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, 97 (4), 937-45

The Elastic Properties of the Cryptococcus Neoformans Capsule

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The Elastic Properties of the Cryptococcus Neoformans Capsule

Susana Frases et al. Biophys J.

Abstract

Microbial capsules are important for virulence, but their architecture and physical properties are poorly understood. The human pathogenic fungus Cryptococcus neoformans has a large polysaccharide capsule that is necessary for virulence and is the target of protective antibody responses. To study the C. neoformans capsule we developed what we believe is a new approach whereby we probed the capsular elastic properties by applying forces using polystyrene beads manipulated with optical tweezers. This method allowed us to determine the Young's modulus for the capsule in various conditions that affect capsule growth. The results indicate that the Young's modulus of the capsule decreases with its size and increases with the Ca(2+) concentration in solution. Also, capsular polysaccharide manifests an unexpected affinity for polystyrene beads, a property that may function in attachment to host cells and environmental structures. Bead probing with optical tweezers provides a new, nondestructive method that may have wide applicability for studying the effects of growth conditions, immune components, and drugs on capsular properties.

Figures

Figure 1
Figure 1
Proposed model for determination of the elastic properties of the C. neoformans capsule. (a) Initial situation where the bead (solid bead) is in its equilibrium position in the trap, ρ = 0, and the capsule is not deformed, z¯ = 0. (b) As the microscope stage moves left under controlled velocity, the cell adhered to the coverslip similarly moves to the left. There appears a “tug of war” between the cell and the optical tweezers. As a result, the trapped bead changes its equilibrium position, from ρ(0) to ρ(V), resulting in Δρ > 0, and the capsule deforms, z¯ > 0, to balance the optical force.
Figure 2
Figure 2
Measurement of the contact angle after attachment of beads to the capsule of C. neoformans. (a) Bright-field image showing four polystyrene beads attached to the C. neoformans capsule. (b) Corresponding immunofluorescence after capsule staining with 18B7 antibody. (c) Merging of the images shown in a and b. (d) A high-magnification look at the boxed zone in c showing the bead-capsule contact angle. Scale bar for ac, 5μm.
Figure 3
Figure 3
Young's modulus determination for the C. neoformans capsule. (a) Schematic representation of the change in bead position, resulting in displacement from its initial equilibrium position, Δρ. (b) A sequence of three distinct frames in the same experiment showing the variation in the bead equilibrium position. Scale bar, 5 μm. (c) A typical Young's modulus experimental curve under control conditions. For this experiment, the slope of the curve Δρ × t is A = 0.021 μm/s. Using the theoretical model described in the Materials and Methods section, the Young's modulus value determined for this experiment was 77 Pa. Arrowheads 1–3 correspond to the three representative frames shown in Fig. 3 b.
Figure 4
Figure 4
Divalent cations influence the elasticity of the C. neoformans polysaccharide capsule. (a) Representative images of C. neoformans after incubation in two different Ca2+ concentrations. Scale bar, 5 μm. (b) Values of Young's modulus after incubation of C. neoformans in different cation Ca2+ concentrations. Data are mean ± SE (n = 20 cells).
Figure 5
Figure 5
Influence of growth time and capsule size on the elastic properties of the C. neoformans capsule. (a) Representative images of C. neoformans cells stained with India ink for each culture interval. Scale bar, 5 μm. (b) Representative bright-field images of C. neoformans cells used in the Young's modulus experiments. Scale bar, 5 μm. (c) Mean ± SE of C. neoformans after 1, 3, and 6 days of growth, confirming that the capsule increases as a function of time. Scale bar, 5 μm. (d) Plot of the Young's modulus as a function of capsule thickness. Data are mean ± SE.
Figure 6
Figure 6
Binding of mAb 18B7 to C. neoformans cells treated with different concentrations of CaCl2 as revealed by indirect immunofluorescence. (a) Fluorescence microscopy revealing mAb 18B7 capsular binding (green, FITC) and the fungal cell wall (blue, UVITEX). Scale bar, 3 μm. (b) Width of the mAb18B7 binding band depended on calcium concentration. The width of 18B7 biding is represented by solid bars. The distance between the cell wall and the edge of mAb binding is represented by open bars. (Inset) Illustration of the measured distances. Significant (p < 0.05) differences in binding between the samples and control conditions were observed.
Figure 7
Figure 7
Binding of mAb 18B7 to PS-coated polystyrene surfaces as a function of Ca2+ and GXM concentrations. The y axis represents absorbance at 405 nm.

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