Cryptococcus neoformans cryoultramicrotomy and vesicle fractionation reveals an intimate association between membrane lipids and glucuronoxylomannan

Cryptococcus neoformans is an encapsulated pathogenic fungus. The cryptococcal capsule is composed of polysaccharides and is necessary for virulence. It has been previously reported that glucuronoxylomannan (GXM), the major capsular component, is synthesized in cytoplasmic compartments and transported to the extracellular space in vesicles, but knowledge on the organelles involved in polysaccharide synthesis and traffic is extremely limited. In this paper we report the GXM distribution in C. neoformans cells sectioned by cryoultramicrotomy and visualized by transmission electron microscopy (TEM) and polysaccharide immunogold staining. Cryosections of fungal cells showed high preservation of intracellular organelles and cell wall structure. Incubation of cryosections with an antibody to GXM revealed that cytoplasmic structures associated to vesicular compartments and reticular membranes are in close proximity to the polysaccharide. GXM was generally found in association with the membrane of intracellular compartments and within different layers of the cell wall. Analysis of extracellular fractions from cryptococcal supernatants by transmission electron microscopy in combination with serologic, chromatographic and spectroscopic methods revealed fractions containing GXM and lipids. These results indicate an intimate association of GXM and lipids in both intracellular and extracellular spaces consistent with polysaccharide synthesis and transport in membrane-associated structures.     

Role of the Apt1 Protein in Polysaccharide Secretion by Cryptococcus neoformans

Flippases are key regulators of membrane asymmetry and secretory mechanisms. Vesicular polysaccharide secretion is essential for the pathogenic mechanisms of Cryptococcus neoformans. On the basis of the observations that flippases are required for polysaccharide secretion in plants and the putative Apt1 flippase is required for cryptococcal virulence, we analyzed the role of this enzyme in polysaccharide release by C. neoformans, using a previously characterized apt1Δ mutant. Mutant and wild-type (WT) cells shared important phenotypic characteristics, including capsule morphology and dimensions, glucuronoxylomannan (GXM) composition, molecular size, and serological properties. The apt1Δ mutant, however, produced extracellular vesicles (EVs) with a lower GXM content and different size distribution in comparison with those of WT cells. Our data also suggested a defective intracellular GXM synthesis in mutant cells, in addition to changes in the architecture of the Golgi apparatus. These findings were correlated with diminished GXM production during in vitro growth, macrophage infection, and lung colonization. This phenotype was associated with decreased survival of the mutant in the lungs of infected mice, reduced induction of interleukin-6 (IL-6) cytokine levels, and inefficacy in colonization of the brain. Taken together, our results indicate that the lack of APT1 caused defects in both GXM synthesis and vesicular export to the extracellular milieu by C. neoformans via processes that are apparently related to the pathogenic mechanisms used by this fungus during animal infection.     

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 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.     

Antibodies to keyhole limpet hemocyanin cross-react with an epitope on the polysaccharide capsule of Cryptococcus neoformans and other carbohydrates: implications for vaccine development

Cryptococcus neoformans causes a life-threatening meningoencephalitis in AIDS patients. Mice immunized with a glycoconjugate vaccine composed of the glucuronoxylomannan (GXM) component of the cryptococcal capsular polysaccharide conjugated to tetanus toxoid produce Abs that can be either protective or nonprotective. Because nonprotective Abs block the efficacy of protective Abs, an effective vaccine must focus the Ab response on a protective epitope. Mice immunized with peptide mimetics of GXM conjugated to keyhole limpet hemocyanin (KLH) with glutaraldehyde developed Abs to GXM. However, control peptides P315 and P24 conjugated to KLH also elicited Abs to GXM. GXM-binding Abs from mice immunized with P315-KLH were inhibited by KLH treated with glutaraldehyde (KLH-g), but not by P315. Furthermore, KLH-g inhibited binding of GXM by serum of mice immunized with GXM-TT, indicating that glutaraldehyde treatment of KLH reveals an epitope(s) that cross-reacts with GXM. Vaccination with KLH-g or unmodified KLH elicited Abs to GXM, but did not confer protection against C. neoformans, suggesting the cross-reactive epitope on KLH was not protective. This was supported by the finding that 4H3, a nonprotective mAb, cross-reacted strongly with KLH-g. Sera from mice immunized with either native KLH or KLH-g cross-reacted with several other carbohydrate Ags, many of which have been conjugated to KLH for vaccine development. This study illustrates how mAbs can be used to determine the efficacy of potential vaccines, in addition to describing the complexity of using KLH and glutaraldehyde in the development of vaccines to carbohydrate Ags.     

An unusual organelle in Cryptococcus neoformans links luminal pH and capsule biosynthesis

Cryptococcus neoformans is a basidiomycete that causes deadly infections in the immunocompromised. We previously generated a secretion mutant in this fungus by introducing a mutation in the SAV1 gene, which encodes a homolog of the Sec4/Rab8 subfamily GTPases. Under restrictive conditions there are two notable morphological changes in the sav1 mutant: accumulation of post-Golgi vesicles and the appearance of an unusual organelle, which we term the sav1 body (SB). The SB is an electron-transparent structure 0.2–1 μm in diameter, with vesicles or other membranous structures associated with the perimeter. Surprisingly, the SB was heavily labeled with anti-glucuronoxylomannan (GXM) antibodies, suggesting that it contains a secreted capsule component, GXM. A structure similar to the SB, also labeled by anti-GXM antibodies, was induced in wild type cells treated with the vacuolar-ATPase inhibitor, bafilomycin A₁. Bafilomycin A₁ and other agents that increase intraluminal pH also inhibited capsule polysaccharide shedding and capsule growth. These studies highlight an unusual organelle observed in C. neoformans with a potential role in polysaccharide synthesis, and a link between luminal pH and GXM biosynthesis.     

A Novel Xylosylphosphotransferase Activity Discovered in Cryptococcus neoformans

Cryptococcus neoformans is a fungal pathogen that causes serious disease in immunocompromised individuals. The organism produces a distinctive polysaccharide capsule that is necessary for its virulence, a predominantly polysaccharide cell wall, and a variety of protein- and lipid-linked glycans. The glycan synthetic pathways of this pathogen are of great interest. Here we report the detection of a novel glycosylphosphotransferase activity in C. neoformans, identification of the corresponding gene, and characterization of the encoded protein. The observed activity is specific for UDP-xylose as a donor and for mannose acceptors and forms a xylose-α-1-phosphate-6-mannose linkage. This is the first report of a xylosylphosphotransferase activity in any system.     

Cryptococcus neoformans Activates RhoGTPase Proteins Followed by Protein Kinase C,Focal Adhesion Kinase,and Ezrin to Promote Traversal across the Blood-Brain Barrier

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis. Previous studies have demonstrated that Cryptococcus binding and invasion of human brain microvascular endothelial cells (HBMEC) is a prerequisite for transmigration across the blood-brain barrier. However, the molecular mechanism involved in the cryptococcal blood-brain barrier traversal is poorly understood. In this study we examined the signaling events in HBMEC during interaction with C. neoformans. Analysis with inhibitors revealed that cryptococcal association, invasion, and transmigration require host actin cytoskeleton rearrangement. Rho pulldown assays revealed that Cryptococcus induces activation of three members of RhoGTPases, e.g. RhoA, Rac1, and Cdc42, and their activations are required for cryptococcal transmigration across the HBMEC monolayer. Western blot analysis showed that Cryptococcus also induces phosphorylation of focal adhesion kinase (FAK), ezrin, and protein kinase C α (PKCα), all of which are involved in the rearrangement of host actin cytoskeleton. Down-regulation of FAK, ezrin, or PKCα by shRNA knockdown, dominant-negative transfection, or inhibitors significantly reduces cryptococcal ability to traverse the HBMEC monolayer, indicating their positive role in cryptococcal transmigration. In addition, activation of RhoGTPases is the upstream event for phosphorylation of FAK, ezrin, and PKCα during C. neoformans-HBMEC interaction. Taken together, our findings demonstrate that C. neoformans activates RhoGTPases and subsequently FAK, ezrin, and PKCα to promote their traversal across the HBMEC monolayer, which is the critical step for cryptococcal brain infection and development of meningitis.     

Cryptococcus neoformans-Derived Microvesicles Enhance the Pathogenesis of Fungal Brain Infection

Cryptococcal meningoencephalitis is the most common fungal disease in the central nervous system. The mechanisms by which Cryptococcus neoformans invades the brain are largely unknown. In this study, we found that C. neoformans-derived microvesicles (CnMVs) can enhance the traversal of the blood-brain barrier (BBB) by C. neoformans in vitro. The immunofluorescence imaging demonstrates that CnMVs can fuse with human brain microvascular endothelial cells (HBMECs), the constituents of the BBB. This activity is presumably due to the ability of the CnMVs to activate HBMEC membrane rafts and induce cell fusogenic activity. CnMVs also enhanced C. neoformans infection of the brain, found in both infected brains and cerebrospinal fluid. In infected mouse brains, CnMVs are distributed inside and around C. neoformans-induced cystic lesions. GFAP (glial fibrillary acidic protein)-positive astrocytes were found surrounding the cystic lesions, overlapping with the 14-3-3-GFP (14-3-3-green fluorescence protein fusion) signals. Substantial changes could be observed in areas that have a high density of CnMV staining. This is the first demonstration that C. neoformans-derived microvesicles can facilitate cryptococcal traversal across the BBB and accumulate at lesion sites of C. neoformans-infected brains. Results of this study suggested that CnMVs play an important role in the pathogenesis of cryptococcal meningoencephalitis.     

Phenotypic heterogeneity in expression of epitopes in the Cryptococcus neoformans capsule

The opportunistic yeast Cryptococcus neoformans is surrounded by a polysaccharide capsule comprised primarily of glucuronoxylomannan (GXM). GXM is a key component of the antigenic character of the capsule. Expression of the epitope that allows for binding of mAbs that require O -acetylation of GXM for mAb recognition was greatly influenced by cell age, growth conditions and serotype. Yeast cells of serotype A grown in vitro under capsule induction conditions showed considerable cell-to-cell variability in binding of two O -acetyl-dependent mAbs, and such mAbs uniformly failed to bind to GXM that covers yeast buds. Expression of the O -acetyl-dependent epitope increased with cell age. In contrast, all serotype A cells harvested from brain tissue bound the same O -acetyl-dependent mAbs. The ability of the cryptococcal capsule to activate the complement cascade and bind C3 occurred uniformly over the surface of all yeast cells, including the bud. Finally, the cell-to-cell variability in binding of O -acetyl-dependent mAbs with strains of serotype A was not found with strains of serotype D; almost all cells of serotype D showed homogeneous binding of O -acetyl-dependent mAbs. These results indicate that variability in expression of antigenic epitopes by GXM should be considered in selection of mAbs used for immunodiagnosis or immunotherapy.     

Binding of the wheat germ lectin to Cryptococcus neoformans suggests an association of chitinlike structures with yeast budding and capsular glucuronoxylomannan

The capsule of Cryptococcus neoformans is a complex structure whose assembly requires intermolecular interactions to connect its components into an organized structure. In this study, we demonstrated that the wheat germ agglutinin (WGA), which binds to sialic acids and beta-1,4-N-acetylglucosamine (GlcNAc) oligomers, can also bind to cryptococcal capsular structures. Confocal microscopy demonstrated that these structures form round or hooklike projections linking the capsule to the cell wall, as well as capsule-associated structures during yeast budding. Chemical analysis of capsular extracts by gas chromatography coupled to mass spectrometry and high-pH anion-exchange chromatography suggested that the molecules recognized by WGA were firmly associated with the cell wall. Enzymatic treatment, competition assays, and staining with chemically modified WGA revealed that GlcNAc oligomers, but not sialic acids, were the molecules recognized by the lectin. Accordingly, treatment of C. neoformans cells with chitinase released glucuronoxylomannan (GXM) from the cell surface and reduced the capsule size. Chitinase-treated acapsular cells bound soluble GXM in a modified pattern. These results indicate an association of chitin-derived structures with GXM and budding in C. neoformans, which may represent a new mechanism by which the capsular polysaccharide interacts with the cell wall and is rearranged during replication.     

A proteomic‐based approach for the identification of immunodominant Cryptococcus neoformans proteins

Cryptococcus neoformans is an opportunistic fungal pathogen that can cause life‐threatening meningoencephalitis in immune compromised patients. Previous, studies in our laboratory have shown that prior exposure to an IFN‐γ‐producing C. neoformans strain (H99γ) elicits protective immunity against a second pulmonary C. neoformans challenge. Here, we characterized the antibody response produced in mice protected against experimental pulmonary C. neoformans infection compared to nonprotected mice. Moreover, we evaluated the efficacy of using serum antibody from protected mice to detect immunodominant C. neoformans proteins. Protected mice were shown to produce significantly more C. neoformans‐specific antibodies following a second experimental pulmonary cryptococcal challenge compared to nonprotected mice. Immunoblot analysis of C. neoformans proteins resolved by 2‐DE using serum from nonprotected mice failed to show any reactivity. In contrast, serum from protected mice was reactive with several cryptococcal protein spots. Analysis of these spots by capillary HPLC‐ESI‐MS/MS identified several cryptococcal proteins shown to be associated with the pathogenesis of cryptococcosis. Our studies demonstrate that mice immunized with C. neoformans strain H99γ produce antibodies that are immune reactive against specific cryptococcal proteins that may provide a basis for the development of immune based therapies that induce protective anticryptococcal immune responses.     

Extracellular vesicles produced by Cryptococcus neoformans contain protein components associated with virulence

Cryptococcus neoformans produces vesicles containing its major virulence factor, the capsular polysaccharide glucuronoxylomannan (GXM). These vesicles cross the cell wall to reach the extracellular space, where the polysaccharide is supposedly used for capsule growth or delivered into host tissues. In the present study, we characterized vesicle morphology and protein composition by a combination of techniques including electron microscopy, proteomics, enzymatic activity, and serological reactivity. Secretory vesicles in C. neoformans appear to be correlated with exosome-like compartments derived from multivesicular bodies. Extracellular vesicles manifested various sizes and morphologies, including electron-lucid membrane bodies and electron-dense vesicles. Seventy-six proteins were identified by proteomic analysis, including several related to virulence and protection against oxidative stress. Biochemical tests indicated laccase and urease activities in vesicles. In addition, different vesicle proteins were recognized by sera from patients with cryptococcosis. These results reveal an efficient and general mechanism of secretion of pathogenesis-related molecules in C. neoformans, suggesting that extracellular vesicles function as “virulence bags” that deliver a concentrated payload of fungal products to host effector cells and tissues.     

Acapsular Cryptococcus neoformans activates the NLRP3 inflammasome

Cryptococcus neoformans (C. neoformans) is an opportunistic fungal pathogen that mainly infects immunocompromised individuals such as AIDS patients. Although cell surface receptors for recognition of C. neoformans have been studies intensively, cytoplasmic recognition of this pathogen remains unclear. As an important detector of pathogen infection, inflammasome can sense and get activated by infection of various pathogens, including pathogenic fungi such as Candida albicans and Aspergillus fumigatus. Our present study showed that acapsular C. neoformans (cap59Δ) activated the NLRP3-, but not AIM2-nor NLRC4- inflammasome. During this process, viability of the fungus was required. Moreover, our in vivo results showed that during the pulmonary infection of cap59Δ, immune cell infiltration into the lung and effective clearance of the fungus were both dependent on the presence of NLRP3 inflammasome. In summary, our data suggest that the capsule of C. neoformans prevents recognition of the fungus by host NLRP3 inflammasome and indicate that manipulation of inflammasome activity maybe a novel approach to control C. neoformans infection.     

Antibody-Mediated Immobilization of Cryptococcus neoformans Promotes Biofilm Formation

Most microbes, including the fungal pathogen Cryptococcus neoformans, can grow as biofilms. Biofilms confer upon microbes a range of characteristics, including an ability to colonize materials such as shunts and catheters and increased resistance to antibiotics. Here, we provide evidence that coating surfaces with a monoclonal antibody to glucuronoxylomannan, the major component of the fungal capsular polysaccharide, immobilizes cryptococcal cells to a surface support and, subsequently, promotes biofilm formation. We used time-lapse microscopy to visualize the growth of cryptococcal biofilms, generating the first movies of fungal biofilm growth. We show that when fungal cells are immobilized using surface-attached specific antibody to the capsule, the initial stages of biofilm formation are significantly faster than those on surfaces with no antibody coating or surfaces coated with unspecific monoclonal antibody. Time-lapse microscopy revealed that biofilm growth was a dynamic process in which cells shuffled position during budding and was accompanied by emergence of planktonic variant cells that left the attached biofilm community. The planktonic variant cells exhibited mobility, presumably by Brownian motion. Our results indicate that microbial immobilization by antibody capture hastens biofilm formation and suggest that antibody coating of medical devices with immunoglobulins must exclude binding to common pathogenic microbes and the possibility that this effect could be exploited in industrial microbiology.Cryptococcus neoformans is a fungal pathogen that is ubiquitous in the environment and enters the body via the inhalation of airborne particles. The C. neoformans cell is surrounded by a layer of polysaccharide that consists predominantly of glucuronoxylomannan (GXM), which forms a protective capsule around the microbe. The capsule has been shown to be essential for virulence in murine models of infection (5-7) and, thus, is considered a key virulence factor. C. neoformans is the causative agent of cryptococcosis, a disease that primarily affects individuals with impaired immune systems, and is a significant problem in AIDS patients (21, 31). The most common manifestation of cryptococcosis is meningoencephalitis.Biofilms are communities of microbes that are attached to surfaces and held together by an extracellular matrix, often consisting predominantly of polysaccharides (8, 10). A great deal is known about bacterial biofilms (3, 9, 24, 30), but fungal biofilm formation is much less studied. Candida albicans is known to synthesize biofilms (11, 28, 29), as is C. neoformans. Biofilm-like structures consisting of innumerable cryptococcal cells encased in a polysaccharide matrix have been reported in human cases of cryptococcosis (32). Biofilm formation confers upon the microbe the capacity for drug resistance, and microbial cells in biofilms are less susceptible to host defense mechanisms (2, 4, 9, 12). In this regard, cells within C. neoformans biofilms are significantly less susceptible to caspofungin and amphotericin B than are planktonic cells (19). The cells within the biofilm are also resistant to the actions of fluconazole and voriconazole and various microbial oxidants and peptides (17, 19).Bacterial and fungal biofilms form readily on prosthetic materials, which poses a tremendous risk of chronic infection (10, 13, 15, 27). C. neoformans biofilms can form on a range of surfaces, including glass, polystyrene, and polyvinyl, and material devices, such as catheters (16). C. neoformans can form biofilms on the ventriculoatrial shunts used to decompress intracerebral pressure in patients with cryptococcal meningoencephalitis (32).The polysaccharide capsule of C. neoformans is essential for biofilm formation (18), and biofilm formation involves the shedding and accumulation of large amounts of GXM into the biofilm extracellular matrix (16). Previously, we reported that antibody to GXM in solution could inhibit biofilm formation through a process that presumably involves interference with polysaccharide shedding (18, 20). However, the effect of antibody-mediated immobilization of C. neoformans cells on cryptococcal biofilm formation has not been explored. In this paper, we report that the monoclonal antibody (MAb) 18B7, which is specific for the capsular polysaccharide GXM, can capture and immobilize C. neoformans to surfaces, a process that promotes biofilm formation. Interestingly, we identified planktonic variant C. neoformans cells that appeared to escape from the biofilm, but whose functions are not known. The results provide new insights on biofilm formation.     

Glucuronoxylomannan in the Cryptococcus species capsule as a target for Chimeric Antigen Receptor T-cell therapy

Background aimsThe genus Cryptococcus comprises two major fungal species that cause clinical infections in humans: Cryptococcus gattii and Cryptococcus neoformans. To establish invasive human disease, inhaled cryptococci must penetrate the lung tissue and reproduce. Each year, about 1 million cases of Cryptococcus infection are reported worldwide, and the infection's mortality rate ranges from 20% to 70%. Many HIV⁺/AIDS patients are affected by Cryptococcus infections, with 220,000 cases of cryptococcal meningitis reported worldwide in this population every year (C. neoformans infection statistics, via the Centers for Disease Control and Prevention, https://www.cdc.gov/fungal/diseases/cryptococcosis-neoformans/statistics.html). To escape from host immune cell attack, Cryptococcus covers itself in a sugar-based capsule composed primarily of glucuronoxylomannan (GXM). To evade phagocytosis, yeast cells increase to a >45-µm perimeter and become titan, or giant, cells. Cryptococci virulence is directly proportional to the percentage of titan/giant cells present during Cryptococcus infection. To combat cryptococcosis, the authors propose the redirection of CD8⁺ T cells to target the GXM in the capsule via expression of a GXM-specific chimeric antigen receptor (GXMR-CAR).ResultsGXMR-CAR has an anti-GXM single-chain variable fragment followed by an IgG4 stalk in the extracellular domain, a CD28 transmembrane domain and CD28 and CD3-? signaling domains. After lentiviral transduction of human T cells with the GXMR-CAR construct, flow cytometry demonstrated that 82.4% of the cells expressed GXMR-CAR on their surface. To determine whether the GXMR-CAR⁺ T cells exhibited GXM-specific recognition, these cells were incubated with GXM for 24 h and examined with the use of brightfield microscopy. Large clusters of proliferating GXMR-CAR⁺ T cells were observed in GXM-treated cells, whereas no clusters were observed in control cells. Moreover, the interaction of GXM with GXMR-CAR⁺ T cells was detected via flow cytometry by using a GXM-specific antibody, and the recognition of GXM by GXMR-CAR T cells triggered the secretion of granzyme and interferon gamma (IFN-γ). The ability of GXMR-CAR T cells to bind to the yeast form of C. neoformans was detected by fluorescent microscopy, but no binding was detected in mock-transduced control T cells (NoDNA T cells). Moreover, lung tissue sections were stained with Gomori Methenamine Silver and evaluated by NanoZoomer (Hamamatsu), revealing a significantly lower number of titan cells, with perimeters ranging from 50 to 130 µm and giant cells >130 µm in the CAR T-cell treated group when compared with other groups. Therefore, the authors validated the study's hypothesis by the redirection of GXMR-CAR⁺ T cells to target GXM, which induces the secretion of cytotoxic granules and IFN-γ that will aid in the control of cryptococcosisConclusionsThus, these findings reveal that GXMR-CAR⁺ T cells can target C. neoformans. Future studies will be focused on determining the therapeutic efficacy of GXMR-CAR⁺ T cells in an animal model of cryptococcosis.     

Chitin-Like Molecules Associate with Cryptococcus neoformans Glucuronoxylomannan To Form a Glycan Complex with Previously Unknown Properties

In prior studies, we demonstrated that glucuronoxylomannan (GXM), the major capsular polysaccharide of the fungal pathogen Cryptococcus neoformans, interacts with chitin oligomers at the cell wall-capsule interface. The structural determinants regulating these carbohydrate-carbohydrate interactions, as well as the functions of these structures, have remained unknown. In this study, we demonstrate that glycan complexes composed of chitooligomers and GXM are formed during fungal growth and macrophage infection by C. neoformans. To investigate the required determinants for the assembly of chitin-GXM complexes, we developed a quantitative scanning electron microscopy-based method using different polysaccharide samples as inhibitors of the interaction of chitin with GXM. This assay revealed that chitin-GXM association involves noncovalent bonds and large GXM fibers and depends on the N-acetyl amino group of chitin. Carboxyl and O-acetyl groups of GXM are not required for polysaccharide-polysaccharide interactions. Glycan complex structures composed of cryptococcal GXM and chitin-derived oligomers were tested for their ability to induce pulmonary cytokines in mice. They were significantly more efficient than either GXM or chitin oligomers alone in inducing the production of lung interleukin 10 (IL-10), IL-17, and tumor necrosis factor alpha (TNF-α). These results indicate that association of chitin-derived structures with GXM through their N-acetyl amino groups generates glycan complexes with previously unknown properties.     

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.     

Structural Characterization by Multistage Mass Spectrometry (MSn) of Human Milk Glycans Recognized by Human Rotaviruses

We have shown that recombinant forms of VP8* domains of the human rotavirus outer capsid spike protein VP4 from human neonatal strains (N155(G10P[11]) and RV3(G3P[6]) and a bovine strain (B223) recognize unique glycans within the repertoire of human milk glycans. The accompanying study by Yu et al.², describes a human milk glycan shotgun glycan microarray that led to the identification of 32 specific glycans in the human milk tagged glycan library that were recognized by these human rotaviruses. These microarray analyses also provided a variety of metadata about the recognized glycan structures compiled from anti-glycan antibody and lectin binding before and after specific glycosidase digestions, along with compositional information from mass analysis by matrix-assisted laser desorption ionization-mass spectrometry. To deduce glycan sequence and utilize information predicted by analyses of metadata from each glycan, 28 of the glycan targets were retrieved from the tagged glycan library for detailed sequencing using sequential disassembly of glycans by ion-trap mass spectrometry. Our aim is to obtain a deeper structural understanding of these key glycans using an orthogonal approach for structural confirmation in a single ion trap mass spectrometer. This sequential ion disassembly strategy details the complexities of linkage and branching in multiple compositions, several of which contained isomeric mixtures including several novel structures. The application of this approach exploits both library matching with standard materials and de novo approaches. This combination together with the metadata generated from lectin and antibody-binding data before and after glycosidase digestions provide a heretofore-unavailable level of analytical detail to glycan structure analysis. The results of these studies showed that, among the 28 glycan targets analyzed, 27 unique structures were identified, and 23 of the human milk glycans recognized by human rotaviruses represent novel structures not previously described as glycans in human milk. The functional glycomics analysis of human milk glycans provides significant insight into the repertoire of glycans comprising the human milk metaglycome.