A synthetic glycan array containing Cryptococcus neoformans glucuronoxylomannan capsular polysaccharide fragments allows the mapping of protective epitopes.
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ABSTRACT:
SUBMITTER: Guazzelli L
PROVIDER: S-EPMC8163368 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC3485937 | biostudies-literature
Project description:The Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) has been conjugated to tetanus toxoid (GXM-TT) as an investigational vaccine. GXM-TT elicits antibodies that are protective in C. neoformans-infected mice. In an effort to characterize the fine specificity and molecular structure of human GXM-TT-elicited antibodies, we generated two GXM monoclonal antibodies (MAbs) from peripheral blood lymphocytes of a volunteer GXM-TT recipient and studied serum GXM antibody idiotype expression in 10 additional vaccinees. The MAbs, 2E9 and 3B6, are the immunoglobulin M(lambda) isotype and bind capsular polysaccharides of C. neoformans serotypes other than the serotype A that was used for immunization. Neither antibody competes with murine GXM MAbs for antigen binding, suggesting that the human MAbs recognize a different epitope. The B-cell superantigen staphylococcal protein A binds both MAbs, and human immunodeficiency virus gp120 binds 2E9. MAb nucleic acid sequence analysis revealed that both antibodies use an identical V lambda 1a-J lambda genetic element with different, somatically mutated, members of the VH3 gene family and different DH and JH gene elements. The gene elements used by both MAbs occur in fetal B-lymphocyte repertoires, autoantibodies, and other polysaccharide antibodies. Post-GXM-TT vaccination GXM antibodies from 10 additional vaccinees expressed a shared idiotype defined by rabbit antiserum raised against MAb 2E9. Our data suggest that the human GXM antibody response is restricted and raise questions regarding the importance of specific variable-region elements and superantigens in the generation of human antibody responses to encapsulated pathogens.
| S-EPMC173409 | biostudies-other
Project description:The capsular components of the human pathogen Cryptococcus neoformans are transported to the extracellular space and then used for capsule enlargement by distal growth. It is not clear, however, how the glucuronoxylomannan (GXM) fibers are incorporated into the capsule. In the present study, we show that concentration of C. neoformans culture supernatants by ultrafiltration results in the formation of highly viscous films containing pure polysaccharide, providing a novel, nondenaturing, and extremely rapid method to isolate extracellular GXM. The weight-averaged molecular mass of GXM in the film, determined using multiangle laser light scattering, was ninefold smaller than that of GXM purified from culture supernatants by differential precipitation with cetyl trimethyl ammonium bromide (CTAB). Polysaccharides obtained either by ultrafiltration or by CTAB-mediated precipitation showed different reactivities with GXM-specific monoclonal antibodies. Viscosity analysis associated with inductively coupled plasma mass spectrometry and measurements of zeta potential in the presence of different ions implied that polysaccharide aggregation was a consequence of the interaction between the carboxyl groups of glucuronic acid and divalent cations. Consistent with this observation, capsule enlargement in living C. neoformans cells was influenced by Ca(2+) in the culture medium. These results suggest that capsular assembly in C. neoformans results from divalent cation-mediated self-aggregation of extracellularly accumulated GXM molecules.
| S-EPMC1951138 | biostudies-literature
Project description:The major virulence factor of the pathogenic fungi Cryptococcus neoformans and C. gattii is the capsule. Glucuronoxylomannan (GXM), the major component of the capsule, is a high-molecular-weight polysaccharide that is shed during cryptococcosis and can persist in patients after successful antifungal therapy. Due to the importance of T cells in the anticryptococcal response, we studied the effect of GXM on the ability of dendritic cells (DCs) to initiate a T-cell response. GXM inhibited the activation of cryptococcal mannoprotein-specific hybridoma T cells and the proliferation of OVA-specific OT-II T cells when murine bone marrow-derived DCs were used as antigen-presenting cells. Inhibition of OT-II T-cell proliferation was observed when either OVA protein or OVA323-339 peptide was used as antigen, indicating GXM did not merely prevent antigen uptake or processing. We found that DCs internalize GXM progressively over time; however, the suppressive effect did not require DCs, as GXM directly inhibited T-cell proliferation induced by anti-CD3 antibody, concanavalin A, or phorbol-12-myristate-13-acetate/ionomycin. Analysis of T-cell viability revealed that the reduced proliferation in the presence of GXM was not the result of increased cell death. GXM isolated from each of the four major cryptococcal serotypes inhibited the proliferation of human peripheral blood mononuclear cells stimulated with tetanus toxoid. Thus, we have defined a new mechanism by which GXM can impart virulence: direct inhibition of T-cell proliferation. In patients with cryptococcosis, this could impair optimal cell-mediated immune responses, thereby contributing to the persistence of cryptococcal infections.
| S-EPMC1635532 | biostudies-literature
Project description:Cryptococcus neoformans capsular polysaccharide is composed of at least two components, glucuronoxylomannan (GXM) and galactoxylomannans (GalXM). Although GXM has been extensively studied, little is known about the location of GalXM in the C. neoformans capsule, in part because there are no serological reagents specific to this antigen. To circumvent the poor immunogenicity of GalXM, this antigen was conjugated to protective antigen from Bacillus anthracis as a protein carrier. The resulting conjugate elicited antibodies that reacted with GalXM in mice and yielded an immune serum that proved useful for studying GalXM in the polysaccharide capsule. In acapsular cells, immune serum localized GalXM to the cell wall. In capsulated cells, immune serum localized GalXM to discrete pockets near the capsule edge. GalXM was abundant on the nascent capsules of budding daughter cells. The constituent sugars of GalXM were found in vesicle fractions consistent with vesicular transport for this polysaccharide. In addition, we generated a single-chain fraction variable fragment antibody with specificity to oxidized carbohydrates that also produced punctate immunofluorescence on encapsulated cells that partially colocalized with GalXM. The results are interpreted to mean that GalXM is a transient component of the polysaccharide capsule of mature cells during the process of secretion. Hence, the function of GalXM appears to be more consistent with that of an exopolysaccharide than a structural component of the cryptococcal capsule.
| S-EPMC2620754 | biostudies-literature
Project description:As part of an ongoing project aimed at identifying protective capsular polysaccharide epitopes for the development of vaccine candidates against the fungal pathogen Cryptococcus neoformans, the synthesis and glycosylation properties of a naphthalenylmethyl (NAP) orthogonally protected trisaccharide thioglycoside, a common building block for construction of serotype B and C capsular polysaccharide structures, were investigated. Ethyl (benzyl 2,3,4-tri-O-benzyl-β-d-glucopyranosyl- uronate)-(1→2)-[2,3,4-tri-O-benzyl-β-d-xylopyranosyl-(1→4)]-6-O-benzyl-3-O-(2-naphthalenylmethyl)-1-thio-α-d-mannopyranoside was prepared and used both as a donor and an acceptor in glycosylation reactions to obtain spacer equipped hexa- and heptasaccharide structures suitable either for continued elongation or for deprotection and printing onto a glycan array or conjugation to a carrier protein. The glycosylation reactions proceeded with high yields and α-selectivity, proving the viability of the building block approach also for construction of 4-O-xylosyl-containing C. neoformans CPS structures.
| S-EPMC4906494 | biostudies-literature
Project description:S. pneumoniae is a major human pathogen with increasing antibiotic resistance. Pneumococcal vaccines consist of capsular polysaccharide (CPS) or their related fragments conjugated to a carrier protein. The repeating unit of S. pneumoniae type 14 CPS shares a core structure with the CPS of Group B Streptococcus (GBS) type III: the only difference is that the latter exhibits a sialic acid unit, with a α-2,3 linkage to galactose. Here, the automated glycan assembly (AGA) of two frameshifts of the repeating unit of S. pneumoniae type 14 is described. The same strategy is used to assemble dimers of the different repeating unit frameshifts. The four structures are assembled with only three commercially available monosaccharide building blocks. We also report an example of how enzymatic sialylation of the compounds obtained with AGA completes a synthetic route for GBS type III glycans. The synthesized structures were tested in competitive ELISA and further confirmed the branched tetrasaccharide Gal-Glc-(Gal-)GlcNAc to be the minimal epitope of S. pneumoniae type 14.
| S-EPMC9054924 | biostudies-literature
Project description:In recent years several groups have shown that isotype switching from IgM to IgG to IgA can affect the affinity and specificity of antibodies sharing identical variable (V) regions. However, whether the same applies to IgE is unknown. In this study we compared the fine specificity of V region-identical IgE and IgA to Cryptococcus neoformans capsular polysaccharide and found that these differed in specificity from each other. The IgE and IgA paratopes were probed by nuclear magnetic resonance spectroscopy with (15)N-labeled peptide mimetics of cryptococcal polysaccharide antigen (Ag). IgE was found to cleave the peptide at a much faster rate than V region-identical IgG subclasses and IgA, consistent with an altered paratope. Both IgE and IgA were opsonic for C. neoformans and protected against infection in mice. In summary, V-region expression in the context of the ϵ constant (C) region results in specificity changes that are greater than observed for comparable IgG subclasses. These results raise the possibility that expression of certain V regions in the context of α and ϵ C regions affects their function and contributes to the special properties of those isotypes.
| S-EPMC4424345 | biostudies-literature
Project description:Antibodies to the Cryptococcus neoformans capsular glucuronoxylomannan (GXM) form the basis of two potential therapeutic intervention strategies, i.e., conjugate vaccines and passive antibody therapy. To better understand the molecular basis of the antibody response, the heavy- and light-chain immunoglobulin variable region (VH and VL, respectively) sequences of seven monoclonal antibodies (MAbs) to GXM were determined. Rabbit anti-idiotypic serum was made to the previously characterized murine MAb 2H1 and used to study MAb 2H1 idiotype expression in other GXM-binding MAbs and immune sera. MAb E1 originated from a C3H/HeJ mouse immunized with C. neoformans serotype A polysaccharide. MAbs 471, 1255, 339, 3C2, 386, and 302 originated from BALB/c mice immunized with polysaccharide of serotypes A, A, B, C, D, and D, respectively, conjugated to sheep erythrocytes. In the E1, VH uses V11 from the T15 gene family and JH3 and has a D segment of three amino acids, and the VL uses a VKSer-like gene family element and JK5. In MAbs 471 and 3C2, the VH uses VH7183-like gene family elements and JH2 and has D segments of seven amino acids, and the VL uses VK5.1 and JK1. In MAbs 1255 and 339, the VH uses VH10-like gene elements and JH4 and has six codon D segments, and the VL uses a VK21-like gene element and JK5. In MAbs 302 and 386, respectively, the VH uses VHGAM-like gene elements and JH2 and JH3 and has six and four codon D segments, and VL uses VK4/5-like gene elements and JK1.VH usage, MAb 2H1 idiotype expression, and fine specificity mapping define a minimum of three GXM epitopes which elicit protective antibodies. The results confirm that the antibody response is highly restricted, suggest a close relationship between molecular structure and serological properties, and provide insight into protein structural motifs important for GXM binding.
| S-EPMC303042 | biostudies-other
Project description: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.
| S-EPMC2292635 | biostudies-literature