FITC-Lectin Avidity of Cryptococcus neoformans Cell Wall and Capsular Components

Foster, Alexander J.; Bird, Roger; Kelly, Steven L.; Nishimura, Kazuko; Poyner, David A.; Taylor, Stephen; Smith, Stephen N.

doi:10.2307/3761980

Cited by 12 publications

(18 citation statements)

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“…The lectin WGA has been used extensively in the last three decades as a probe to study surface components of different organisms, including C. neoformans (12). The lectin has affinity for sialic acids and ␤-1,4-GlcNAc oligomers (1,26,32).…”

Section: Discussionmentioning

confidence: 99%

“…As detailed above, the potential targets of WGA binding in C. neoformans include sialic acids, chitin, chitin oligomers, and hyaluronic acid, all of them previously described as surface components of cryptococci (2,3,12,16,37). To evaluate whether the binding of WGA to C. neoformans involved sialic acids, yeast cells were incubated in the presence of tetramethyl rhodamine isocyanate-labeled, succinylated WGA (EY Laboratories) under the same conditions described above for the Alexa 594 conjugate.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, it has been demonstrated that chitosan, the de-O-acetylated form of chitin, is required for cell wall integrity and maintenance of the correct assembly of the pigment melanin and the cryptococcal capsule (2). The presence of cell wall chitinlike oligomers in C. neoformans was suggested by the reactivity of yeast cells with the wheat germ agglutinin (WGA) (12).…”

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confidence: 99%

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Binding of the Wheat Germ Lectin to Cryptococcus neoformans Suggests an Association of Chitinlike Structures with Yeast Budding and Capsular Glucuronoxylomannan

Alvarez²,

et al. 2008

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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 ␤-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.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Binding of the Wheat Germ Lectin to Cryptococcus neoformans Suggests an Association of Chitinlike Structures with Yeast Budding and Capsular Glucuronoxylomannan

Alvarez²,

et al. 2008

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“…Concanavalin A (ConA), which binds internal ␣-linked mannose in glycoprotein core structures, also showed spore-specific binding. This lectin had been discovered previously to bind the bud scars of C. neoformans yeast cells (19,21). However, like DSL, ConA binds the entire surface of the spore but does not bind filaments or basidia (Fig.…”

mentioning

confidence: 99%

Isolation and Characterization of Cryptococcus neoformans Spores Reveal a Critical Role for Capsule Biosynthesis Genes in Spore Biogenesis

Botts¹,

Giles²,

Gates

et al. 2009

Eukaryot Cell

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Spores are essential particles for the survival of many organisms, both prokaryotic and eukaryotic. Among the eukaryotes, fungi have developed spores with superior resistance and dispersal properties. For the human fungal pathogens, however, relatively little is known about the role that spores play in dispersal and infection. Here we present the purification and characterization of spores from the environmental fungus Cryptococcus neoformans. For the first time, we purified spores to homogeneity and assessed their morphological, stress resistance, and surface properties. We found that spores are morphologically distinct from yeast cells and are covered with a thick spore coat. Spores are also more resistant to environmental stresses than yeast cells and display a spore-specific configuration of polysaccharides on their surfaces. Surprisingly, we found that the surface of the spore reacts with antibodies to the polysaccharide glucuronoxylomannan, the most abundant component of the polysaccharide capsule required for C. neoformans virulence. We explored the role of capsule polysaccharide in spore development by assessing spore formation in a series of acapsular strains and determined that capsule biosynthesis genes are required for proper sexual development and normal spore formation. Our findings suggest that C. neoformans spores may have an adapted cell surface that facilitates persistence in harsh environments and ultimately allows them to infect mammalian hosts.

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“…However, the marked fluorescence obtained from both these entities, more than double the number of log channel values of some other microbial eucaryotes (Smith et al 1999(Smith et al , 2001Foster et al 2004), may well indicate a particularly rich pigment and nucleotide nature, as the quiescent nature of cysts would discount the association of autofluorescence with metabolic activity as potentially occurs in monocytes and macrophages.…”

Section: Fitc-lectin Concentration and Binding By Trophozoites And Cystsmentioning

confidence: 99%

Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC-lectin binding and fluorescence evaluation

et al. 2004

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Aims: Characterization of the representative protozoan Acanthamoeba polyphaga surface carbohydrate exposure by a novel combination of flow cytometry and ligand-receptor analysis. Methods and Results: Trophozoite and cyst morphological forms were exposed to a panel of FITC-lectins. Population fluorescence associated with FITC-lectin binding to acanthamoebal surface moieties was ascertained by flow cytometry. Increasing concentrations of representative FITC-lectins, saturation binding and determination of K d and relative B max values were employed to characterize carbohydrate residue exposure. FITC-lectins specific for N-acetylglucosamine, N-acetylgalactosamine and mannose/glucose were readily bound by trophozoite and cyst surfaces. Minor incremental increases in FITC-lectin concentration resulted in significant differences in surface fluorescence intensity and supported the calculation of ligand-binding determinants, K d and relative B max , which gave a trophozoite and cyst rank order of lectin affinity and surface receptor presence. Conclusions: Trophozoites and cysts expose similar surface carbohydrate residues, foremost amongst which is N-acetylglucosamine, in varying orientation and availability. Significance and Impact of the Study: The outlined versatile combination of flow cytometry and ligandreceptor analysis allowed the characterization of surface carbohydrate exposure by protozoan morphological forms and in turn will support a valid comparison of carbohydrate exposure by other single-cell protozoa and eucaryotic microbes analysed in the same manner.

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FITC-Lectin Avidity of Cryptococcus neoformans Cell Wall and Capsular Components

Cited by 12 publications

References 0 publications

Binding of the Wheat Germ Lectin to Cryptococcus neoformans Suggests an Association of Chitinlike Structures with Yeast Budding and Capsular Glucuronoxylomannan

Binding of the Wheat Germ Lectin to Cryptococcus neoformans Suggests an Association of Chitinlike Structures with Yeast Budding and Capsular Glucuronoxylomannan

Isolation and Characterization of Cryptococcus neoformans Spores Reveal a Critical Role for Capsule Biosynthesis Genes in Spore Biogenesis

Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC-lectin binding and fluorescence evaluation

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