Structural studies of the capsular polysaccharide of a non-neoformans Cryptococcus species identified as C. laurentii, which was reclassified as Cryptococcus flavescens, from a patient with AIDS

Ikeda, Reiko; Maeda, Toshihiro

doi:10.1016/j.carres.2003.11.015

Cited by 23 publications

(18 citation statements)

References 23 publications

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“…We established that GXM was susceptible to partial acid hydrolysis using conditions developed for the hydrolysis of polysaccharides from other cryptococcal species (0.3 M TFA at 95°C for 5 h) ( Fig. 2) (15). However, MS analysis of the hydrolyzed GXM using those conditions revealed only ions with m/z values that corresponded to oligosaccharides composed only of Man and GlcA.…”

Section: Partial Acid Hydrolysis Of Gxmmentioning

confidence: 95%

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Capsule Structural Heterogeneity and Antigenic Variation in Cryptococcus neoformans

McFadden¹,

Fries²,

Wang

et al. 2007

Eukaryot Cell

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Cryptococcus neoformans is a human pathogenic fungus with a capsule composed primarily of glucuronoxylomannan (GXM) that is important for virulence. Current views of GXM structure postulate a polymer composed of repeating mannose trisaccharide motifs bearing a single ␤(1,2) glucuronic acid with variable xylose and O-acetyl substitutions to form six triads. GXM from different strains is notoriously variable in triad composition, but it is not known if the polymer consists of one or more motif-repeating units. We investigated the polymeric organization of GXM by using mass spectrometry to determine if its compositional motif arrangement was similar to that of bacterial capsular polysaccharides, namely, a polymer of a single repeating unit. The results were consistent with, and confirmatory for, the current view that the basic unit of GXM is a repeating mannose trisaccharide motif, but we also found evidence for the copolymerization of different GXM repeating units in one polysaccharide molecule. Analysis of GXM from isogenic phenotypic switch variants suggested structural differences caused by glucuronic acid positional effects, which implied flexibility in the synthetic pathway. Our results suggest that cryptococcal capsule synthesis is fundamentally different from that observed in prokaryotes and employs a unique eukaryotic approach, which theoretically could synthesize an infinite number of structural combinations. The biological significance of this capsule construction scheme is that it is likely to confer a powerful avoidance strategy for interactions with the immune system and phagocytic environmental predators. Consistent with this premise, the antigenic variation of a capsular epitope recognized by a nonprotective antibody was observed under different growth conditions.

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Section: Partial Acid Hydrolysis Of Gxmmentioning

confidence: 95%

“…The sample was vacuum dried and then dissolved in 20 mM ammonium bicarbonate. Partial hydrolysis was confirmed by the presence of multiple products visible by fluorophore-assisted carbohydrate electrophoresis (FACE) (14) and by thin-layer chromatography (15). For FACE, samples were run on a 30% polyacrylamide gel.…”

Section: Methodsmentioning

confidence: 99%

Capsule Structural Heterogeneity and Antigenic Variation in Cryptococcus neoformans

McFadden¹,

Fries²,

Wang

et al. 2007

Eukaryot Cell

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“…Following dialysis, the polysaccharides were hydrolyzed with 0.5 M HCl at room temperature for 12 h, neutralized with NaOH, dialyzed against water, and evaporated to dryness. A 1 H nuclear magnetic resonance (NMR) spectrum of the product was recorded using a GSX-400 NMR spectrometer (JEOL) in 99.9% D 2 O at 90°C; sodium 3-(trimethylsilyl)-1-propanesulfonate (DSS) was used as the external standard (26,27).…”

Section: Methodsmentioning

confidence: 99%

“…The interaction of GXM with protein fraction obtained from S. aureus was analyzed by surface plasmon resonance (SPR) by using a Biacore 3000 (Biacore, Uppsala, Sweden). For immobilization of GXM on CM5 sensor chips, the acidic GXM was (67) reduced to neutral polysaccharide by the method described previously (26,27), because native GXM was not immobilized perhaps due to its charge. Reduced GXM (500 g) was dissolved in 500 l of 0.1 M acetate buffer (pH 5.5), and 10 l of 50 mM NaIO 4 in 100 mM acetate buffer (pH 5.5) was added.…”

Section: Methodsmentioning

confidence: 99%

Contribution of the Mannan Backbone of Cryptococcal Glucuronoxylomannan and a Glycolytic Enzyme ofStaphylococcus aureusto Contact-Mediated Killing ofCryptococcus neoformans

et al. 2007

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The fungal pathogen Cryptococcus neoformans is killed by the bacterium Staphylococcus aureus, and the killing is inhibited by soluble capsular polysaccharides. To investigate the mechanism of killing, cells in coculture were examined by scanning and transmission electron microscopy. S. aureus attached to the capsule of C. neoformans, and the ultrastructure of the attached C. neoformans cells was characteristic of dead cells. To identify the molecules that contributed to the fungal-bacterial interaction, we treated each with NaIO 4 or protease. Treatment of C. neoformans with NaIO 4 promoted adherence. It was inferred that cleavage of xylose and glucuronic acid side chains of glucuronoxylomannan (GXM) allowed S. aureus to recognize mannose residues in the backbone, which resisted periodate oxidation. On the other hand, treatment of S. aureus with protease decreased adherence, suggesting that protein contributed to attachment in S. aureus. In confirmation, side chain-cleaved polysaccharide or defined ␣-(133)-mannan inhibited the killing at lower concentrations than native GXM did. Also, these polysaccharides reduced the adherence of the two species and induced clumping of pure S. aureus cells. ␣-(133)-Mannooligosaccharides with a degree of polymerization (DP) of >3 induced cluster formation of S. aureus in a dose-dependent manner. Surface plasmon resonance analyses showed interaction of GXM and surface protein from S. aureus; the interaction was inhibited by oligosaccharides with a DP of >3. Conformations of ␣-(133) oligosaccharides were predicted. The three-dimensional structures of mannooligosaccharides larger than triose appeared curved and could be imagined to be recognized by a hypothetical staphylococcal lectin. Native polyacrylamide gel electrophoresis of staphylococcal protein followed by electroblotting, enzyme-linked immunolectin assay, protein staining, and N-terminal amino acid sequencing suggested that the candidate protein was triosephosphate isomerase (TPI). The enzymatic activities were confirmed by using whole cells of S. aureus. TPI point mutants of S. aureus decreased the ability to interact with C. neoformans. Thus, TPI on S. aureus adheres to the capsule of C. neoformans by recognizing the structure of mannotriose units in the backbone of GXM; we suggest that this contact is required for killing of C. neoformans.

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“…Although branching has not been described for C. neoformans GXM, other microbes have more complicated backbone substitutions. In this regard the GXM of Cryptococcus flavescens may have side chains consisting of more than two xylose residues (23).…”

mentioning

confidence: 99%

Radiological Studies Reveal Radial Differences in the Architecture of the Polysaccharide Capsule ofCryptococcus neoformans

Bryan¹,

Zaragoza

Zhang³

et al. 2005

Eukaryot Cell

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The polysaccharide capsule of the pathogenic fungus Cryptococcus neoformans is an important virulence factor, but relatively little is known about its architecture. We applied a combination of radiological, chemical, and serological methods to investigate the structure of this polysaccharide capsule. Exposure of C. neoformans cells to gamma radiation, dimethyl sulfoxide, or radiolabeled monoclonal antibody removed a significant part of the capsule. Short intervals of gamma irradiation removed the outer portion of the cryptococcal capsule without killing cells, which could subsequently repair their capsules. Survival analysis of irradiated wild-type, acapsular mutant, and complemented mutant strains demonstrated that the capsule contributed to radioprotection and had a linear attenuation coefficient higher than that of lead. The capsule portions remaining after dimethyl sulfoxide or gamma radiation treatment were comparable in size, 65 to 66 m 3 , and retained immunoreactivity for a monoclonal antibody to glucuronoxylomannan. Simultaneous or sequential treatment of the cells with dimethyl sulfoxide and radiation removed the remaining capsule so that it was not visible by light microscopy. The capsule could be protected against radiation by either of the free radical scavengers ascorbic acid and sorbitol. Sugar composition analysis of polysaccharide removed from the outer and inner parts of the capsule revealed significant differences in glucuronic acid and xylose molar ratios, implying differences in the chemical structure of the constituent polysaccharides. Our results provide compelling evidence for the existence of two zones in the C. neoformans capsule that differ in susceptibility to dimethyl sulfoxide and radiation and, possibly, in packing and composition.

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Structural studies of the capsular polysaccharide of a non-neoformans Cryptococcus species identified as C. laurentii, which was reclassified as Cryptococcus flavescens, from a patient with AIDS

Cited by 23 publications

References 23 publications

Capsule Structural Heterogeneity and Antigenic Variation in Cryptococcus neoformans

Capsule Structural Heterogeneity and Antigenic Variation in Cryptococcus neoformans

Contribution of the Mannan Backbone of Cryptococcal Glucuronoxylomannan and a Glycolytic Enzyme ofStaphylococcus aureusto Contact-Mediated Killing ofCryptococcus neoformans

Radiological Studies Reveal Radial Differences in the Architecture of the Polysaccharide Capsule ofCryptococcus neoformans

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