abyssinica), but this trait was not immediately associated with virulence . Later studies by Kwon-Chung's laboratory proposed an association with virulence based on a number of classical genetics studies. In the first, a UV-generated mel− mutant, 92t-1, in serotype D strain B-3502 and its progeny from a cross to B-3501, were injected into Swiss albino mice. The three Mel+ strains killed mice with a mean survival of 23, 46 and 29 days, respectively, while no mice inoculated with the mel− strains died. All strains tested appeared to have identical morphology and growth rates in rich media . In a follow-up study, Rhodes et al.  used a spontaneous mel− mutant of B-3502 crossed to the Mel+ B-3501 to produce three Mel+ and three mel− progeny. When injected into mice, the cumulative mortality was greater in the Mel+ than the mel− group and some mel−C. neoformans strains were found to have reverted to Mel+ suggesting a protective melanin-associated phenotype.
The above studies, while suggesting a role for melanin in virulence, were limited because, at the time, the strains could not be molecularly characterized. Identification of the laccase gene, CNLAC1, later provided the means to construct targeted knockout strains to test the role of laccase in melanin formation and virulence [14,15]. One such strain, 10S, was found to contain a 4-kb deletion of the 5′ end of the CNLAC1 gene, which resulted in absent laccase transcription and white colonies on Niger seed agar. Congenic Mel+ and mel− strains, 10S-BUC4 and 10S-BU4, were then made by complementation with either wild-type CNLAC1 or the transformation marker, URA5 and backcrossed five times. Chromosomal patterns and growth rates of the strains were identical. The mel− mutant 10S-BU4 was found to exhibit attenuated virulence using an intravenous mouse model, which was restored by complementation with wild-type CNLAC1. In the same experiment, an additional set of isogenic Mel+ and mel− mutants was constructed by complementing a previously described UV-derived mel− mutant with either intact wild-type CNLAC1 or the selection marker Ura5 alone. Virulence of the mel− strain was significantly reduced from that of Mel+ strain, consistent with the results from the 10S series . These experiments were recently repeated using an intratracheal mouse model which showed that laccase activity was required for extrapulmonary dissemination but not for pulmonary persistence of the organism . These data all strongly support the concept that laccase and its enzyme products are responsible for the pigment-associated virulence observed from earlier studies.
3Structural analysis and cellular location of the laccase enzyme from C. neoformans
Laccase of C. neoformans was initially referred to as a phenol oxidase or diphenol oxidase because of its ability to make colored products from a wide variety of phenolic compounds having two hydroxyl groups, but not tyrosine . However, a number of enzymes including tyrosinases, peroxidases and the yeast iron transporter, Fet3, also share these substrate activities, thus requiring additional studies to identify the enzyme as a laccase . Purification and cloning of the cryptococcal enzyme allowed its identification as a laccase based on several properties . Atomic absorption measurements showed the enzyme to contain 4 mol/mol of copper and to have an absorbance at 610 and 320 nm, characteristic of type I and III copper of laccases, respectively. These sites were corroborated by the presence of several consensus copper-binding domains from amino acid sequence derived from CNLAC1 cDNA (Fig. 2(a)). Molecular modeling of cryptococcal laccase based on X-ray data of the Coprinus cinereus laccase  shows the intimate binding of four copper centers as well as a predominant β-pleaded sheet motif within the protein (Fig. 2(b)). Purified cryptococcal laccase was also shown by HPLC and mass spectroscopy to produce an important melanin intermediate o-dopachrome (DAQ) from dopamine. The enzyme also exhibited a high substrate specificity towards catecholamines which may contribute to optimization of cryptococcal laccase for the neurotropism of C. neoformans[16,19].
Cell localization studies have shown that laccase is covalently linked to the carbohydrate cell wall. Using a monoclonal antibody to laccase, immuno-electron microscopy demonstrated the enzyme within the cell wall matrix (Fig. 2(c)) which was also evident by confocal epi-fluorescence using a green-fluorescent-tagged laccase expression construct (Fig. 2(d)). This peripheral location allows laccase to access small substrates such as catecholamines and metals that permeate through the porous capsule to allow effective participation in events leading to immune modulation .
4C. neoformans contains two laccase genes: CNLAC1 and CNLAC2
Earlier studies suggested the presence of only one genomic copy of the laccase gene in C. neoformans serotype D strain B-3501 based on Southern blot analysis . However, genome projects using several different strains have revealed a second laccase homolog present in the genome of C. neoformans (Stanford Genome Technology Center C. neoformans Genome Project, http://sequence-www.stanford.edu/group/C.neoformans/index.html; MIT Center for Genome Research, http://www.broad.mit.edu/annotation/fungi/cryptococcus_neoformans). In either serotype A (H99) or D (JEC21), the two laccase genes appear to be in a tandem repeat about 5.3 kb apart in the same orientation with CNLAC1 located downstream from a second paralog, CNLAC2 (Fig. 3(a)). The two genes exhibit 65% nucleotide identity and 72% amino acid sequence identity with conservation of important laccase copper-binding sites using a CLUSTAL W algorithm (Fig. 2(b); ). The paralogs appear to show many of the features typical for permanent preservation of duplicate genes . For example, the promoter region of the two copies shares little similarity suggesting that the regulation of CNLAC2 has become distinct from CNLAC1. Indeed, deletion of 2.5 kb of the CNLAC1 ORF abolishes laccase enzyme activity under glucose starvation conditions (Fig. 3(b)), suggesting that CNLAC1 alone expresses the dominant laccase enzyme activity under these conditions. Neither gene appears to be a non-functional pseudogene, however, as transcripts of both have been identified from mRNA combined from cells grown under seven different conditions as described in the TIGR JEC21 EST database (http://www.tigr.org/tdb/e2k1/cna1/).
5Roles of laccase-derived products in the virulence of C. neoformans
The production of melanin by C. neoformans during infection is a compelling story given the availability of potential melanin-producing substrates in the central nervous system such as the neurotransmitters, dopamine, nor-epinephrine (NE) and epinephrine. Indeed, in vitro, black melanin pigments have been shown to be protective for C. neoformans against oxidants, microbiocidal proteins and antibiotics and to alter the cellular charge of fungal cells making them resistant to phagocytosis [23–29]. However, a controversy exists regarding the presence of these melanin pigments during brain infection. To understand the issues surrounding this controversy, it is important to consider principals related to the enzymatic production of melanin by laccase. Laccase initiates the conversion of catecholamines such as dopamine into melanin by oxidizing it to the highly reactive o-quinone (DAQ), which then non-enzymatically condenses with either itself or with other metabolites when present in sufficient concentrations. For example, when the yeast is incubated on agar or in solutions containing large amounts (100 mg l−1) of dopamine and deficient in competing DAQ scavengers such as proteins or carbohydrates, typical black pigments are readily produced (Fig. 4(a), first column). These black pigments are produced by uninhibited polymerization of DAQ and have been shown to be typical eumelanin pigments, both by chemical degradative methods [30,31] and by use of a monoclonal antibody developed against melanin pigments  (see scheme Fig. 4(b)). However, as cells are incubated with progressively lower concentrations of catecholamines approaching that present in murine brains (1 mg l−1; ) or if DAQ scavengers such as proteins or carbohydrates are present in significant amounts [34,35], production of melanin pigment becomes less with the same amount of laccase enzyme present (Fig. 4(a), last column and Fig. 4(b)– low DA concentration). Indeed, isolation of laccase-producing C. neoformans strains from mouse brains during an acute 2-week infection yields completely white cells, despite the identification of DAQ products, suggesting the formation of something other than a typical eumelanin pigment . A lack of melanin pigments was also found on a genetically altered laccase-positive strain of C. neoformans that caused a chronic 3-month infection in mice (unpublished observation). However, other investigators have shown that similar laccase-positive cells isolated from both animal and human brain exhibited immunoreactivity with a monoclonal antibody developed against melanin and showed increased stability to acid hydrolysis, consistent with a true melanin polymer [37,38]. One explanation of these apparently contradictory results is that antibodies derived from a DAQ–DAQ polymer (melanin) also may recognize structurally similar DAQ–protein adducts or DAQ–carbohydrate crosslinks, the latter of which would also be expected to convey increased acid stability to the fungal cell wall. Indeed, given the heterogeneity of the extracellular matrix of brain during cryptococcal infection, there may be a number of dopamine-derived products produced by laccase during brain infection. However, it is important to note that previous immunological experiments using cells containing heavy black pigments produced in the presence of large quantities of dopamine may have to be interpreted cautiously, as the cells studied may not accurately mimic the non-pigmented cells that are present during brain infection.
Dopamine products may not be the only laccase-derived products to be important in the pathogenesis of cryptococcosis, and recent studies are beginning to explore the rich diversity of laccase enzyme activities that could play a role in virulence. For example, the presence of laccase has been shown to result in protection against murine alveolar macrophages without the addition of catecholamines, suggesting an intrinsic protective effect of laccase. In these studies, the finding of a novel iron oxidase activity of cryptococcal laccase as well as abolition of the laccase-protective effect by mannitol and accentuation by iron suggested that laccase may modulate macrophage-dependent Fenton reactions . Another interesting laccase activity is the ability to produce oxygenated lipids from fatty acids . One of the most important groups of these lipids are the prostaglandins and leukotrienes which are potent immunomodulators of the immune response and have recently been shown to be produced by C. neoformans. Studies are currently underway to dissect the role of laccase in the production of such novel immunodulatory compounds (G. Huffnagle, personal communications).
6Molecular regulation of laccase and signal transduction pathways
Laccase activity of C. neoformans is induced by a number of environmental stimuli including the metals calcium, iron and copper, and repressed by nutrients such as glucose and nitrogen as well as by elevated temperature (see Table 1). Although virulence factors such as laccase are often thought of as intrinsic attributes of an opportunistic pathogen, it is important to realize that these factors and their regulatory pathways have evolved under environmental rather than under host pressures. Nevertheless, these cellular pathways define the pathogen's ability to cause host damage because the responses generated during evolution to allow survival in the environment are also uniquely appropriate for survival and pathogenesis in the mammalian host. For example, glucose deprivation stimulates laccase expression as a foraging response to degrade cellulose in the hollows of trees and also leads to laccase expression under low glucose conditions present in the brain during infection (Fig. 6) [15,42,43]. In the same way, induction of laccase by metals may have evolved under environmental pressures, but has been beneficial to Cryptococcus during pathogenesis. In the environment, induction of fungal laccases by iron has been proposed to modulate iron-catalyzed Fenton reactions during cellulose depolymerization . In the mammalian host, iron within the phagolysosome may induce iron oxidase activity of laccase, which, in turn, reduces potentially toxic Fenton reactants . Other regulatory stimuli are more difficult to understand in relationship to pathogenesis but provide key insights into the regulation of cryptococcal virulence. For example, elevated host temperatures (37 °C) are a well-known repressor of laccase (Fig. 6)  which makes sense from an environmental point of view, since the free-radical-generating capability of laccases may contribute to cellular toxicity at elevated temperatures, but appears counter-intuitive to the concept of a pathogen that would appear to benefit most from increased expression of virulence-related attributes. This apparent contradiction reminds us of the complexity of virulence regulation and provides caution against developing generalized intuitive models of virulence regulation.
Table 1. Inducers and repressors of laccase in C. neoformans
To begin to identify transcriptional regulators of laccase, Zhang et al.  conducted a reporter dissection of a 2-kb 5′-UTR of CNLAC1 from serotype D under glucose starvation conditions. These studies utilized a newly developed cryptococcal reporter plasmid, pVEW, and electromobility shift assay (EMSA) techniques adapted for C. neoformans, and were the first systematic structural and functional studies of a 5′-enhancer region of a basidiomycete fungus. As diagramed in Fig. 5(a), two groups of interactive enhancer regions, located over a range of 1.5 kb from the mRNA start site, were found to be involved in CNLAC1 regulation (region 2: −1721 to −1615 and region 7) in addition to a TATA promoter at position −539. Region 2 contains a consensus GC-rich site, a heat-shock transcription factor (HSF) consensus site and region 7 contains a consensus E2F site, each of which showed significant binding to nuclear proteins under derepressed conditions. The formation of additional larger EMSA complexes using DNA sequences spanning the GC-rich and the HSF consensus site of region 2 suggested cooperativity in the interaction between transcription factors binding these two sites. Two regions of repression were also evident under derepressed conditions (region 5: −1351 to −1207 and region 8: −991 to −971). The presence of multiple interactive enhancer sites over a fairly large upstream range suggests that CNLAC1 transcriptional regulation exhibits features often associated with higher eukaryotic regulation. The relatively large number of enhancer and repressor sites also suggest that the enzyme is tightly regulated to respond to a number of environmental and host conditions that may allow for expression to vary within different environments or tissues. Efforts are currently underway to identify these transcription factors and to relate them to the regulation of laccase under specific conditions. Identification of laccase-associated regulators may also allow for the identification of additional down-stream regulatory targets that may act in concert with laccase to produce the full laccase-associated virulence phenotype suggested by the original melanin-negative mouse studies of Kwon-Chung.
Metal induction of laccase has been best studied with respect to copper. Quantities as low as 5 μM resulted in significant induction of laccase transcription and enzyme activity (Fig. 6) . Copper induction of laccase was also found to occur through region 2, which contains two metal response elements similar to the binding site for the copper-responsive transcription factor Cuf1 in Schizosaccharomyces pombe (see Fig. 5(a)). We are currently investigating mechanisms of copper induction and its relationship to virulence.
Components of signal pathways have also been reported to be required for laccase expression . A G-protein α-subunit homolog, GPA1, from C. neoformans was disrupted via homologous targeting resulting in significantly attenuated virulence and laccase expression . The authors also found that exogenous cAMP suppressed the gpa1 mutant phenotype and restored laccase expression. Disruption of two downstream targets in the signal transduction pathway, an adenylyl cyclase gene which is the target of Gα and a cAMP-dependent protein kinase subunit gene (PKA1) which is activated by cAMP, also reduced laccase activity in a separate experiment. These results showed that the Gα-cAMP-PKA signaling pathway modulates laccase expression in C. neoformans, sensing extracellular stimuli such as hormones and nutrients to regulate cell development and adaptation (see Fig. 5(b)). In other studies, Chang et al. [52,53] demonstrated that homologs of the MAP kinase cascade target Ste12a (a-mating-type-specific) and Ste12α (α-specific) in C. neoformans are both required for wild-type laccase activity. However, Yue et al.  reported that disruption of Ste12α in a serotype A strain H99 had no effect on laccase expression. These disparate observations suggest that regulation of laccase expression may be strain-dependent. Also, Wang et al.  found that two cyclophilin A homologs from H99 are required for laccase activity. Unlike most organisms that only have one copy of the cyclophilin A gene, C. neoformans has two cyclophilin homologs. Knockout of either of the genes has no obvious effect on laccase activity. However, after a double knockout of both copies, melanin biosynthesis decreased. Cyclophilin A is the target of the immunosuppressant and antimicrobial drug cyclosporin A. The drug-cyclophilin A complex virtually inhibits a Ca2+ calmodulin-activated protein phosphatase calcineurin. This result supports an earlier observation that Ca+ ion increases laccase activity, though the precise mechanism remains unknown. These results show the complexity of laccase regulation in C. neoformans and the intimacy to which laccase is associated with virulence and stress response in this organism.
7Probing the biosynthetic machinery of virulence in C. neoformans
While much research has centered on the regulation of virulence, less is known about the cellular machinery involved in the production of these traits. To study these processes, an insertional mutagenesis method was developed and used to isolate genes required for laccase expression . The first gene identified was Vph1, which encodes a subunit of a vacuolar proton pump H+-ATPase . H+-ATPases are large complexes of at least 13 subunits and are localized to intracellular membranes of vesicles including the Golgi, vacuole and secretory vesicles. Its function is to maintain low pH in the membrane compartments . The cryptococcal Δvph1 mutant was defective in laccase enzyme activity, capsule and virulence but laccase transcription was preserved. Further studies of laccase in the Δvph1 mutant revealed that the defect in vesicular acidification led to ineffective metaliation of the laccase enzyme that could be restored by the addition of exogenous copper . Interestingly, another cryptococcal mutant also contained an insertion within a vesicular pump, this time a chloride pump, CLC-A. CLC-A shares high homology (63%) to GEF1, a chloride channel identified in S. cerevisiae. Gef1 is required for cation homeostasis and copper metaliation of the related multi-copper oxidase involved in iron uptake, Fet3 . Surprisingly, in the cryptococcal Δclc-a mutant, not only was laccase enzyme activity defective, but laccase mRNA was undetectable . Again, copper addition to the media restored both laccase transcription and activity. These results suggest a role for chloride-dependent metaliation of factor(s) required to induce laccase transcription. In addition, the pleotropic effects on laccase, capsule and temperature-dependent growth of the Δvph1 mutation suggest that vesicular pump targets may make suitable drug targets for future development.
8Future directions of laccase research
Despite a tremendous amount of work, many fundamental questions about the biological functions of laccase remain unanswered, suggesting several exciting areas of research in a number of disciplines. The development of molecular–biological techniques in the fungus has now allowed the identification of regulators of laccase by methods such as insertional mutagenesis and complementation of mutants. Tools such as microarrays, dependent on new data generated from three genomic sequencing projects, will help to identify gene products regulated in concert with laccase that may play a synergistic or additive role in the virulence of the organism. At the protein level, recent studies showing an expanded spectrum of enzyme activity of the enzyme, such as ferroxidase activity, may suggest additional products that serve immunomodulatory or protective roles for the fungus. Finally, newly developed methods recently applied to the study of melanin in vitro such as solid-state NMR  may allow for the molecular characterization of dopamine-derived products in the brain.
We thank Dr. G. Mauk for work on the modeling of the cryptococcal laccase. This work was supported, in part, by United States Public Health Service Grant NIH-AI49371, A145995, and an American Heart Association Fellowship.