Cryptococcus neoformans virulence is enhanced after growth in the genetically malleable host Dictyostelium discoideum

Abstract

Cryptococcus neoformans is an encapsulated, environmental fungus that can cause life-threatening meningitis. Pathogenicity of C. neoformans for macrophages and vertebrate hosts may be a mechanism selected in evolution for protection against environmental predators. In this study, we investigated whether Dictyostelium discoideum could serve as an alternate host for C. neoformans. D. discoideum has a defined genetic system which provides significant advantages for the study of fungus-amoeba interactions. Our results show that D. discoideum is susceptible to infection with C. neoformans and that the interactions are similar to those described previously for this fungus with macrophages and Acanthamoeba castellanii. Acapsular C. neoformans cells did not replicate when coincubated with D. discoideum. However, incubation of acapsular C. neoformans with D. discoideum mutants defective in myosin VII synthesis resulted in infection, validating the concept that avirulent organisms can be virulent in impaired hosts even at the unicellular level. Phagocytosis of C. neoformans by D. discoideum could be inhibited with capsule-specific antibodies and various sugars. Passage of an encapsulated C. neoformans strain through D. discoideum cultures increased virulence and was accompanied by larger capsules and faster time to melanization. These results add to the evidence implicating soil ameboid predators as important factors for the maintenance of C. neoformans virulence in the environment and suggest that D. discoideum promises to be an extremely useful system for studying the interaction of C. neoformans with phagocytic cells.

FIG. 1.

Fungal CFU after incubation of C. neoformans with or without D. discoideum. Solid bars denote CFU at 0 h, open bars denote CFU at 24 h, and hatched bars denote CFU at 48 h. The error brackets represent one standard deviation. The differences between the CFU of C. neoformans strains 24067, H99, and 3501 incubated with D. discoideum and the corresponding fungal cells in PBS at 48 h were significant (P ≤ 0.001). Numbers of CFU for C. neoformans Cap67 were similar in PBS and D. discoideum for each time interval. Numbers of CFU for C. neoformans strains 24067 (serotype A) (A), H99 (serotype D) (B), 3501 (serotype A) (C), Cap67 (acapsular variant of 3501) (D), and F7 (pseudohyphal variant of 24067) (E) are shown. Each experiment was repeated with similar results.

FIG. 2.

Viability of D. discoideum after incubation with C. neoformans. Solid bars represent viability at 0 h, open bars represent viability at 24 h, and hatched bars represent viability at 48 h. Error brackets denote one standard deviation. (A) Percentages of amoebae that are trypan blue positive. At 48 h, P values were ≤0.001 for comparisons of amoebae incubated with any of the C. neoformans strains with D. discoideum to amoebae alone. (B) Numbers of PFU representing the total numbers of viable D. discoideum cells. At 48 h a significant decrease in PFU of D. discoideum cells was measured after incubation C. neoformans strains 24067, H99, and 3501 (P ≤ 0.001). The number of PFU of D. discoideum incubated with C. neoformans Cap67 was unchanged throughout the assay. The experiment was done twice with similar results.

FIG. 3.

Phagocytosis of C. neoformans strains 24067, 3501, H99, F7, and Cap67 by D. discoideum. Bars represent the numbers of phagocytic events by D. discoideum, and error brackets denote 1 standard deviation. The phagocytosis index was determined by counting the total number of internalized fungal cells per 100 amoebae. For each experimental condition the number of repetitions was five. This experiment was repeated on different days and yielded similar results.

FIG. 4.

Transmission electron micrographs of C. neoformans strain 3501 cells interacting with D. discoideum. (A) C. neoformans is being engulfed by the pseudopods of a D. discoideum cell 2 h postinfection. (B) Two individual phagocytic events by one D. discoideum cell 2 h postinfection. In one event the C. neoformans cells are in membrane-bound vacuoles. The second event shows a budding C. neoformans cell in a vacuole. (Magnifications: ×24,000 [A] and ×18,000 [B]). The micrographs shown are representative of what was observed under the microscope.

FIG. 5.

Interaction of C. neoformans strains with D. discoideum myoi and rtoA mutants. (A) Phagocytosis of C. neoformans Cap67 cells by D. discoideum myoi or wild-type cells. Wild-type D. discoideum cells phagocytosed significantly more fungal cells than myoi null cells (P = 0.042). Solid bars represent the numbers of phagocytic events by D. discoideum, and error brackets denote one standard deviation. The phagocytosis index was determined by counting the number of D. discoideum cells with at least one internalized C. neoformans cell. For each experimental condition the number of repetitions was five. (B) Number of CFU of Cap67 after incubation with myoi null D. discoideum cells (▪), wild-type D. discoideum cells (♦), and alone (▴). Error bars denote one standard deviation. (C) Number of CFU of 3501 after incubation with myoi null D. discoideum cells (▪), wild-type D. discoideum cells (♦), and alone (▴). Error bars denote one standard deviation. (D) Phagocytosis of C. neoformans H99 cells by D. discoideum rtoA or wild-type cells. Wild-type D. discoideum cells phagocytosed significantly more fungal cells than rtoA null cells (P ≤ 0.001). Solid bars represent the numbers of phagocytic events by D. discoideum, and error brackets denote one standard deviation. For each experimental condition the number of repetitions was five. (E) C. neoformans H99 cell counts after incubation with either wild-type or rtoA D. discoideum cells. Bars represent numbers of CFU at different times; solid bars denote CFU at 0 h, open bars denote CFU at 24 h, and hatched bars denote CFU at 48 h. The error brackets represent one standard deviation. There are no significant differences between C. neoformans growth with wild-type and rtoA D. discoideum cells. These experiments were repeated on different days and yielded similar results.

FIG. 6.

Inhibition of D. discoideum phagocytosis of C. neoformans 24067 cells by 1 M mannose and MAb 18B7. Solid bars represent the number of phagocytic events by D. discoideum, and error brackets denote one standard deviation. The phagocytosis index was determined by counting the number of D. discoideum cells with at least one internalized C. neoformans cell. For each experimental condition the number of repetitions was five.

FIG. 7.

Survival of A/J mice infected with either 10⁷ C. neoformans cells grown with live D. discoideum (−▪−) (n = 15), 10⁷ C. neoformans cells grown with killed D. discoideum (−○−) (n = 15), 10⁷ C. neoformans cells (-▴-) (n = 15), or 10⁷ live D. discoideum cells (-×-) (n = 5). The graph shows that C. neoformans 24067 primed by growth with live D. discoideum is more lethal than C. neoformans grown either alone or with killed D. discoideum (P ≤ 0.005 for both). Also, D. discoideum alone was not pathogenic. There were no significant differences in the survival of mice infected with C. neoformans alone and C. neoformans grown with killed D. discoideum (P = 0.974). This experiment was performed twice with similar results.

FIG. 8.

Phenotypic differences between D. discoideum passaged or unpassaged C. neoformans cells. (A) Capsule area of C. neoformans cells grown at 37 or 30°C after passage. Bars represent capsule area, and error brackets denote one standard deviation. At 37°C there is a difference between D. discoideum passaged C. neoformans cells and unpassaged cells (P ≤ 0.001). (B) Growth curves at 37 and 30°C of passaged or unpassaged C. neoformans cells. Growth curves are similar for passaged C. neoformans grown at 37°C (×) compared to unpassaged C. neoformans at 37°C (▪) and for passaged C. neoformans at 30°C (*) compared to unpassaged C. neoformans at 30°C (▴). This experiment was repeated with similar results.