EDTA Inhibits Biofilm Formation,Extracellular Vesicular Secretion,and Shedding of the Capsular Polysaccharide Glucuronoxylomannan by Cryptococcus neoformans

The fungal pathogen Cryptococcus neoformans can grow as a biofilm on a range of synthetic and prosthetic materials. Cryptococcal biofilm formation can complicate the placement of shunts used to relieve increased intracranial pressure in cryptococcal meningitis and can serve as a nidus for chronic infection. Biofilms are generally advantageous to pathogens in vivo, as they can confer resistance to antimicrobial compounds, including fluconazole and voriconazole in the case of C. neoformans. EDTA can inhibit biofilm formation by several microbes and enhances the susceptibility of biofilms to antifungal drugs. In this study, we evaluated the effect of sublethal concentrations of EDTA on the growth of cryptococcal biofilms. EDTA inhibited biofilm growth by C. neoformans, and the inhibition could be reversed by the addition of magnesium or calcium, implying that the inhibitory effect was by divalent cation starvation. EDTA also reduced the amount of the capsular polysaccharide glucuronoxylomannan shed into the biofilm matrix and decreased vesicular secretion from the cell, thus providing a potential mechanism for the inhibitory effect of this cation-chelating compound. Our data imply that the growth of C. neoformans biofilms requires the presence of divalent metals in the growth medium and suggest that cations are required for the export of materials needed for biofilm formation, possibly including extracellular vesicles.     

A Putative P-Type ATPase,Apt1, Is Involved in Stress Tolerance and Virulence in Cryptococcus neoformans

The export of virulence factors, such as the capsule polysaccharide, to the cell surface is a critical aspect of the pathogenicity of Cryptococcus neoformans. A view of capsule export via exocytosis and extracellular vesicles is emerging, but the molecular mechanisms underlying virulence factor transport pathways remain to be established. In this study, we characterized the APT1 gene, which encodes a predicted integral membrane P-type ATPase belonging to the type IV, Drs2 family of aminophospholipid translocases (flippases) (APTs). APTs maintain the phospholipid asymmetry that is critical in membrane fusion events for trafficking and in establishing cell polarity. Deletion of the APT1 gene resulted in phenotypes consistent with similar roles in C. neoformans. These included altered actin distribution, increased sensitivity to stress conditions (oxidative and nitrosative stress) and to trafficking inhibitors, such as brefeldin A and monensin, a reduction in exported acid phosphatase activity, and hypersensitivity to the antifungal drugs amphotericin B, fluconazole, and cinnamycin. However, there was no difference in growth, capsule size, or melanin production between the wild type and the apt1 mutant strains at either 30°C or 37°C. Despite the absence of an influence on these major virulence factors, Apt1 was required for survival during interactions with macrophages, and apt1 mutants exhibited attenuated virulence in a mouse inhalation model of cryptococcosis. Therefore, Apt1 contributes to virulence and the stress response in C. neoformans through apparent functions in membrane fusion and trafficking that do not influence the deposition of major virulence factors, such as capsule and melanin, outside the cell.The opportunistic fungal pathogen Cryptococcus neoformans causes life-threatening meningoencephalitis in immunocompromised individuals (44). One million cases of cryptococcosis are estimated to occur each year, and approximately two-thirds of these are fatal (43). Key virulence traits for the fungus include growth at the mammalian host temperature, production of a polysaccharide capsule, deposition of laccase-synthesized melanin in the cell wall, secretion of enzymes, and resistance to host defenses, such as oxidative and nitrosative killing (44).The polysaccharide capsule is a key virulence factor and is both cell associated and released during infection (4). The two species of polysaccharide in the capsule, an abundant glucuronoxylomannan (GXM) and a minor galactoxylomannan (GalXM), cause a number of deleterious effects in mammalian hosts (4, 44). Extracellular vesicles (exosomes) containing capsule polysaccharide are present in culture supernatants, in lysates of macrophages containing C. neoformans, and in association with fungal cells during murine infection (41, 49, 50, 54). These so-called “virulence factor delivery bags” are thought to pass through the cell wall to deliver material outside the cell (50). Proteomic analysis of the vesicles identified 76 proteins, and many of these are associated with virulence, including urease, laccase, heat shock proteins, superoxide dismutase, thiol-specific antioxidants, and catalases (49).The mechanisms of trafficking of capsule polysaccharide and laccase are being actively pursued. For example, analysis of a mutant with a defect in the exocyst GTPase Sec4/Rab8 (designated Sav1) revealed the accumulation of intracellular vesicles containing capsule polysaccharide, thus providing support for intracellular synthesis and secretion via exocytosis (60). In addition, reduced expression of the exocyst protein Sec6 due to RNA interference (RNAi) resulted in partial attenuation of virulence as well as defects in melanin production and the export of urease and soluble capsule polysaccharide (42). The RNAi strains were also completely defective in the production of extracellular exosomes but retained wild-type (WT) levels of cell-associated capsule. Trafficking of the laccase required for melanin production and virulence has also been examined. Hu et al. (25) showed that C. neoformans lacking Vps34 (vacuolar protein sorting 34) had a marked reduction in melanin formation, suggesting that laccase-containing vesicles are derived from the endocytic pathway. Overall, the current evidence suggests that exocytic, endocytic, and specialized extracellular vesicles mediate the export of capsule and other virulence factors in C. neoformans (42, 49, 60).We demonstrated previously that vesicle trafficking functions in C. neoformans are regulated by the cAMP signal transduction pathway, which also controls the elaboration of both the capsule and melanin (28). We found that treatment of C. neoformans with inhibitors of Golgi apparatus-mediated transport (e.g., brefeldin A or monensin) or with lithium chloride results in inhibition of capsule expression (28). In addition, we found that cAMP-dependent protein kinase regulated the expression of a predicted phospatidylethanolamine binding protein, Ova1, which negatively influences capsule and melanin formation. These findings focused our attention on the roles of intracellular trafficking functions and phospholipids in virulence factor expression.In the context of phospholipid trafficking, some aminophospholipid translocases within the P-type ATPases are known to play roles in fungal virulence. For example, the aminophospholipid translocase MgApt2 is required for exocytosis during plant infection by the rice blast pathogen Magnaporthe grisea (18). P-type ATPases are a large family of multitransmembrane domain, ATP-dependent transporters, and three subfamilies are found in eukaryotes (29): (i) heavy metal ion ATPases (e.g., copper transporters), (ii) non-heavy-metal ion ATPases (e.g., Ca²⁺, H⁺, Na⁺, and K⁺ ATPases), and (iii) aminophospholipid translocases (APTs/flippases of the type IV or Drs2 family). APTs maintain the asymmetrical distribution of aminophospholipids in membranes by translocating phosphatidylserine (PS) and/or phosphatidylethanolamine (PE) from one leaflet of the bilayer to the other. Phospholipid asymmetry is important in membrane fusion events (vesicle budding and docking) at the plasma membrane and in the trans-Golgi network (3). Thus, APTs are required for efficient Golgi function and play roles in both endocytosis and exocytosis. Some disorders in humans have been linked or attributed to genes from the APT subfamily, including familial intrahepatic cholestasis and Angelman syndrome (32, 55).Previously, we constructed a deletion of the APT1 gene, encoding a putative aminophospholipid translocase, as part of a study to examine disomy at chromosome 13 in C. neoformans (27). Our preliminary phenotypic analysis suggested a connection to nitrosative stress and prompted further investigation of virulence-related functions. In the present study, we show that Apt1 is functionally related to Drs2 in Saccharomyces cerevisiae and has roles in membrane trafficking and sensitivity to stress (oxidative and nitrosative) and drugs targeting ergosterol biosynthesis and secretion. Importantly, loss of Apt1 does not influence capsule and melanin formation, but the protein is required for intracellular growth in macrophages and for full virulence in mice.     

Subcellular Localization Directs Signaling Specificity of the Cryptococcus neoformans Ras1 Protein

In the human fungal pathogen Cryptococcus neoformans, Ras signaling mediates sexual differentiation, morphogenesis, and pathogenesis. By studying Ras prenylation and palmitoylation in this organism, we have found that the subcellular localization of this protein dictates its downstream signaling specificity. Inhibiting C. neoformans Ras1 prenylation results in the defective general membrane targeting of this protein and the loss of all Ras function. In contrast, palmitoylation mediates localization of Ras1 to the plasma membrane and is required for normal morphogenesis and survival at high temperatures. However, palmitoylation and plasma membrane localization are not required for Ras-dependent sexual differentiation. Likely as a result of its effect on thermotolerance, Ras1 palmitoylation is also required for the pathogenesis of C. neoformans. These data support an emerging paradigm of compartmentalized Ras signaling. However, our studies also demonstrate fundamental differences between the Ras pathways in different organisms that emphasize the functional flexibility of conserved signaling cascades.     

Anti-Chemotactic Activity of Capsular Polysaccharide of Cryptococcus neoformans In Vitro

In this study, we demonstrated the anti-chemotaetic activity of the capsular polysaccharides (CPSs) isolated from each of the heavily (H)- and weakly (W)-encapsulated strains of Cryptococcus neoformans in vitro. The capacity for activation of the alternative complement pathway (ACP) of cells of the two C. neoformans strains in fresh human sera was comparable to that of zymosan (insoluble control), whereas the capacity for generation of the chemotactic factor (CF) of the cells of the two strains in fresh murine sera was markedly lower in the order H- < W-strain than that of zymosan. Conversely, the capacities for ACP activation and CF generation of the CPSs were extremely lower than those of lipopolysaccharide (LPS, soluble control). When zymosan-activated murine serum was incubated with CPS, both CPSs inhibited CF activity dose dependently. When zymosan-activated serum was incubated with heat-killed cells of each strain of C. neoformans, H and W, the CF activity of the treated sera decreased significantly, suggesting that CPS per se did not affect the neutrophils directly, but CPS absorbed CF. On the other hand, both CPSs were shown to possess the O-acetyl groups in their molecules by ¹H-nuclear magnetic resonance spectroscopy. The de-O-acetylation of both CPSs increased the capacity for ACP activation to a level similar to that of LPS, and the de-O-acetylated CPS of both strains exhibited a lower ability to inhibit CF than did native CPS. Collectively, these results suggest that the anti-chemotactic activity of CPS accounts for its ability to absorb the CF which was mostly generated at the sites around the cell wall of whole cells via the ACP, thus suppressing the inflammatory response by preventing dispersal of CF to the extracellular space; and also that the O-acetyl group is partly, if any, involved in the mechanism for incompetence in ACP activation as well as the inhibition of CF.     

Role of conserved active site residues in catalysis by phospholipase B1 from Cryptococcus neoformans

Phospholipase B1 (PLB1), secreted by the pathogenic yeast Cryptococcus neoformans, has an established role in virulence. Although the mechanism of its phospholipase B, lysophospholipase, and lysophospholipase transacylase activities is unknown, it possesses lipase, subtilisin protease aspartate, and phospholipase motifs containing putative catalytic residues S146, D392, and R108, respectively, conserved in fungal PLBs and essential for human cytosolic phospholipase A2 (cPLA2) catalysis. To determine the role of these residues in PLB1 catalysis, each was substituted with alanine, and the mutant cDNAs were expressed in Saccharomyces cerevisiae. The mutant PLB1s were deficient in all three enzymatic activities. As the active site structure of PLB1 is unknown, a homology model was developed, based on the X-ray structure of the cPLA2 catalytic domain. This shows that the two proteins share a closely related fold, with the three catalytic residues located in identical positions as part of a single active site, with S146 and D392 forming a catalytic dyad. The model suggests that PLB1 lacks the "lid" region which occludes the cPLA2 active site and provides a mechanism of interfacial activation. In silico substrate docking studies with cPLA2 reveal the binding mode of the lipid headgroup, confirming the catalytic dyad mechanism for the cleavage of the sn-2 ester bond within one of two separate binding tracts for the lipid acyl chains. Residues specific for binding arachidonic and palmitic acids, preferred substrates for cPLA2 and PLB1, respectively, are identified. These results provide an explanation for differences in substrate specificity between lipases sharing the cPLA2 catalytic domain fold and for the differential effect of inhibitors on PLB1 enzymatic activities.     

利用蛋白质组学技术筛选隐球菌作用血管内皮细胞后差异表达蛋白

目的筛选新生隐球菌孵育后血管内皮细胞与正常细胞的差异蛋白质谱。方法利用二维凝胶电泳获得新生隐球菌孵育后血管内皮细胞与正常细胞差异表达蛋白质点,并对部分差异蛋白点进行质谱鉴定分析。结果Peroxiredoxin1及Calpactin I light chain等13个蛋白的表达水平在两组细胞间发生明显改变。结论Peroxiredoxin1及Calpactin I lightchain等蛋白可能参与了新生隐球菌对血管内皮细胞屏障的侵袭过程。     

Influence of Molecular Sizes of Cryptococcus neoformans Capsular Polysaccharide on Phagocytosis

The role of capsular polysaccharides (CPS) of Cryptococcus neoformans in phagocytosis by murine alveolar macrophages was investigated in four strains of C. neoformans serotype A, YC-11, YC-5, YC-27 and YC-13. Phagocytosis rates increased markedly after adding 10% mouse serum, compared to fetal calf serum. The reverse relation between capsular thickness of C. neoformans and phagocytosis by alveolar macrophages was observed except in YC-27, which had thin capsules and high virulence. The phagocytosis rate in mice serum was 17.3% in YC-11 (capsule thickness 2.8-3.5 μm), 39.8% in YC-5 (capsule size 0.8-1.5 μm), 20.3% in YC-27 (capsule size 0.6-1.1 μm), and 62.8% in YC-13 (capsule not detected microscopically). The CPS of YC-11, YC-5, and YC-27 analyzed by gel-filtration using CL-2B showed high molecular fractions near the void volume. However, the CPS of YC-13 showed only low molecular fractions. The widely eluted CPS of YC-11 was separated into 3 fractions and each fraction was added in the phagocytosis assay of YC-13. Phagocytosis was markedly suppressed particularly by the addition of a higher molecular fraction. These results suggest that phagocytosis of C. neoformans by alveolar macrophages is influenced by the molecular sizes of the CPS.     

The Role of Ceramide Synthases in the Pathogenicity of Cryptococcus neoformans

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Role of laccase in the biology and virulence of Cryptococcus neoformans

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.     

Role of laccase in the biology and virulence of Cryptococcus neoformans

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.     

Impact of Protein Palmitoylation on the Virulence Potential of Cryptococcus neoformans

The localization and specialized function of Ras-like proteins are largely determined by posttranslational processing events. In a highly regulated process, palmitoyl groups may be added to C-terminal cysteine residues, targeting these proteins to specific membranes. In the human fungal pathogen Cryptococcus neoformans, Ras1 protein palmitoylation is essential for growth at high temperature but is dispensable for sexual differentiation. Ras1 palmitoylation is also required for localization of this protein on the plasma membrane. Together, these results support a model in which specific Ras functions are mediated from different subcellular locations. We therefore hypothesize that proteins that activate Ras1 or mediate Ras1 localization to the plasma membrane will be important for C. neoformans pathogenesis. To further characterize the Ras1 signaling cascade mediating high-temperature growth, we have identified a family of protein S-acyltransferases (PATs), enzymes that mediate palmitoylation, in the C. neoformans genome database. Deletion strains for each candidate gene were generated by homogenous recombination, and each mutant strain was assessed for Ras1-mediated phenotypes, including high-temperature growth, morphogenesis, and sexual development. We found that full Ras1 palmitoylation and function required one particular PAT, Pfa4, and deletion of the PFA4 gene in C. neoformans resulted in altered Ras1 localization to membranes, impaired growth at 37°C, and reduced virulence.     

Regulatory Diversity of TUP1 in Cryptococcus neoformans

Cryptococcus neoformans serotype A strains, the major cause of cryptococcosis, are distributed worldwide, while serotype D strains are more concentrated in Central Europe. We have previously shown that deletion of the global regulator TUP1 in serotype D isolates results in a novel peptide-mediated, density-dependent growth phenotype that mimics quorum sensing and is not known to exist in other fungi. Unlike for tup1Δ strains of serotype D, the density-dependent growth phenotype was found to be absent in tup1Δ strains of serotype A which had been derived from several different genetic clusters. The serotype A H99 tup1Δ strain showed less retardation in the growth rate than tup1Δ strains of serotype D, but the mating efficiency was found to be similar in both serotypes. Deletion of TUP1 in the H99 strain resulted in significantly enhanced capsule production and defective melanin formation and also revealed a unique regulatory role of the TUP1 gene in maintaining iron/copper homeostasis. Differential expression of various genes involved in capsule formation and iron/copper homeostasis was observed between the wild-type and tup1Δ H99 strains. Furthermore, the H99 tup1Δ strain displayed pleiotropic effects which included sensitivity to sodium dodecyl sulfate, susceptibility to fluconazole, and attenuated virulence. These results demonstrate that the global regulator TUP1 has pathobiological significance and plays both conserved and distinct roles in serotype A and D strains of C. neoformans.The fungal Tup1 proteins function as global repressors which regulate a large number of genes associated with growth, morphological differentiation, and sexual and asexual reproduction. As a consequence, tup1 mutants are known to display numerous phenotypes (9, 19, 42). The deletion of TUP1 in Candida albicans results in constitutive filamentous growth with no budding yeast cells and is accompanied by loss of virulence (2, 32). In Penicillium marneffei, the only dimorphic species known in the genus Penicillium, deletion of the TUP1 homolog, tupA, confers reduced filamentation and abnormality in yeast morphogenesis (38). In the filamentous fungi Aspergillus nidulans and Neurospora crassa, deletion of the TUP1 homologs, rcoA and rco-1, respectively, severely affects growth and sexual and asexual reproduction (12, 46).Cryptococcus neoformans is a bipolar heterothallic basidiomycetous yeast with two serotypes, A and D, and the function of Tup1 has been studied only for serotype D strains (26, 27). While disruption of TUP1 in strains of serotype D did not affect yeast or hyphal cell morphology, it resulted in mating-type-dependent differences, including temperature-dependent growth, sensitivity to 0.8 M KCl, and expression of genes in several other biological pathways (26). Most importantly, tup1Δ strains displayed a peptide-mediated quorum-sensing-like phenomenon in both mating types of serotype D strains which has not been reported for any other fungal species (27).According to genome sequence data, the serotype A reference strain H99 shares 95% sequence identity with the serotype D reference strain JEC21 (29). However, serotype-specific differences between the two strains have been demonstrated in two major signaling pathways, the pheromone-responsive Cpk1 mitogen-activated protein kinase and cyclic AMP (cAMP) (5, 13, 41, 47). In addition, the high-osmolarity glycerol (HOG) pathway also showed regulatory disparity between the two serotypes (1, 8). Since the regulation of peptide-mediated quorum sensing by TUP1 is reported only for serotype D strains, we sought to determine whether the deletion of TUP1 in serotype A strains would have similar consequences. Surprisingly, we found striking differences in the phenotypes manifested by tup1Δ strains of the two serotypes. We report here the serotype-specific differences in TUP1 regulation between A and D strains and the novel regulatory role of TUP1 in maintaining iron/copper homeostasis in C. neoformans.     

Polysaccharide diversity in VNI isolates of Cryptococcus neoformans from Roraima,Northern Brazil

Species of the Cryptococcus genus comprise environmental, encapsulated fungal pathogens that cause lethal meningitis in immunosuppressed individuals. In humans, fungal uptake of hypocapsular Cryptococcus by macrophages was associated with high fungal burden in the cerebrospinal fluid and long-term patient survival. On the basis of the key role of the cryptococcal capsule in disease, we analyzed the diversity of capsular structures in 23 isolates from pigeon excreta collected in the cities of Boa Vista, Bonfim and Pacaraima, in the state of Roraima (Northern Brazil). All isolates were identified as Cryptococcus neoformans (VNI genotype) by MALDI-TOF mass spectrometry. Through a combination of fluorescence microscopy, flow cytometry, ELISA and spectrophotometric methods, each isolate was characterized at the phenotypical level, which included measurements of growth rates at 30 and 37 °C, pigmentation, cell body size, capsular dimensions, serological reactivity, urease production and ability to produce extracellular glucuronoxylomannan (GXM), the main capsular component of C. neoformans. With the exception of melanization, a formidable diversity was observed considering all parameters tested in our study. Of note, hyper and hypo producers of GXM were identified, in addition to isolates with hyper and hypo profiles of reactivity with a polysaccharide-binding monoclonal antibody. Capsular dimensions were also highly variable in the collection of isolates. Extracellular GXM production correlated positively with capsular dimensions, urease activity and cell size. Unexpectedly, GXM concentrations did not correlate with serological reactivity with the cryptococcal capsule. These results reveal a high diversity in the ability of environmental C. neoformans to produce capsular components, which might impact the outcome of human cryptococcosis.     

Role of Cryptococcus neoformans Rho1 GTPases in the PKC1 Signaling Pathway in Response to Thermal Stress

To initiate and establish infection in mammals, the opportunistic fungal pathogen Cryptococcus neoformans must survive and thrive upon subjection to host temperature. Primary maintenance of cell integrity is controlled through the protein kinase C1 (PKC1) signaling pathway, which is regulated by a Rho1 GTPase in Saccharomyces cerevisiae. We identified three C. neoformans Rho GTPases, Rho1, Rho10, and Rho11, and have begun to elucidate their role in growth and activation of the PKC1 pathway in response to thermal stress. Western blot analysis revealed that heat shock of wild-type cells resulted in phosphorylation of Mpk1 mitogen-activated protein kinase (MAPK). Constitutive activation of Rho1 caused phosphorylation of Mpk1 independent of temperature, indicating its role in pathway regulation. A strain with a deletion of RHO10 also displayed this constitutive Mpk1 phosphorylation phenotype, while one with a deletion of RHO11 yielded phosphorylation similar to that of wild type. Surprisingly, like a rho10Δ strain, a strain with a deletion of both RHO10 and RHO11 displayed temperature sensitivity but mimicked wild-type phosphorylation, which suggests that Rho10 and Rho11 have coordinately regulated functions. Heat shock-induced Mpk1 phosphorylation also required the PKC1 pathway kinases Bck1 and Mkk2. However, Pkc1, thought to be the major regulatory kinase of the cell integrity pathway, was dispensable for this response. Together, our results argue that Rho proteins likely interact via downstream components of the PKC1 pathway or by alternative pathways to activate the cell integrity pathway in C. neoformans.     

Role for Chitin and Chitooligomers in the Capsular Architecture of Cryptococcus neoformans

Molecules composed of β-1,4-linked N-acetylglucosamine (GlcNAc) and deacetylated glucosamine units play key roles as surface constituents of the human pathogenic fungus Cryptococcus neoformans. GlcNAc is the monomeric unit of chitin and chitooligomers, which participate in the connection of capsular polysaccharides to the cryptococcal cell wall. In the present study, we evaluated the role of GlcNAc-containing structures in the assembly of the cryptococcal capsule. The in vivo expression of chitooligomers in C. neoformans varied depending on the infected tissue, as inferred from the differential reactivity of yeast forms to the wheat germ agglutinin (WGA) in infected brain and lungs of rats. Chromatographic and dynamic light-scattering analyses demonstrated that glucuronoxylomannan (GXM), the major cryptococcal capsular component, interacts with chitin and chitooligomers. When added to C. neoformans cultures, chitooligomers formed soluble complexes with GXM and interfered in capsular assembly, as manifested by aberrant capsules with defective connections with the cell wall and no reactivity with a monoclonal antibody to GXM. Cultivation of C. neoformans in the presence of an inhibitor of glucosamine 6-phosphate synthase resulted in altered expression of cell wall chitin. These cells formed capsules that were loosely connected to the cryptococcal wall and contained fibers with decreased diameters and altered monosaccharide composition. These results contribute to our understanding of the role played by chitin and chitooligosaccharides on the cryptococcal capsular structure, broadening the functional activities attributed to GlcNAc-containing structures in this biological system.Cryptococcus neoformans is the etiologic agent of cryptococcosis, a disease still characterized by high morbidity and mortality despite antifungal therapy (3). Pathogenic species belonging to the Cryptococcus genus also include Cryptococcus gattii, which causes disease mostly in immunocompetent individuals (24). A unique characteristic of Cryptococcus species is the presence of a polysaccharide capsule, which is essential for virulence (7-9, 19, 25, 33).C. neoformans has a complex cell surface. The thick fungal cell wall is composed of polysaccharides (29), pigments (11), lipids (35), and proteins (36). External to the cryptococcal cell wall, capsular polysaccharides form a capsule (19). Seemingly, the assembly of the surface envelope of C. neoformans requires the interaction of cell wall components with capsular elements. Some of the cryptococcal cell wall-capsule connectors have been identified, including the structural polysaccharide α-1,3-glucan and chitooligomers (29, 30, 32).Chitin-like molecules in fungi are polymerized by chitin synthases, which use cytoplasmic pools of UDP-GlcNAc (N-acetylglucosamine) to form β-1,4-linked oligosaccharides and large polymers. In C. neoformans, the final cellular site of chitin accumulation is the cell wall. The polysaccharide is also used for chitosan synthesis through enzymatic deacetylation (1). Eight putative cryptococcal chitin synthase genes and three regulator proteins have been identified (2). The chitin synthase Chs3 and regulator Csr2 may form a complex with chitin deacetylases for conversion of chitin to chitosan (1). Key early events in the synthesis of chitin/chitosan require the activity of glucosamine 6-phosphate synthase, which promotes the glutamine-dependent amination of fructose 6-phosphate to form glucosamine 6-phosphate, a substrate used for UDP-GlcNAc synthesis (23).In a previous study, we demonstrated that β-1,4-linked GlcNAc oligomers, which are specifically recognized by the wheat germ agglutinin (WGA), form bridge-like connections between the cell wall and the capsule of C. neoformans (32). In fact, other reports indicate that molecules composed of GlcNAc or its deacetylated derivative play key roles in C. neoformans structural biology. For example, mutations in the genes responsible for the expression of chitin synthase 3 or of the biosynthetic regulator Csr2p caused the loss of the ability to retain the virulence-related pigment melanin in the cell wall (1, 2). These cells were also defective in the synthesis of chitosan, which has also been demonstrated to regulate the retention of cell wall melanin (1). Treatment of C. neoformans acapsular mutants with chitinase affected the incorporation of capsular components into the cell wall (32). Considering that melanin and capsular components are crucial for virulence, these results strongly suggest that GlcNAc-derived molecules are key components of the C. neoformans cell surface. The expression of GlcNAc-containing molecules is likely to be modulated during infection since chitinase expression by host cells is induced during lung cryptococcosis (37).In this study, we used β-1,4-linked GlcNAc oligomers and an inhibitor of UDP-GlcNAc synthesis to evaluate the role played by GlcNAc-containing molecules in the surface architecture of C. neoformans. The results point to a direct relationship between the expression of GlcNAc-containing molecules and capsular assembly, indicating that chitin and chitooligomers are required for capsule organization in C. neoformans.     

Melanogenesis in Cryptococcus neoformans

Melanogenesis in Cryptococcus neoformans begins with the oxidation of dihydroxyphenylalanine by the enzyme phenol oxidase. The succeeding steps are very rapid. Two intermediates, dopachrome and 5,6-dihydroxyindole, have been isolated and characterized by high performance liquid chromatography. A pathway of melanin formation in C. neoformans is proposed, based on the presence of these intermediates.     

Non-specific immunosuppression by Cryptococcus neoformans infection

Cryptococcus neoformans-infected animals were found to be immunosuppressed when tested by a variety of assays for immune competence. Primary humoral immune responses and delayed-type hypersensitivity reactions to sheep erythrocytes were suppressed in animals which had been infected for two weeks. Lymphocyte proliferation (LP) assays to sRBC stroma were also significantly diminished at two weeks of infection. Spleen cells of infected mice suppressed the LP response of sRBC immunized, normal mice in vitro. At least a part of the suppression could be attributed to a nylon wool non-adherent cell. Suppressor cells continued to be present in spleen cell suspensions following treatment with anti-T cell serum or anti-immunoglobulin and complement. When infected spleen cells were separated by adherence to plastic, both the adherent and non-adherent fractions exhibited suppressive activity. Incubation of infected spleen cells in tissue culture for 48 hr resulted in the elaboration of soluble immunosuppreessive factors into the tissue culture medium. These data indicated that immune suppression in cryptococcosis can occur as a result of infection with Cryptococcus neoformans, and that at least one mechanism involved is the induction of adherent and non-adherent suppressor cells in the spleens of infected mice.