Phenotypic switching in the human pathogenic fungus Cryptococcus neoformans is associated with changes in virulence and pulmonary inflammatory response in rodents

Abstract

High-frequency reversible changes in colony morphology were observed in three strains of Cryptococcus neoformans. For one strain (SB4, serotype A), this process produced three colony types: smooth (S), wrinkled (W), and serrated (C). The frequency of switching between colony types varied for the individual colony transitions and was as high as 10(-3). Mice infected with colony type W died faster than those infected with other colony types. The rat inflammatory response to infection with colony types S, W, and C was C > S > W and ranged from intense granulomatous inflammation with caseous necrosis for infection with type C to minimal inflammation for infection with type W. Infection with the various colony types was associated with different antibody responses to cryptococcal proteins in rats. Analysis of cellular characteristics revealed differences between the three colony types. High-frequency changes in colony morphology were also observed in two additional strains of C. neoformans. For one strain (24067A, serotype D) the switching occurred between smooth and wrinkled colonies. For the other strain (J32A, serotype A), the switching occurred between mucoid and nonmucoid colonies. The findings indicate that C. neoformans undergoes phenotypic switching and that this process can affect virulence and host inflammatory and immune responses. Phenotypic switching may play a role in the ability of this fungus to escape host defenses and establish chronic infections.

Figure 1

Colony types of strain SB4. (A) Colony types W. (B) Colony type S revertants from colony type W. (Inset) W/S-sectored colony. (C) Colony type W revertants from colony type C. (D) Colony type C with colony type S revertant. Colony types of strain 24067A illustrating colony type S revertant from colony type W (E) and colony type W revertant from colony type S (F). (Inset) W/S-sectored colony. (A and C–F, about ×4; B, about ×2.5.)

Figure 2

Switching frequencies for SB4, 24067A, and J32A for cells grown at 30°C on SDA. Frequencies for each strain were determined twice with similar results. Italicized numbers are frequencies after UV irradiation.

Figure 3

Scanning electron microscopy of SB4 cells from colony types W (A), S (B); and C (C). (×3,000. Bar = 10 μm.) This experiment was done twice using different S, W, and C colonies to ensure the reproducibility of our findings. Hematoxylin and eosin staining of lung tissue form rats infected with SB4 cells from colony types W (D), S (E), and C (F). (×100.) Arrows point to CN. Staining for cryptococcal polysaccharide demonstrates localization of polysaccharide in rats infected with cells from colony types W (G), S (H), and C (I). (×50.) Arrows point to CN and arrowheads point to polysaccharide material inside mononuclear cells.

Figure 4

Western blots of rats infected with cells from the various colony types. The serum of rats (n = 3) infected with each colony type from day 30 of infection was reacted against the protein extracts obtained from colony types S, W, or C.