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, 106 (4), 1228-33

Capsule of Cryptococcus Neoformans Grows by Enlargement of Polysaccharide Molecules

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Capsule of Cryptococcus Neoformans Grows by Enlargement of Polysaccharide Molecules

Susana Frases et al. Proc Natl Acad Sci U S A.

Abstract

The human pathogenic fungus Cryptococcus neoformans has a distinctive polysaccharide (PS) capsule that enlarges during infection. The capsule is essential for virulence, but the mechanism for capsular growth is unknown. In the present study, we used dynamic light scattering (LS) analysis of capsular PS and optical tweezers (OT) to explore the architecture of the capsule. Analysis of capsular PS from cells with small and large capsules by dynamic LS revealed a linear correlation between PS effective diameter and microscopic capsular diameter. This result implied that capsule growth was achieved by the addition of molecules with larger effective diameter, such that some molecules can span the entire diameter of the capsule. Measurement of polystyrene bead penetration of C. neoformans capsules by using OT techniques revealed that the outer regions were penetrable, but not the inner regions. Our results provide a mechanism for capsular enlargement based on the axial lengthening of PS molecules and suggest a model for the architecture of a eukaryotic microbial capsule.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Multimodal size distribution analysis of PS fractions; exo-PS (A) and capsular-PS (B) obtained from strain 24067 (serotype D). The x axis represents size distribution by particle diameter; y axis corresponds to the values of percentage intensity weighted sizes obtained from the NNLS algorithm (27).
Fig. 2.
Fig. 2.
Multimodal size distribution analysis of capsular-PS from strains H99, B3501, 24067 (C. neoformans), NIH191, and NIH198 (C. gattii) in noninducing and inducing media. The x axis represents particles size distribution measured as a diameter in nanometers; y axis corresponds to the values of percentage intensity weighted sizes obtained from the NNLS algorithm (27). (Scale bars, 2 μm.)
Fig. 3.
Fig. 3.
Relationship between capsule size and effective diameter. Correlation between capsule size and PS fragments in Cryptococcus (Upper). Linear regression curve fitting (Y = 0.9884x + 267.48, R2 = 0.9297) obtained from the average of capsular PS size measured by India ink negative stain and multimodal size distribution analysis of capsular PS from strains H99, B3501, 24067, 191, and 198 incubated in noninducing or capsule-inducing media. The x axis represents particles size distribution measured by India ink; y axis corresponds with average size values to the highest intensity weighted sizes obtained from the NNLS algorithm (27) (Lower).
Fig. 4.
Fig. 4.
Multimodal size distribution analysis of capsular-PS from C. neoformans (strain H99) in capsule-inducing media at different time points. The x axis represents particles size distribution measured as a diameter in nanometers; y axis corresponds to the values of percentage intensity weighted sizes obtained from the NNLS algorithm (27). (Scale bars, 2 μm.)
Fig. 5.
Fig. 5.
Proposed model for Cryptococcus neoformans architecture. Capsule assemblage model (A). We provide a schematic representation that incorporates several observations made in this study and in recent reports from various laboratories, including ours (14, 16, 19, 28). GXM fibers are attached to cell wall glucans through noncovalent interactions (12, 28). To account for the observation that capsular PS density decreases as a function of radial distance (16, 19), we propose that the 2 major populations of GXM molecules such that the larger ones span the length of the India ink visible capsule and serve as a scaffold for the attachment of smaller fibrils. GXM-GXM interactions are thought to be mediated by the formation of divalent cation bridges (14). A small population of very large fibrils serves to extend the effective diameter of the capsule past the zone of India ink exclusion. Although the diagram shows GXM-GXM interactions limited to the inner layer only, we surmise that this interaction can occur throughout the capsule diameter with the caveat that they are more frequent in the inner layers of the capsule where they account for the higher density of those regions. Also, we have drawn the PS fibrils as linear molecules for simplicity, fully aware of the important caveat that scanning electron microscopy reveals that such molecules are tangled with apparent branching (13), and that such molecules have significant secondary structure (25). (B) Scanning electron microscopic image of a C. neoformans cell where zones I and II of the capsule are apparent by the density of fibrils. (C) C. neoformans cell suspended in India Ink. According to the model proposed in A, zone II ends at the exclusion boundary of India Ink particles. There is a fuzzy edge to the capsule, which we represent as occurring within zone III.
Fig. 6.
Fig. 6.
Penetration of PS capsule by polystyrene bead. (A) Early stage in capsule growth showing a cell with a small capsule, confirmed by India ink negative stain and the bead adjacent to the cell wall. (B) Later stages in capsule growth showing a C. neoformans with a large capsule and the bead distant from the cell wall, represented by measured distance (z). (C) Imunofluorescence staining of capsular PS confirming that the beads penetrate the capsule (zone II), but do not touch the cell wall. (D) Schematic representation of measurements in which a is the radius of the bead and z is the zone I length in C. neoformans. (E) Filled circles, plot of the z during time; open circles, capsule size measurements by India ink stain. The x axis represents time point in days and y axis the length in micrometers. (Scale bars A–C, 3 μm.)

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