Medically important Cryptococcus species

1.     CLASSIFICATION
Kingdom:      Fungi
Phylum:        Basidiomycota
Class:            Tremellomycetes
Order:           Tremellales
Family:         Tremellaceae
Genus:          Cryptococcus
Species:        neoformans

2. INTRODUCTION
The genus Cryptococcus comprises more than 50 species and the medically significant species includes only Cryptococcus neoformans and Cryptococcus gattii which are considered principal pathogens in humans. Previously, C. neoformans was defined as having two varieties-var neoformans and var gattii. However, based on the elucidation of the genomic sequences, C. neoformans and C. gattii are now considered two distinct species. These two species have 5 serotypes based on antigenic specificity of the capsular polysaccharide; these include serotypes A, D, and AD (C. neoformans) and serotypes B and C (C. gattii).
C. neoformans is the most common species in the United States and other temperate climates throughout the world and is found in aged pigeon droppings. Until recently, C gattii was found principally in tropical and subtropical climates. C gattii is not associated with birds but grows in litter around certain species of eucalyptus trees (ie, Eucalyptus camaldulensis, Eucalyptus tereticornis). A 2016 epidemiological study performed in Bogota, Columbia, showed that C. neoformans also has a preference for eucalyptus tree species, similar to C. gattii. 

C. neoformans serotype A causes most cryptococcal infections in immunocompromised patients, including patients infected with HIV. For unknown reasons, C. gattii rarely infects persons with HIV infection and other immunosuppressed patients. Patients infected with C gattii are usually immunocompetent, respond slowly to treatment, and are at risk for developing intracerebral mass lesions (eg, cryptococcomas). A 2016 epidemiological study revealed that Cryptococcus tetragattii (AFLP7/VGIV), one of the five recognized genotypes of C. gattii sensu lato, is associated with a higher prevalence of cryptococcal meningitis among HIV-infected patients in Zimbabwe. 

C neoformans reproduces by budding and forms round yeastlike cells that are 3-6 µm in diameter. Within the host and in certain culture media, a large polysaccharide capsule surrounds each cell. C neoformans forms smooth, convex, yellow or tan colonies on solid media at 20-37°C (68-98.6°F). This fungus is identified based on its microscopic appearance, biochemical test results, and ability to grow at 37°C (98.6°F); most nonpathogenic Cryptococcus strains do not grow at this temperature. In addition, C neoformans does not assimilate lactose and nitrates or produce pseudomycelia on cornmeal or rice-Tween agar.

Most strains of C neoformans can use creatinine as a nitrogen source, which may partially explain the growth of the organism in creatinine-rich avian feces. Another useful biochemical characteristic of C neoformans, which distinguishes it from nonpathogenic strains, is its ability to produce melanin. The fungal enzyme phenol oxidase acts on certain substrates (eg, dihydroxyphenylalanine, caffeic acid) to produce melanin.

C. gattii contains genotypes VGI and the more commonly identified VGIIa and VDIIb. Cryptococcus species can reproduce via same-sex mating, and VGIIa may have arisen from the same-sex mating of VGIIb and another strain that has yet to be identified. In 1976, Kwon-Chung described the perfect (ie, sexual, teleomorphic) form of C neoformans, which was named Filobasidiella neoformans. Prior to the identification of F. neoformans, which is mycelial, C neoformans was considered a monomorphic yeast. F. neoformans results from the mating of suitable strains of serotypes A and D. The perfect state of C gattii is Filobasidiella bacillispora and results from the mating of serotypes B and C. Some strains of A and D can mate with strains of B and C.

Cryptococcus neoformans is an encapsulated yeast. In 1894, Busse, a pathologist, first described the yeast in a paper he presented to the Greifswald Medical Society. Busse isolated the yeast from the tibia of a 31-year-old woman, noted its resistance to sodium hydroxide, and published the case report that same year.  The following year, a surgeon named Buschke reported the same isolate from the same patient, thus establishing the early eponym of Busse-Buschke disease.  This single case served to identify a new yeast and to prove its pathogenic potential.

Since the initial reports, researchers have identified the diverse spectrum of host responses to cryptococcal infection. The responses range from a harmless colonization of the airways and asymptomatic infection in laboratory workers (resulting in only a positive skin test finding) to meningitis or disseminated disease. Although virulence in animals and, possibly, humans varies among strains of cryptococci, virulence probably plays a relatively small role in the outcome of an infection. The crucial factor is the immune status of the host.

The most serious infections usually develop in patients with defective cell-mediated immunity. For example, patients with AIDS, patients undergoing organ transplantation, patients with reticuloendothelial malignancy, patients undergoing corticosteroid treatment (but not those with neutropenia or immunoglobulin deficiency), and patients with sarcoidosis develop the most serious cryptococcal infections.

With the global emergence of AIDS, the incidence of cryptococcosis is increasing and now represents a major life-threatening fungal infection in these patients.

3. EPIDEMIOLOGY

C. neoformans is distributed worldwide. Most cases of cryptococcosis involve serotypes A and D. Serotypes B and C, C. gattii, are most common in tropical and subtropical areas and can be isolated from certain species of eucalyptus trees and the air beneath them. C. neoformans, which is recovered from aged pigeon feces, bird nests, and guano, is invariably serotype A or D. Although serotypes A and D exist in high concentrations in the pigeon feces, the fungus does not infect the birds. In moist or desiccated pigeon excreta, C neoformans may remain viable for 2 years or longer. In saprobic environments, C neoformans grows unencapsulated; however, unencapsulated strains regain their virulence following reacquisition of their polysaccharide capsule. C gattii usually causes disease in patients w ith intact cell-mediated immunity.
Naturally occurring cryptococcosis occurs in both animals and humans, but neither animal-to-human transmission nor person-to-person respiratory transmission via the respiratory route has been documented. Transmission via organ transplantation has been reported when infected donor organs were used. C. neoformans causes the vast majority of cryptococcal infections in immunosuppressed hosts, including patients with AIDS, whereas C gattii causes 70%-80% of cryptococcal infections among immunocompetent hosts. AIDS, decompensated liver cirrhosis, cellular mediated immunity suppressive regimens, and autoimmune disorders have been established as possible risk factors in the causation of invasive C neoformans disease. 

Although C neoformans is found worldwide, C gattii is usually identified in subtropical areas such as Australia, South America, Southeast Asia, and Central and sub-Saharan Africa. In the United States, C gattii is found in Southern California and more recently in the states of Washington and Oregon.

C. gattii may be found in association with several different trees, such as river red gum trees (E camaldulensis) and forest red gum trees (E tereticornis). Infection is acquired by inhalation of air-borne propagules that infect the lungs and may result in fungemia, leading to CNS involvement.

In 1999, C gattii emerged on Vancouver Island, British Columbia, Canada. Infections were reported among residents and visitors to the island, as well as among domesticated and wild animals. Disease has been most often identified in cats, dogs and ferrets. Marine mammals have also been infected. Vectors can disperse the spores from an endemic area to a previously unaffected area. This may have been the route of spread in the case of Vancouver Island. Since 2003, cryptococcal disease has become a provincially notifiable infection in British Columbia. Isolates have been identified in coastal Douglas fir and coastal western hemlock bioclimatic zones. C gattii has been identified subsequently in the states of Washington and Oregon.

The incidence of infection related to age, race, or occupation does not significantly differ. Healthy persons with a history of exposure to pigeons or bird feces and laboratory workers exposed to an aerosol of the organism have a higher rate of positive delayed hypersensitivity skin reactions to cryptococcal antigen or cryptococci. Occasionally, laboratory accidents result in transmission of C neoformans, but pulmonary and disseminated disease is rare in this setting. Accidental cutaneous inoculation with C. neoformans causes localized cutaneous disease.

A report from British Columbia, Canada indicates that risk factors for infection by C. gattii include steroid use and underlying pulmonary disease. The authors also report a predilection for individuals older than 50 years, current smokers, and those with immunosuppression due to HIV or invasive malignancy.

The advent and increased use of anti-TNF and anti-CD 54 has posed an increased risk of contracting cryptococcosis, along with an increased risk of delayed diagnosis.

A report presented in 2016 emphasized in ocular complications of CM caused by C. gattii AFLP4/VGI under continuation of AFT in Taiwan. In general, C. gattii AFLP4/VGI tends to produce larger and more numerous cryptococcomas in the central nervous system. Seaton et al. suggested that the high rate of visual loss in immunocompetent patients with C. gattii AFLP4/VGI infections may reflect immune-mediated optic nerve dysfunction in C. gattii meningitis caused by either compression due to arachnoid adhesions or edema and inflammatory cell-mediated damage. The optic nerve lesion can be due to direct destruction by this pathogen or indirectly caused by increased intracranial pressure. Use of corticosteroids could be recommended for immunocompetent patients with severe C. gattii sensu stricto (AFLP4/VGI) meningitis. CM due to C. gattii AFLP4/VGI can cause significant neurological morbidities, including ocular complications.


4. DISINFECTION
C. neoformans is susceptible to 70% ethanol, 0.5% chlorhexidine, 1.2% sodium hypochlorite, iodophors (e.g., betadine), phenolic disinfectants, glutaraldehyde and formaldehyde. Hydrogen peroxide (3%) or a preparation containing 0.05% chlorhexidine were not effective in one of the studies. In biofilms, only 0.5% chlorhexidine and extremely high concentrations of sodium hypochlorite were reported to be fungicidal. C. neoformans can also be killed by moist heat of 121°C for a minimum of 20 minutes or dry heat of 165-170°C for 2 hours. Although ultraviolet light reduced the number of viable organisms in one of the experiments, it was not considered to be fungicidal, as there was less than a hundred-fold decrease in the fungal load.

5. INCUBATION PERIOD
The incubation period for C. neoformans infections is uncertain, as this organism is ubiquitous and it is often impossible to determine when the person was exposed. Some clinical cases can occur months or years after exposure.

The C. gattii organisms responsible for the Vancouver Island outbreak have a distinctive molecular type, which has allowed the incubation period to be determined in visitors to the island. Illnesses caused by this organism have appeared 6 weeks to 13 months after exposure, with an estimated median incubation period of 6-7 months.

6. VIRULENCE FACTORS
First classical genetic studies on virulence determinants concentrated on the distinctive phenotypic characteristics that were often used for diagnostic purposes including polysaccharide capsule formation (Jacobson et al. 1982; Rhodes et al. 1982; Kwon-Chung and Rhodes 1986) and melanin formation (Kwon-Chung et al. 1982; Rhodes et al. 1982). The third trait essential for virulence is the ability to grow at mammalian body temperature (Petter et al. 2001; Perfect 2006). Molecular genetic studies using gene-deletion strains have established the role of these three traits in cryptococcal virulence (Chang and Kwon-Chung 1994; Salas et al. 1996; Odom et al. 1997; Janbon et al. 2001). In fact, the molecular study into the role of capsule as the fungal virulence factor in C. neoformans was the first fulfillment of the molecular Koch’s postulates (Chang and Kwon-Chung 1994).

a) Capsule
The cryptococcal capsule is affixed by the cell wall, which is composed of glucans, chitin, chitosan, and glycoprotein (Doering 2009; Gilbert et al. 2011). The capsule is composed primarily of two large virulence-implicated repeating polysaccharides (Kumar et al. 2011), glucuronoxylomannan (GXM, ~1–7 million Da) and glucuronoxylomannogalactan (GXMGal,
~100,000 Da), whose structures are shown in Figure 1. The capsule may also include mannoproteins, hyaluronic acid, and sialic acid. The capsule is displayed on the cryptococcal surface and component polysaccharides are also shed into the environment. Although it is likely that both forms play roles in pathogenesis, most structural work has been done on the shed material, which may differ from that associated with the cell (Frases et al. 2008; Kumar et al. 2011). Capsule polysaccharides are likely made in the Golgi (Yoneda and Doering 2006), requiring synthesis of activated sugar donors, transport of these compounds into the Golgi, polymerization of capsule components, export of product polysaccharides, and assembly at the cell surface (Doering 2009).  Based on the capsule structures (Fig. 1), the sugar donors are expected to include GDP mannose, UDP-glucuronate, UDP-xylose, UDP galactofuranose, and UDP-galactopyranose. Enzymes required for synthesis of the first four have been biochemically characterized, and genes encoding synthetic machinery for all five have been identified and deleted to assess the impact on cells (Bar-Peled et al. 2001, 2004; Wills et al. 2001; Moyrand et al. 2002, 2007, 2008; Griffith et al. 2004; Moyrand and Janbon 2004; Beverley et al. 2005; Doering 2009).

The lack of these proteins reduces capsule production and virulence in animal models, except for the enzyme responsible for UDP-galactofuranose synthesis; this may be because galactofuranose is a minor component of capsule polysaccharides (Heiss et al. 2013). Two nucleotide sugar transporters for GDP-mannose and one for UDP-galactose have also been investigated (Cottrell et al. 2007; Moyrand et al. 2007; Wang et al. 2014).


Unencapsulated yeast are readily phagocytosed and destroyed, whereas encapsulated organisms are more resistant to phagocytosis. The cryptococcal polysaccharide capsule has antiphagocytic properties and may be immunosuppressive. The antiphagocytic properties of the capsule block recognition of the yeast by phagocytes and inhibit leukocyte migration into the area of fungal replication.


b) Melanin Formation  
Melanin is produced by a wide variety of fungal species and the pigment deposited in the cell wall is known to play an important protective role against environmental stress (Nosanchuk and Casadevall 2006). C. neoformans produces eumelanin only in the presence of substrates such as 3,4-dihydroxyphenylalanine (DOPA) and other di/polyphenolic compounds (Chasakes 1975; Polacheck et al. 1982). The early observations, using melanin-lacking mutants isolated by UV irradiation, suggesting the importance of melanin as a virulence factor (Kwon-Chung et al. 1982; Rhodes et al. 1982) was confirmed by the use of LAC1 gene-deletion mutants (Salas et al. 1996). C. neoformans contains two laccase genes, LAC1 and LAC2, in the genome but only LAC1 is expressed significantly under most conditions and virulence is reduced only when the LAC1 gene is deleted (Zhu and Williamson 2004; Pukkila-Worley et al. 2005).The cryptococcal laccase, a member of the multicopper oxidases is localized in the cell walls (Zhu et al. 2001; Waterman et al. 2007) and its transport to the cell wall is Sec6 dependent (Panepinto et al. 2009). Melanization of Cryptococci require numerous additional genes such as the copper transporter Ccc2, the copper chaperone Atx1, the chitin synthase Chs3, the transcriptional coactivator Mbf1, the chromatin remodeling enzyme Snf5 (Walton et al. 2005), the transcription factor Rim101, and its regulatory gene Rim20 (Liu et al. 2008). As in the case of capsule formation, melanization is regulated by number of different pathways (Liu et al. 2008).

There have been numerous studies regarding the role of cryptococcal melanin in protection from phagocytosis (Wang et al. 1995; Liu et al. 1999), killing by host cells (Blasi et al. 1995; Wang et al. 1995), oxidants (Wang and Casadevall 1994; Blasi et al. 1995; Jacobson and Hong 1997), and microbicidal peptides (Doering et al. 1999). In addition, melanin is reported to protect cryptococci from antifungal agents including amphotericin B (Ikeda et al. 2003; Martinez and Casadevall 2006), caspofungin (van Duin et al. 2002; Martinez and Casadevall 2006), and azoles (van Duin et al. 2002, 2004; Ikeda et al. 2003).

c) Growth at Mammalian Body Temperature
Although the ability to grow at 37°C is not sufficient by itself to be a mammalian pathogen, it is essential for any microbial pathogen to be able to cause invasive disease. The primary reason that both C. neoformans and C. gattii are the only successful pathogens among the more than 70 Cryptococcus species is their ability to grow robustly at physiological temperatures. All the remaining cryptococcal species produce a polysaccharide capsule with or without melanin but fail to grow or grow poorly at 37°C (Petter et al. 2001; Fonseca et al. 2011). The first molecular study related to the cryptococcal growth at mammalian body temperature has identified calcineurin, the highly conserved Ca2þ/ calmodulin-activated serine/threonine-specific phosphatase encoded by CNA1, to be essential for growth at 37°C but not at 24°C. As a consequence, CNA1 disrupted mutant strains are avirulent (Odom et al. 1997; Fox et al. 2001) and it was proposed that the signaling cascade involving calcineurin is required for cryptococcal pathogenesis (Odom et al. 1997). The importance of calcineurin for cryptococcal growth at 37°C but not at 24°C also explained the reason why mice treated with cyclosporin A (CsA), which inhibits signal transduction at 37°C but not at 24°C, are protected from cryptococcosis (Mody et al. 1988) and that CsA is toxic to C. neoformans only at 37°C but not at 24°C (Odom et al. 1997). Subsequently, many genes required for cryptococcal growth at physiological temperatures and the genes that are significantly up-regulated during growth at elevated temperatures have been identified (Perfect 2006) using various molecular methods such as complementation cloning (Chung et al. 2003), insertional library (Idnurm et al. 2004), genomic- DNA microarrays (Kraus et al. 2004), serial analysis of gene expression (SAGE) (Steen et al. 2002), signature-tagged mutagenesis (Liu et al. 2008), and representational difference analysis (Rosa e Silva et al. 2008). The functions of these genes varied ranging from cell-wall assembly, stress signaling, membrane integrity, basic metabolism, pre-mRNA splicing, chromatin remodeling, and others (Steen et al. 2002; Chung et al. 2003; Kraus et al. 2004; Liu et al. 2008; Rosa e Silva et al. 2008). Functional studies of the genes identified as important for growth at 37°C, using targeted disruption, showed correlation with low to no infectivity (Kraus et al. 2004; Liu et al. 2008). However, temperature regulated genes did not necessarily associate with a temperature-sensitive phenotype (Akhter et al. 2003; Cox et al. 2003).

d) Degradation Enzymes
C. neoformans produces many degradation enzymes, some of them have been established as virulence determinants. Urease (Cox et al. 2000, 2001; Osterholzer et al. 2009; Shi et al. 2010; Bahn and Jung 2013; Singh et al. 2013) and phospholipase B (Cox et al. 2001; Ganendren et al. 2006; Wright et al. 2007; Chayakulkeeree et al. 2011) are the two most studied degradation enzymes that have a role in cryptococcal pathogenicity. The functions of these enzymes promote intracellular survival of the yeasts (Wright et al. 2007), hydrolysis of host cell membranes to penetrate into tissue (Chen et al. 1997), immunomodulation (Noverr et al. 2003; Osterholzer et al. 2009), and the enhancement of fungal dissemination from the lung to the brain (Cox et al. 2000; Noverr et al. 2003; Wright et al. 2007; Shi et al. 2010; Singh et al.2013). Unlike the polysaccharide capsule, however, a lack of these enzymes results in reduced rather than a complete loss of virulence. The correlation between in vitro phospholipase B activity of cryptococcal strains and virulence in mice was first demonstrated in 1997 (Chen et al. 1997) and subsequent work with phospholipase B gene (PLB1)-deletion strains confirmed the importance of the enzyme as a virulence determinant (Cox et al. 2001; Noverr et al. 2003). Phospholipase B is transported to the cell surface in vesicles (Eisenman et al. 2009) and its secretion is dependent upon Sec14, a phosphatidylinositol transfer protein (Chayakulkeeree et al. 2011). Transport of the enzyme to the cell surface enhances adhesion of the cryptococcal cells to human lung epithelial cells, the first step toward initiation of interstitial pulmonary cryptococcosis (Ganendren et al. 2006). It also disrupts the host cell membranes by hydrolysis of the ester linkages on membrane phospholipids, which enables penetration into the host tissue (Chen et al. 2000). Plb1 also supports the intracellular survival of Cryptococci within macrophages in connection with lipid metabolism (Wright et al. 2007), which is key for the eventual dissemination of cryptococci to the brain. Cryptococcal urease activity is important for fungal propagation in the lungs as the enzyme promotes accumulation of immature dendritic cells as well as the non-protective T2 immune response (Osterholzer et al. 2009). Urease activity is also important in the fungus’ ability to cross the blood–brain barrier (Olszewski et al. 2004; Shi et al. 2010) by enhancing sequestration of the yeast cells within microcapillary beds (Olszewski et al. 2004). The mechanism of urease activation has been studied extensively in bacteria and plants but rarely in fungi. The cryptococcal urease activation system has recently been interpreted, which showed that the factors required for activation of the urease apoenzyme encoded by URE1 resemble plants more than bacteria (Singh et al. 2013). As with other ureases, the cryptococcal urease is a nickel enzyme and requires the accessory proteins, Ure4, Ure6, and Ure7, which are homologs of the bacterial accessory proteins UreD, UreF, and UreG, respectively. The cryptococcal genome lacks a homolog of bacterial UreE, a nickel chaperone. However, Ure7 (the homolog of bacterial UreG) appears to combine the functions of bacterial UreE and UreG. Strains harboring an intact URE1 but disrupted accessory proteins disseminated to the brain at rates similar to the ure1 mutant, which indicates that it is the urease activity and not the Ure1 protein that is a virulence factor in C. neoformans (Singh et al. 2013). The enzyme appears to require SEC6 for secretion to the surface of the yeast cells (Panepinto et al. 2009).

e) Sensing and responding to environmental variables
More recently, other virulence factors have emerged that are being investigated from the perspective of sensing and signaling, often affecting the classical virulence traits. Three recent developments have been described.

The first is metal homeostasis. A role of calcium in signaling in C. neoformans has been well studied in the context of calcineurin signaling (Steinbach et al. 2007), so is not covered further. Iron and copper are the next best-studied metals. Both are relevant to pathogenesis because of their availability to the pathogen, the interconnection between oxygen availability, metal uptake, heme (with iron as a cofactor), and heme dependent sterol biosynthesis. Thus, altered Fe or Cu homeostasis affects multiple aspects of C. neoformans biology that are clinically relevant, including antifungal drug efficacy. Iron can be either limiting or in excess in a host depending on health status. Effects of iron limitation on C. neoformans have been known since the early 1990s, because one consequence is the enhancement of capsule formation (Vartivarian et al. 1993). C. neoformans senses iron levels, and regulates a suite of genes using the Cir1 transcription factor (Jung et al. 2006). Those genes include an adjacent pair of genes for iron oxidation and high-affinity uptake, which are also required for full virulence (Jung et al. 2008, 2009). Other factors, such as the siderophore transporter Sit1, are not required for pathogenesis although this may reflect gene redundancy (Tangen et al. 2007). Another source of iron for C. neoformans is heme, yet use of this iron source is currently less well established (Jung et al. 2010; Cadieux et al. 2013). The link between copper and melanization has also been known for two decades. For example, mutants impaired in melanin synthesis can be rescued by the addition of copper to the medium (Torres-Guererro and Edman 1994; Walton et al. 2005). Copper is a cofactor for multi-copper oxidases, which include the ferroxidase Cfo1 for iron uptake and laccase for melanin synthesis. Altered copper homeostasis influences these two properties. The MAC1 gene was identified as naturally variable in populations, affecting mating efficiency and melanization (Lin et al. 2006). MAC1 (CUF1) encodes a copper-response transcription factor that regulates target genes (Ding et al. 2011).A fascinating discovery is that the effects of mutation that impair two totally opposite responses to copper (i.e., to low or high Cu concentrations) are the same. That is, loss of the Ctr4 transporter for uptake under low Cu concentrations (Waterman et al. 2007, 2012) or loss of the metallothioneins used in sequestration of Cu under toxic high concentrations (Ding et al. 2013) both reduced virulence. Two more recently studied metals are nickel and zinc. Nickel is a cofactor for urease. Like urease deficient strains, those impaired in nickel homeostasis have defects in dissemination after pulmonary infection (Olszewski et al. 2004; Singh et al. 2013). Disruption in C. gattii of the ZAP1 gene, which encodes a zinc sensor and transcription factor, reduces virulence (Schneider et al. 2012). Zinc is a cofactor in many proteins, including zinc finger transcription factors, so loss of virulence may reflect an impact on a number of key processes.

The second example is the impact of gases on pathogenesis. O2 and CO2 vary in concentration in different parts of the body, lower or higher, respectively, than the natural environment in which Cryptococcus species reside. CO2 sensing uses carbonic anhydrase and adenlyl cyclase. Carbonic anhydrases are zinc proteins that convert CO2 to bicarbonate (HCO3–). Two homologs are expressed in C. neoformans, with Can2 having the major function. Deletion of CAN2 prevents growth under low CO2 concentrations; however, virulence is unaffected presumably because of the high CO2 concentrations within the host (Bahn et al. 2005). The other CO2 sensor, adenylyl cyclase (Klengel et al. 2005; Mogensen et al. 2006), synthesizes the signaling molecule cyclic AMP, and is also part of the Ga signaling pathway, and the cac1 mutants are nonpathogenic (Alspaugh et al. 2002).
Concentrations of O2 are sensed by Scp1, a candidate endoplasmic reticulum peptidase.Scp1 cleaves the inactive Sre1 into an active transcription factor that is shuttled into the nucleus. Deletion of either gene reduces the ability of C. neoformans to grow at 3% oxygen levels, and reduces virulence (Chang et al. 2007; Chun et al. 2007). Genes regulated by hypoxia include those for sterol biosynthesis and metal homeostasis. The deleterious effects of low O2 levels are likely in part through altered membrane fluidity.

The third example is the potential role of light sensing in pathogenesis. Deletion of genes that encode a blue-light-sensing complex reduces virulence (Idnurm and Heitman 2005; Zhu et al. 2013). It is unknown whether darkness represents a specific signal and the relevant genes are also unknown. Although genes for heme biosynthesis and iron uptake are induced by light, the light-sensing mutants do not have phenotypes that indicate any change in these properties (Idnurm and Heitman 2010). Hence, the three best-established virulence traits and growth or stress phenotypes are unaffected, and further analysis may enable discoveries about virulence factors operating in Cryptococcus species.

7. PATHOGENESIS
Of the more than 50 species that comprise the genus Cryptococcus, human disease is primarily associated with C. neoformans and C. gattii. Animal models provide much of the understanding of the pathogenesis and the host defense mechanisms involved in cryptococcal infections. The organism is primarily transmitted via the respiratory route, but not directly from human to human.

Cryptococcus neoformans and Cryptococcus gattii are environmental, basidiomycetous yeasts. Unlike other pathogenic fungi, these yeast cells possess large polysaccharide capsules (Figure 2). C neoformans occurs worldwide in nature and is isolated readily from dry pigeon feces, as well as trees, soil, and other sites. C gattii is less common and typically associated with trees in tropical areas. Both species cause cryptococcosis, which follows inhalation of desiccated yeast cells or possibly the smaller basidiospores.

As mentioned earlier, cryptococcosis begins with inhalation of desiccated airborne yeast cells, or possibly sexually produced basidiospores, into the lungs. Because the propagules are small (1.5–3.5 mm), they reach the distal airways and come into contact with alveolar macrophages. Following inhalation, the yeast spores are deposited into the pulmonary alveoli, where they must survive the neutral-to-alkaline pH and physiologic concentrations of carbon dioxide before they are phagocytized by alveolar macrophages. From the lungs, these neurotropic yeasts typically migrate to the central nervous system where they cause meningoencephalitis (Figure 2 ).




Figure 2. Transmission of Infection of Cryptococcus

However, they also have the capacity to infect many other organs (eg, skin, eyes, prostate). Glucosylceramide synthase (GCS) has been identified as an essential factor in the survival of C neoformans in this extracellular environment. Although GCS is a critical factor in extracellular survival of the yeast, the yeast no longer requires GCS to survive the intracellular, more acidic, environment within the macrophage once it is phagocytized by alveolar macrophages.

The host response to cryptococcal infection includes both cellular and humoral components. Animal models demonstrate that natural killer cells participate in the early killing of cryptococci and, possibly,antibody-dependent cell-mediated killing. In vitro monocyte-derived macrophages, natural killer cells, and T lymphocytes can inhibit or kill cryptococci. A successful host response includes an increase in helper T-cell activity, skin test conversion, and a reduction in the number of viable organisms in the tissues. In addition to cellular mechanisms, anticryptococcal antibodies and soluble anticryptococcal factors have been described. Antibodies to cryptococcal antigens play a critical role in enhancing the macrophage- and lymphocyte-mediated immune response to the organism. Researchers have used monoclonal antibodies to capsular polysaccharide to passively immunize mice against C. neoformans.

 Serologic evidence indicates that cryptococcal infection in humans is prevalent (Goldman et al. 2001) but disease is rare. Activated alveolar macrophages recruit other immune cells through cytokines and chemokines and elicit a proper Th1 response and granulomatous inflammation. In a normal host, an effective immune response eliminates most inhaled cryptococci. In contrast, in an immunocompromised host, the cryptococcal cells proliferate, hematogenously disseminate to the brain by crossing the blood–brain barrier (Chang et al. 2004; Shi et al. 2010), and adapt to the suboptimal levels of oxygen and nutritional conditions of the brain to multiply and cause meningoencephalitis (Chang et al. 2007; Chun et al. 2007). Although virtually every organ in the body can be involved, infection of the central nervous system (CNS) is the most common clinical manifestation of cryptococcosis and the most common cause of death.

C neoformans occurs in immunocompetent persons but more often in patients with HIV/AIDS, hematogenous malignancies, and other immunosuppressive conditions. Cryptococcosis due to C gattii is rarer and usually associated with apparently normal hosts. Overall, approximately one million new cases of cryptococcosis occur annually, and the mortality approaches 50%. More than 90% of these infections are caused by C neoformans. Although C gattii is less prevalent globally, for the past decade, there has been an expanding outbreak of infections with this species in the Pacific Northwest. Untreated CNS infection is uniformly fatal (Kwon-Chung and Bennett 1992; Casadevall and Perfect 1998; Perfect 2010). Another commonly occurring cryptococcal infection is the formation of a small lung–lymph complex where yeasts remain viable but dormant and these patients remain clinically asymptomatic (Salyer et al. 1974; Baker 1976) until loss of local immunity resulting from various causes such as corticosteroid treatment, progression of an HIV infection, or other immunosuppressive conditions (Perfect 2010). Upon this loss of immunity, the dormant yeast cells are activated and begin to multiply in the pulmonary–lymph node complex and disseminate into extrapulmonary sites. This reactivation of latent infection has been seen mainly with C. neoformans infection as the most common incidence in AIDS patients, contributing to the definition of cryptococcosis as an AIDS defining illness (Dromer 2011). In contrast, infection caused by C. gattii occurs more often in immunocompetent patients with or without any known underlying conditions (Sorrell et al. 2011).

Figure 3. Route of cryptococcalmeningoencephalitis.

Although C. neoformans primarily presents as meningoencephalitis, pulmonary infection is considerably more common with C. gattii infection (Chen et al. 2000; Galanis et al. 2010). Animal studies supported these differences in the primary target organs between the two species; mice infected with C. neoformans succumbed to infection by CNS infection, whereas mice infected with C. gattii died by pulmonary infection (Ngamskulrungroj et al. 2012).

8. CLINICAL MANIFESTATION
The consequences of infection with C. neoformans or C. gattii range from asymptomatic colonization of the airways to respiratory signs of varying severity, or disseminated infections that may involve the CNS, eye, skin and other organs. While there seem to be some differences between the syndromes caused by C. neoformans and C. gattii, both species can affect any organ. In immunosuppressed hosts, C. neoformans may cause little inflammation, and the symptoms can be mild even with extensive disease. Only a small percentage of the people exposed to either organism become ill.

In most patients, Cryptococus spp. enter the body via the respiratory tract and replicate first in the lungs. Many pulmonary infections are asymptomatic in both immunocompetent and immunosuppressed hosts, although lesions may be apparent on x-ray. In clinical cases, the signs vary from a nonspecific cough alone, to more significant symptoms that can include dyspnea or shortness of breath, pleuritic chest pain or hemoptysis. Other signs may include low-grade fever, weight loss, anorexia and malaise. Pleural effusions can occur, but are uncommon, and adult respiratory distress syndrome has been reported. C. neoformans can cause an asymptomatic pulmonary infection followed later by the development of meningitis, which is often the first indication of disease. If limited to the lungs, C neoformans infection may cause pneumonia, poorly defined mass lesions, pulmonary nodules, and, rarely, pleural effusion. Although immune defects are common in patients with meningitis or disseminated infection, patients with disease that is confined to the lungs are usually immunocompetent. Serious respiratory syndromes and progressive pulmonary disease are more likely to occur in immunocompromised patients. Many infections in healthy patients may be self-limited.

From the lungs, Cryptococcus spp. may spread to other organ systems, particularly in immunosuppressed patients. Respiratory symptoms can either precede or occur concurrently with other syndromes. Disseminated disease can also be seen in individuals who had asymptomatic pulmonary infections.

CNS disease is the most common form of disseminated cryptococcosis. The typical syndromes are subacute or chronic meningitis and meningoencephalitis, or mass lesions (cryptococcomas) in the brain. The development of the illness is often insidious, with initial signs such as headache, fatigue, drowsiness or changes in behavior. A persistent headache, often of several weeks’ duration, is a common presentation. Although some patients may have a fever, body temperature can also be only slightly elevated or normal. Neck stiffness is often minimal or absent. Other signs, such as abnormalities in vision, seizures, vomiting, impaired consciousness and paralysis, can develop with the progression of the disease. Cranial nerve paralysis is common. Cryptococcomas may cause focal signs such as aphasia, cerebellar syndrome or paresis, especially in immunocompetent patients.

Cerebrospinal fluid pressure and protein may be increased and the cell count elevated, whereas the glucose is normal or low. Patients may complain of headache, neck stiffness, and disorientation. Elevated cerebrospinal fluid (CSF) pressure from cryptococcomas or chronic meningoencephalitis can lead to hydrocephalus and further neurological signs, including dementia. Other syndromes, such as spinal cord lesions or ischemic stroke, have also been seen. In addition, there may be lesions in skin, lungs, or other organs. The course of cryptococcal meningitis may fluctuate over long periods, but all untreated cases are ultimately fatal. Globally, about 5–8% of patients with AIDS develop cryptococcal meningitis. The infection is not transmitted from person to person.

C neoformans infection is usually characterized by little or no necrosis or organ dysfunction until late in the disease. Organ damage may be accelerated in persons with heavy infections. The lack of identifiable endotoxins or exotoxins may be partly responsible for the absence of extensive necrosis early in cryptococcal infections. Organ damage is primarily due to tissue distortion secondary to the expanding fungal burden. Extensive inflammation or fibrosis is rare. The characteristic lesion of C neoformans consists of a cystic cluster of yeast with no well-defined inflammatory response. Well-formed granulomas are generally absent.

The eye is also a common site of dissemination, resulting in lesions such as optic neuritis, chorioretinitis and endophthalmitis. Ocular signs, including vision loss, can also be caused by intracranial hypertension from CNS disease.

Direct inoculation into the skin (primary cutaneous cryptococcosis) is an uncommon presentation, and typically results in a localized lesion such as a nodule, tubercle or abscess at the inoculation site. The lesions of primary cutaneous cryptococcosis sometimes regress spontaneously.  Dissemination of organisms to the skin can cause a variety of lesions, which may mimic other diseases. Papules, which may ulcerate or evolve to other forms, are often seen initially. Other reported lesions include pustules, vesicles, bullae, ulcers, palpable purpura, superficial granulomas, plaques, subcutaneous tumor-like masses, cellulitis, abscesses or sinus tracts, and even rare cases of necrotizing fasciitis. AIDS patients may have umbilicated papules that resemble molluscum contagiosum. Cutaneous involvement often occurs concurrently with cryptococcosis in the brain or other organs.

Less frequent or rare syndromes include osteomyelitis, septic arthritis, myocarditis, lymphadenitis, hepatitis, peritonitis, abdominal cryptococcomas, gastrointestinal involvement, renal abscesses, prostatitis, myositis, endocarditis and septic shock. In AIDS patients, invasion of the adrenal glands may cause adrenal insufficiency. Urogenital involvement is often asymptomatic.

9. LABORATORY DIAGNOSIS
Specimen
Specimens for the laboratory diagnosis of cryptococcosis include cerebrospinal fluid (CSF), tissue, exudates, sputum, blood, cutaneous scrapings, and urine. Spinal fluid is centrifuged before microscopic examination and culture. For direct microscopy, specimens are often examined in wet mounts, both directly and after mixing with India ink, which delineates the capsule.

Figure4 Cryptococcus species in Meningoencephalitis

i) Culture
A definitive diagnosis can also be obtained by culture. Colonies develop within a few days on most media at room temperature or 37°C. Media with cycloheximide inhibit Cryptococcus and should be avoided. Cultures can be identified by growth at 37°C and detection of urease. Alternatively, on an appropriate diphenolic substrate, the phenol oxidase (or laccase) of C. neoformans and C. gattii produces melanin in the cell walls and colonies develop a brown pigment. Both C. neoformans and C. gattii produce melanin and usually form brown colonies on Niger (birdseed) agar. Canavanine-glycine-bromthymol blue agar can distinguish C. gattii from C. neoformans.

Although C. neoformans and C. gattii can form colonies on most media, growth is best on fungal media such as Sabouraud dextrose agar without cycloheximide. Colonies usually appear within 2 to 5 days, but growth may be delayed in samples with few organisms. The organism is identified by its appearance, ability to grow at 37°C and biochemical tests; by molecular methods such as DNA sequencing; or with commercial yeast identification systems.


ii) Microscopy
Cryptococcus spp. can sometimes be found in clinical samples by direct observation. C. neoformans and C. gattii are round to oval yeasts, surrounded by large capsules that stain strongly with Mayer’s mucicarmine. In an India ink preparation, the capsule appears as a clear halo around the yeast cell. Unless budding is observed, it can be confused with a fat droplet or other artifact. Other useful stains include Alcian blue, Gomori methenamine silver, colloidal iron, periodic acid-Schiff (PAS), Masson-Fontana silver stain, Gram’s stain, new methylene blue and Wright’s stain. Cryptococcus spp. can also be identified in the tissues by immunofluorescence or immunohistochemistry. Some species of Cryptococcus do not appear to stain well with certain antibodies. Microscopy may detect non-viable yeasts, which appear intact, in the tissues for several months after treatment. During culture, human diagnostic laboratories do not always differentiate C. gattii from C. neoformans.

iii) Serology
Cryptococcosis is usually diagnosed by detecting the organism or its antigens in blood, or in tissues and fluids from affected sites (e.g., cerebrospinal fluid, bronchial washings, and urine). The latex slide agglutination test or enzyme immunoassay (EIA) for cryptococcal antigen is positive in 90% of patients with cryptococcal meningitis. With effective treatment, the antigen titer drops except in AIDS patients, who often maintain high antigen titers for long periods (Table 1).

The newest test for GXM is a lateral flow assay (LFA), in which monoclonal antibodies to GXM are prepared in an EIA format on a dipstick that can be placed in serum, CSF, or urine and produces a positive test color change within 20 minutes. This LFA test has been used extensively as a point of care screen for cryptococcosis in sub-Saharan Africa. Serology to detect specific antibodies is not generally used in diagnosis, as healthy people are often seropositive.
Table 1  Showing Test for Cryptococcal antigen in various sample
Sample
Antigen
Test
Sensitivity
%
Specificity
%
Comments
Serum
GXM
LA, EIA


LFA
90



87-91
95-100



87-100
Cross-reactions with Trichosporon,
Stomatococcus, Capnocytophaga
CSF

LA, EIA
LFA
97

99
86-100

99

Urine

LFA
70-80
92
Rapid, 20 min


iv) Imaging Technique
Other helpful tests include CT and MRI in patients with CNS disease, and X-rays in patients with pulmonary signs.

v) Molecular Methods
Differential media can also aid identification. If needed for epidemiological analyses, genetic types can be identified by techniques such as multilocus sequence typing (MLST) or amplified fragment length polymorphism (AFLP). However, veterinary diagnostic laboratories may not identify Cryptococcus even to the species level.

10. TREATMENT
Combination therapy of amphotericin B and flucytosine has been considered the standard treatment for cryptococcal meningitis, though the benefit from adding flucytosine remains controversial. Amphotericin B (with or without flucytosine) is curative in most non-AIDS patients. Inadequately treated AIDS patients will almost always relapse when amphotericin B is withdrawn and require suppressive therapy with fluconazole, which offers excellent penetration of the central nervous system.

Pulmonary cryptococcosis resolves without specific therapy in most immunocompetent patients. Antifungal therapy is necessary for the following:
·         Pulmonary cryptococcosis in immunosuppressed hosts
·         CNS cryptococcosis
·         Disseminated nonpulmonary non-CNS cryptococcosis

Treatment for cryptococcal meningitis in patients with AIDS is as follows:
·         Amphotericin B deoxycholate, 0.7-1 mg/kg/day for 2 weeks, with or without
·         Flucytosine, 100 mg/kg/day in 4 divided doses for 2 weeks
·         Flucytosine speeds clearance of viable yeast from CSF but is potentially toxic, especially in patients with renal dysfunction
·         After 2 weeks, fluconazole at 400 mg/day for a minimum of 8-10 weeks

Alternative initial therapies include the following:
·         Liposomal amphotericin B 3-4 mg/kg/day for at least 2 weeks in patients at risk for renal dysfunction; in patients who have failed therapy or have a high fungal burden of disease, liposomal amphotericin B 6 mg/kg/day IV has been given safely
·         In patients intolerant of amphotericin B products, fluconazole 800-1200 mg/day plus flucytosine 100 mg/kg/day for at least 6 weeks can be used. Initial therapy should be considered successful only after CSF culture is negative for cryptococcal organisms and the patient has had significant clinical improvement. 

11. PREVENTION
Complete prevention of exposure is probably impossible. C. neoformans is ubiquitous, while C. gattii has now been identified in a variety of climates, in and around many species of trees. Despite the frequency of exposure, most people do not become ill.
In some circumstances, it might be possible to decrease the level of exposure from some environmental sources, such as bird droppings (especially pigeon droppings), trees during logging and cutting, eucalyptus trees in bloom, and soil disturbances. Removal of guano should be preceded by chemical decontamination or wetting with water or oil to decrease aerosolization.
Although no cases of animal-to-human transmission have been reported (except via avian feces in the environment), it is wise to use caution when handling animals with cryptococcosis. People handling such animals should use appropriate barrier precautions, including avoidance of accidental inoculation into breaks in the skin. Cages and litter boxes should be decontaminated regularly. Targeted screening of immunosuppressed individuals, using tests that detect cryptococcal antigens, might identify disseminated infections in the early stages when they are most readily treated.


REFERENCE

1.     Akhter S, McDade HC, Gorlach JM, Heinrich G, Cox GM, Perfect JR. (2003). Role of alternative oxidase gene in pathogenesis of Cryptococcus neoformans. Infect Immun 71:5794–5802.
2.      Alspaugh JA, Pukkila-Worley R, Harashima T, Cavallo LM, Funnell D, Cox GM, Perfect JR, Kronstad JW, Heitman J. (2002). Adenylyl cyclase functions downstream of the Ga protein Gpa1 and controls mating and pathogenicity of Cryptococcus neoformans. Eukaryot Cell 1: 75–84.
3.      Bahn Y-S, Cox GM, Perfect JR, Heitman J. (2005). Carbonic anhydrase and CO2 sensing during Cryptococcus neoformans growth, differentiation, and virulence. Curr Biol 15: 2013–2020.
4.      Bahn YS, Jung KW. (2013). Stress signaling pathways for the pathogenicity of Cryptococcus. Eukaryot Cell 12: 1564–1577.
5.      Baker RD. (1976). The primary pulmonary lymph node complex of crytptococcosis. Am J Clin Pathol 65: 83–92.
6.      Bar-Peled M, Griffith CL, Doering TL. (2001). Functional cloning and characterization of a UDP-glucuronic acid decarboxylase: The pathogenic fungus Cryptococcus neoformans elucidates UDP-xylose synthesis. Proc Natl Acad Sci 98: 12003–12008.
7.      Bar-Peled M, Griffith CL, Ory JJ, Doering TL. (2004). Biosynthesis of UDP-GlcA, a key metabolite for capsular polysaccharide synthesis in the pathogenic fungus Cryptococcus neoformans. Biochem J 381: 131–136.
8.      Beverley SM, Owens KL, Showalter M, Griffith CL, Doering TL, Jones VC, McNeil MR. (2005). Eukaryotic UDP-galactopyranose mutase (GLF gene) in microbial and metazoal pathogens. Eukaryot Cell 4: 1147–1154.
9.      Blasi E, Barluzzi R,Mazzolla R, Tancini B, Saleppico S, Puliti M, Pitzurra L, Bistoni F. (1995). Role of nitric oxide and melanogenesis in the accomplishment of anticryptococcal activity by the BV-2 microglial cell line. J Neuroimmunol 58: 111–116.
10.  Carroll K.C. and et al. (2016). Jawetz, Melnick, & Adelberg’s Medical Microbiology. Twenty-Seventh Edition. Mc Graw Hill Education.
11.  Casadevall A, Perfect JR. (1998). Cryptococcus neoformans. ASM, Washington, DC.
12.  Chang YC, Kwon-Chung KJ. (1994). Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence. Mol Cell Biol 14: 4912–4919.
13.  Chaskes S, Tyndall RL. (19750. Pigment production by Cryptococcus neoformans frompara- and ortho-diphenols: Effect of the nitrogen source. J Clin Microbiol 1: 500–514.
14.  Chayakulkeeree M, Johnston SA, Oei JB, Lev S, Williamson PR,Wilson CF, Zuo X, Leal AL, Vainstein MH,MeyerW, et al. (2011). SEC14 is a specific requirement for secretion of phospholipase B1 and pathogenicity of Cryptococcus neoformans. Mol Microbiol 80: 1088–1101.
15.  Chen SC, Muller M, Zhou JZ, Wright LC, Sorrell TC. (19970. Phospholipase activity in Cryptococcus neoformans: A new virulence factor? J Infect Dis 175: 414–420.
16.  Chung S, Mondon P, Chang YC, Kwon-Chung KJ. (2003). Cryptococcus neoformans with a mutation in the tetratricopeptide repeat-containing gene, CCN1, causes subcutaneous lesions but fails to cause systemic infection. Infect Immun 71: 1988–1994.
17.   Cottrell TR, Griffith CL, Liu H, Nenninger AA, Doering TL. (2007). The pathogenic fungus Cryptococcus neoformans expresses two functional GDP-mannose transporters with distinct expression patterns and roles in capsule synthesis. Eukaryot Cell 6: 776–785.
18.  Cox GM, Harrison TS, McDade HC, Taborda CP, HeinrichG, Casadevall A, Perfect JR. (2003). Superoxide dismutase influences the virulence of Cryptococcus neoformans by affecting growth within macrophages. Infect Immun 71:173–180.
19.  Cox GM, McDade HC, Chen SC, Tucker SC, Gottfredsson M, Wright LC, Sorrell TC, Leidich SD, Casadevall A, Ghannoum MA, et al. (2001). Extracellular phospholipase activity is a virulence factor for Cryptococcus neoformans. Mol Microbiol 39: 166–175.
20.  Cox GM, Mukherjee J, Cole GT, Casadevall A, Perfect JR. (2000). Urease as a virulence factor in experimental cryptococcosis. Infect Immun 68: 443–448.
21.  Ding C, Festa RA, Chen YL, Espart A, Palacios O, Espin J, Capdevila M, Atrian S, Heitman J, Thiele DJ. (2013). Cryptococcus neoformans copper detoxification machinery is critical for fungal virulence. Cell Host Microbe 13: 265–276.
22.  Ding C, Yin J, Tovar EM, FitzpatrickDA, Higgins DG, Thiele DJ. (2011). The copper regulon of the human fungal pathogen Cryptococcus neoformans H99. Mol Microbiol 81: 1560–1576.
23.  Doering TL. (2009). How sweet it is! Cell wall biogenesis and polysaccharide capsule formation in Cryptococcus neoformans. Annu Rev Microbiol 63: 223–247.
24.  Dromer F, Casadevall A, Perfect JR, Sorrell T. (2011). Cryptococcus neoformans: Latency and disease. In Cryptococcus: From human pathogen to model yeast (ed. Heitman J, et al.), pp. 431–439. ASM, Washington, DC.
25.  Eisenman HC, Frases S, Nicola AM, Rodrigues ML, Casadevall A. (2009). Vesicle-associated melanization in Cryptococcus neoformans. Microbiol 155: 3860–3867.
26.  Fonseca A, Boekhout T, Fell JW. (2011). Cryptococcus Vuillemin (1901). In The yeasts: A taxonomic study, Vol. 3 (ed.Kurtzman CP, Fell JW, Boekhout T), pp. 1661–1745. Elsevier, Amsterdam.
27.  Fox DS, Cruz MC, Sia RA, Ke H, Cox GM, Cardenas ME, Heitman J. (2001). Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12-FK506 in Cryptococcus neoformans. Mol Microbiol 39: 835–849.
28.  Frases S, Nimrichter L, Viana NB, Nakouzi A, Casadevall A. (2008). Cryptococcus neoformans capsular polysaccharide and exopolysaccharide fractions manifest physical, chemical, and antigenic differences. Eukaryot Cell 7: 319–327.
29.  Ganendren R, Carter E, Sorrell T,Widmer F,Wright L. (2006). Cryptococcus gattii, BritishColumbia, Canada, 1999–2007. Emerg Infect Dis 16: 251–257.
30.  Griffith CL, Klutts JS, Zhang L, Levery SB, Doering TL. (2004). UDP-glucose dehydrogenase plays multiple roles in the biology of the pathogenic fungus Cryptococcus neoformans. J Biol Chem 279: 51669–51676
31.  Heiss C, Skowyra ML, Liu H, Klutts JS, Wang Z, Williams M, Srikanta D, Beverley SM, Azadi P, Doering TL. (2013). Unusual galactofuranose modification of a capsule polysaccharide in the pathogenic yeast Cryptococcus neoformans. J Biol Chem 288: 10994–11003.
32.  Idnurm A, Heitman J. (2005). Light controls growth and development via a conserved pathway in the fungal kingdom. PLoS Biol 3: 615–626.
33.  Idnurm A, Heitman J. (2010). Ferrochelatase is a conserved downstream target of the blue light-sensing white collar complex in fungi. Microbiol 156: 2393–2407.
34.  Idnurm A, Reedy JL, Nussbaum JC, Heitman J. (2004). Cryptococcus neoformans virulence gene discovery through insertional mutagenesis. Eukaryot Cell 3: 420–429.
35.  Ikeda R, Sugita T, Jacobson ES, Shinoda T. 2003. Effects of melanin upon susceptibility of Cryptococcus to antifungals. Microbiol Immunol 47: 271–277.
36.  Jacobson ES, Ayers DJ, Harrell AC, Nicholas CC. (1982). Genetic and phenotypic characterization of capsule mutants of Cryptococcus neoformans. J Bacteriol 150: 1292–1296.
37.  Jacobson ES, Hong JD. (1997). Redox buffering by melanin and Fe(II) in Cryptococcus neoformans. J Bacteriol 179: 5340–5346.
38.  Janbon G, HimmelreichU,Moyrand F, Improvisi L, Dromer F. (2001). Cas1p is a membrane protein necessary for the O-acetylation of the Cryptococcus neoformans capsular polysaccharide. Mol Microbiol 42: 453–467.
39.  Jung WH, Saikia S, Hu G, Wang J, Fung CK, D’Souza C, White R, Kronstad JW. (2010). HapX positively and negatively regulates the transcriptional response to iron deprivation in Cryptococcus neoformans. PLoS Pathog 6:e1001209.
40.  Klengel T, Liang W-J, Chaloupka J, Ruoff C, Schroppel K, Naglik JR, Eckert SE,Mogensen EG, Haynes K, Tuite MF, et al. (2005). Fungal adenylyl cyclase integrates CO2 sensing with cAMP signaling and virulence. Curr Biol 15: 2021– 2026.
41.  Kraus PR, Boily MJ, Giles SS, Stajich JE, Allen A, Cox GM, Dietrich FS, Perfect JR, Heitman J. (2004). Identification of Cryptococcus neoformans temperature-regulated genes with a genomic-DNA microarray. Eukaryot Cell 3: 1249–1260.
42.  Kumar P, Yang M, Haynes BC, Skowyra ML, Doering TL. (2011). Emerging themes in cryptococcal capsule synthesis. Curr Opin Struct Biol 21: 597–602.
43.  Kwon-Chung K J,  Fraser J A, Doering T L , Wang Z , Janbon G, Idnurm A, and Bahn Y S (2014). Cryptococcus neoformans and Cryptococcus gattii, the Etiologic Agents of Cryptococcosis Cold Spring Harb Perspect Med
44.  Kwon-Chung KJ, Polacheck I, Popkin TJ. (1982). Melaninlacking mutants of Cryptococcus neoformans and their virulence for mice. J Bacteriol 150: 1414–1421.
45.  Kwon-Chung KJ, Rhodes JC. (1986). Encapsulation and melanin formation as indicators of virulence in Cryptococcus neoformans. Infect Immun 51: 218–223.
46.  Lin X, Huang JC, Mitchell TG, Heitman J. (2006). Virulence attributes and hyphal growth of Cryptococcus neoformans are quantitative traits and the MATa allele enhances filamentation. PLoS Genet 2: e187.
47.  Liu L, Tewari RP, Williamson PR. (1999). Laccase protects Cryptococcus neoformans from antifungal activity of alveolar macrophages. Infect Immun 67: 6034–6039.
48.  Liu OW, Chun CD, Chow ED, Chen C, Madhani HD, Noble SM. (2008). Systematic genetic analysis of virulence in the human fungal pathogen Cryptococcus neoformans. Cell 135: 174–188.
49.  Martinez LR, Casadevall A. (2006). Susceptibility of Cryptococcus neoformans biofilms to antifungal agents in vitro. Antimicrob Agents Chemother 50: 1021–1033.
50.  Mogensen EG, Janbon G, Chaloupka J, Steegborn C, Fu MS, Moyrand F, Klengel T, Pearson DS, Geeves MA, Buck J, et al. (2006). Cryptococcus neoformans senses CO2 through the carbonic anhydrase Can2 and the adenylyl cyclase Cac1. Eukaryot Cell 5: 103–111.
51.  Moyrand F, Fontaine T, Janbon G. (2007). Systematic capsule gene disruption reveals the central role of galactose metabolism on Cryptococcus neoformans virulence. Mol Microbiol 64: 771–781.
52.  Moyrand F, Klaproth B, Himmelreich U, Dromer F, Janbon G. (2002). Isolation and characterization of capsule structure mutant strains of Cryptococcus neoformans. MolMicrobiol 45: 837–849.
53.  Moyrand F, Lafontaine I, Fontaine T, Janbon G. (2008). UGE1 and UGE2 regulate the UDP-glucose/UDP-galactose equilibrium in Cryptococcus neoformans. Eukaryot Cell 7: 2069–2077.
54.  Nosanchuk JD, Casadevall A. (2006). Impact of melanin on microbial virulence and clinical resistance to antimicrobial compounds. Antimicrob Agents Chemother 50:3519–3528.
55.  Noverr MC, Cox GM, Perfect JR, Huffnagle GB. (2003). Role of PLB1 in pulmonary inflammation and cryptococcal eicosanoid production. Infect Immun 71: 1538–1547.
56.  Odom A,Muir S, Lim E, Toffaletti DL, Perfect J, Heitman J. (1997). Calcineurin is required for virulence of Cryptococcus neoformans. EMBO J 16: 2576–2589.
57.  Osterholzer JJ, Surana R, Milam JE,Montano GT, Chen GH, Sonstein J, Curtis JL,Huffnagle GB, Toews GB, Olszewski MA. (2009). Cryptococcal urease promotes the accumulation of immature dendritic cells and a non-protective T2 immune response within the lung. Am J Pathol 174: 932–943.
58.  Panepinto J, Komperda K, Frases S, Park YD, Djordjevic JT, Casadevall A, Williamson PR. (2009). Sec6-dependent sorting of fungal extracellular exosomes and laccase of Cryptococcus neoformans. Mol Microbiol 71: 1165–1176.
59.  Perfect JR. 2006. Cryptococcus neoformans: The yeast that likes it hot. FEMS Yeast Res 6: 463–468.
60.  Petter R, Kang BS, Boekhout T, Davis BJ, Kwon-Chung KJ. (2001). A survey of  heterobasidiomycetous yeasts for the presence of the genes homologous to virulence factors of Filobasidiella neoformans, CNLAC1 and CAP59. Microbiol 147: 2029–2036.
61.  Petter R, Kang BS, Boekhout T, Davis BJ, Kwon-Chung KJ. (2001). A survey of heterobasidiomycetous yeasts for the presence of the genes homologous to virulence factors of Filobasidiella neoformans, CNLAC1 and CAP59. Microbiol 147: 2029–2036.
62.  Polacheck I, Hearing VJ, Kwon-Chung KJ. (1982). Biochemical studies of phenoloxidase and utilization of catecholamines in Cryptococcus neoformans. J Bacteriol 150:1212–1220.
63.  Pukkila-Worley R, Gerrald QD, Kraus PR, Boily MJ, Davis MJ, Giles SS, Cox GM, Heitman J, Alspaugh JA. (2005). Transcriptional network of multiple capsule and melanin genes governed by the Cryptococcus neoformans cyclic AMP cascade. Eukaryot Cell 4: 190–201.
64.  Rhodes JC, Polacheck I, Kwon-Chung KJ. (1982). Phenoloxidase activity and virulence in isogenic strains of Cryptococcus neoformans. Infect Immun 36: 1175–1184.
65.  Rosa e Silva LK, Staats CC, Goulart LS, Morello LG, Pelegrinelli Fungaro MH, Schrank A, Vainstein MH. (2008). Identification of novel temperature-regulated genes in the human pathogen Cryptococcus neoformans using representational difference analysis. Res Microbiol 159: 221–229.
66.  Salas SD, Bennett JE, Kwon-Chung KJ, Perfect JR,Williamson PR. (1996). Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans. J Exp Med 184:377–386.
67.  Salyer WR, Salyer DC, Baker RD. (1974). Primary complex of Cryptococcus and pulmonary lymph nodes. J Infect Dis 130: 74–77.
68.  Schneider RdO, Fogac¸a NdS S, Kmetzsch L, Schrank A, Vainstein MH, Staats CC. (2012). Zap1 regulates zinc homeostasis and modulates virulence in Cryptococcus gattii. PLoS ONE 7: e43773.
69.  Shi M, Li SS, Zheng C, Jones GJ, Kim KS, Zhou H, Kubes P,Mody CH. (2010). Real-time imaging of trapping and urease-dependent transmigration of Cryptococcus neoformans in mouse brain. J Clin Invest 120: 1683–1693.
70.  Singh A, Panting RJ, Varma A, Saijo T,Waldron KJ, Jong A,Ngamskulrungroj P, Chang YC, Rutherford JC, Kwon-Chung KJ. (2013). Factors required for activation of urease as a virulence determinant in Cryptococcus neoformans. mBio 4: e00220-13.
71.  Sorrell TC, Chen SCA, Phillips P, Marr KA. (2011). Clinical perspectives on Cryptococcus neoformans and Cryptococcus gattii: Implications for diagnosis and management. In Cryptococcus: From human pathogen to model yeast (ed. Steen BR, Lian T, Zuyderduyn S,MacDonald WK, MarraM, Jones SJ, Kronstad JW. 2002. Temperature-regulated transcription in the pathogenic fungus Cryptococcus neoformans. Genome Res 12: 1386–1400.
72.  Steinbach WJ, Reedy JL, Cramer RA Jr., Perfect JR, Heitman J. (2007). Harnessing calcineurin as a novel anti-infective agent against invasive fungal infections. Nat Rev Microbiol 5: 418–430.
73.  Torres-GuererroH, Edman JC. (1994). Melanin-deficient mutants of Cryptococcus neoformans. J Med Vet Mycol 32: 303–313.
74.  Vaishnav VV, Bacon BE, O’Neill M, Cherniak  R. (1998). Structural characterization of the galactoxylomannan of Cryptococcus neoformans Cap67. Carbohydr Res 306: 315–330.
75.  van Duin D, Casadevall A, Nosanchuk JD. (2002). Melanization of Cryptococcus neoformans and Histoplasma capsulatum reduces their susceptibilities to amphotericin B and caspofungin. Antimicrob Agents Chemother 46:3394–3400.
76.  van DuinD, CleareW, ZaragozaO, Casadevall A,Nosanchuk JD. (2004). Effects of voriconazole on Cryptococcus neoformans. Antimicrob Agents Chemother 48: 2014–2020.
77.  Vartivarian SE, Anaissie EJ, Cowart RE, Sprigg HA, Tingler MJ, Jacobson ES. (1993). Regulation of cryptococcal capsular polysaccharide by iron. J Infect Dis 167: 186–190.
78.  Walton FJ, Idnurm A, Heitman J. (2005). Novel gene functions required for melanization of the human pathogen Cryptococcus neoformans. Mol Microbiol 57: 1381–1396.
79.  Walton FJ, Idnurm A, Heitman J. (2005). Novel gene functions required for melanization of the human pathogen Cryptococcus neoformans. Mol Microbiol 57: 1381–1396.
80.  Wang Y, Aisen P, Casadevall A. (1995). Cryptococcus neoformans melanin and virulence:Mechanism of action. Infect Immun 63: 3131–3136.
81.  Wang ZA, Griffith CL, Skowyra ML, Salina N, Williams M, Maier EJ, Gish SR, Liu H, Brent MR, Doering TL. (2014). The dual GDP-mannose transporters of Cryptococcus neoformans and their role in biology and virulence. Eukaryot Cell 13: 832–842.
82.  Waterman SR, Hacham M, Hu G, Zhu X, Park Y-D, Shin S, Panepinto J, Valyi-Nagy T, Beam C,Husain S, et al. (2007). Role of a CUF1/CTR4 copper regulatory axis in the virulence of Cryptococcus neoformans. J Clin Invest 117:794–802.
83.  Waterman SR, Hacham M, Panepinto J, Hu G, Shin S, Williamson PR. (2007). Cell wall targeting of laccase of Cryptococcus neoformans during infection of mice. Infect Immun 75: 714–722.
84.  Waterman SR, Park Y-D, Raja M, Qiu J, Hammoud DA, O’Halloran TV, Williamson PR. (2012). Role of CTR4 in the virulence of Cryptococcus neoformans. mBio 3:e00285-12.
85.  Wills EA, Roberts IS, Del Poeta M, Rivera J, Casadevall A,Cox GM, Perfect JR. 2001. Identification and characterization of the Cryptococcus neoformans phosphomannose isomerase-encoding gene, MAN1, and its impact on pathogenicity. Mol Microbiol 40: 610–620.
86.  Wright LC, Santangelo RM, Ganendren R, Payne J, Djordjevic JT, Sorrell TC. (2007). Cryptococcal lipid metabolism:Phospholipase B1 is implicated in transcellular metabolism of macrophage-derived lipids. Eukaryot Cell 6:37–47.
87.  Yoneda A, Doering TL. (2006). A eukaryotic capsular polysaccharide is synthesized intracellularly and secreted via exocytosis. Mol Biol Cell 17: 5131–5140.
88.  Zhu X, Gibbons J, Garcia-Rivera J, Casadevall A,Williamson PR. (2001). Laccase of Cryptococcus neoformans is a cellwall associated virulence factor. Infect Immun 69: 5589–5596.

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