Interaction of Blastomyces dermatitidis, Sporothrix schenckii, and Histoplasma capsulatum with Acanthamoeba castellanii

Abstract

Several dimorphic fungi are important human pathogens, but the origin and maintenance of virulence in these organisms is enigmatic, since an interaction with a mammalian host is not a requisite for fungal survival. Recently, Cryptococcus neoformans was shown to interact with macrophages, slime molds, and amoebae in a similar manner, suggesting that fungal pathogenic strategies may arise from environmental interactions with phagocytic microorganisms. In this study, we examined the interactions of three dimorphic fungi with the soil amoeba Acanthameobae castellanii. Yeast forms of Blastomyces dermatitidis, Sporothrix schenckii, and Histoplasma capsulatum were each ingested by amoebae and macrophages, and phagocytosis of yeast cells resulted in amoeba death and fungal growth. H. capsulatum conidia were also cytotoxic to amoebae. For each fungal species, exposure of yeast cells to amoebae resulted in an increase in hyphal cells. Exposure of an avirulent laboratory strain of H. capsulatum to A. castellanii selected for, or induced, a phenotype of H. capsulatum that caused a persistent murine lung infection. These results are consistent with the view that soil amoebae may contribute to the selection and maintenance of certain traits in pathogenic dimorphic fungi that confer on these microbes the capacity for virulence in mammals.

FIG. 1.

Phagocytosis of H. capsulatum, B. dermatitidis, and S. schenckii cells by A. castellanii cells and J774.16 macrophages. Bars represent the phagocytic index by either amoebae (solid bars) or macrophages (hatched bars), and each error bar denotes one standard deviation. The phagocytosis index was determined by counting the total number of fluorescent fungal cells per 100 amoebae or macrophages. For each experimental condition, the number of repetitions was five. This experiment was repeated on different days and yielded similar results.

FIG. 2.

TEM of H. capsulatum cells interacting with A. castellanii. (A and B) Two separate phagocytic events at 2 h postincubation with amoebae. (C and D) Yeast cells in a membrane-bound vacuole surrounding the fungal cell 2 h after infection of the amoeba suspension with fungal cells. (E) Two individual H. capsulatum fungal cells in separate phagocytic compartments indicating two independent phagocytic events. Magnification, ×15,000 (A, B, and E) and ×12,000 (C and D).

FIG. 3.

TEM of B. dermatitidis cells interacting with A. castellanii cells. (A) An apparently intact A. castellanii cell with pseudopodia interacting with a B. dermatitidis cell. (B) B. dermatitidis cells engaged with an amoeba manifest reduced cytoplasmic electron density and electron-dense mitochondria in amoeba cells at 24 h postinfection. Magnification, ×9,000 (A) and ×12,000 (B).

FIG. 4.

Transformation of dimorphic fungi from yeast forms to pseudohyphal or hyphal forms. (A) TEM (magnification, ×15,000) of a representative hyphal form from a 24-h coculture of B. dermatitidis (Bd) yeast with A. castellanii (Ac). Pseudohyphal (arrowhead) and hyphal forms (asterisk) were present in cocultures of H. capsulatum yeast with A. castellanii (Ac) after 24 h (B) and 48 h (C). The cells were stained with Gram's crystal violet (magnification, ×1,000).

FIG. 5.

Fungal cell counts after incubation with or without amoebae in PBS. Bars represent CFU at different times: solid bars denote CFU at 0 h and hatched bars denote CFU at 48 h. The error bars each represent one standard deviation. There are significant differences (P ≤ 0.001) between each of the fungi incubated with amoebae and the corresponding cells in PBS at 48 h. (A) H. capsulatum CIB 1980 (an attenuated strain); (B) H. capsulatum ATCC G217B; (C) B. dermatitidis; (D) S. schenckii; (E) C. albicans; (F) S. cerevisiae. Initial numbers of CFU vary, since the experiment with all fungi was done simultaneously. Each experiment was done at least twice with similar results.

FIG. 6.

Viability of amoebae (Ac) after incubation with three different dimorphic fungi, B. dermatitidis (Bd), S. schenckii (Ss), and H. capsulatum (Hc) strains CIB 1980 and ATCC G217B. Amoeba cell viability was inferred by the ability of the cell to exclude trypan blue dye. Error bars each denote one standard deviation. At 24 and 48 h, the P value was ≤0.001 when comparing amoebae incubated with any of the dimorphic fungal species to amoebae alone. The experiment was done at least twice with similar results.

FIG. 7.

Viability of amoebae (Ac) after incubation in wells where B. dermatitidis (Bd), H. capsulatum (Hc), or S. schenckii (Ss) and amoeba cells were separated by permeable inserts (A) or where amoebae were separated from coincubated fungal and amoeba cells (B). There were no significant differences in viability for A. castellanii cells incubated in PBS alone and under the other conditions.

FIG. 8.

Interactions of H. capsulatum conidia with amoebae. (A) TEM illustrating A. castellanii phagocytosing a conidial cell after 2 h of incubation. (B) Micrograph depicting a conidial cell and a yeast cell in proximity to an amoeba cell after 24 h of incubation. (C) Micrograph illustrating an internalized H. capsulatum yeast cell surrounded by a membrane-bound vacuole after 24 h of incubation. (D) Trypan blue exclusion assay results illustrating the percentage of dead amoebae after incubation with and without H. capsulatum conidia at 0, 24, and 28 h. Solid bars indicate amoebae in PBS only, open bars represent amoebae with J1 conidia, and hatched bars represent J2 conidia with amoebae. Error bars each represent one standard deviation. At 24 h, the P value was 0.028 when comparing amoebae alone to both J1 and J2 conidia with amoebae, and at 48 h, death of amoebae incubated with J1 and J2 was also significant compared to amoebae alone (P = 0.015 and 0.022, respectively). (E) Changes in CFU of J1 and J2 conidia after incubation with amoebae. Solid bars represent CFU at 0 h, open bars represent CFU at 24 h, and hatched bars represent CFU at 48 h. Error bars each denote one standard deviation. Both the 24- and 48-h results for J2 conidia with amoebae are significant compared to J2 in PBS alone (P ≤ 0.001 for both). (F and G) Micrograph depicting morphology changes of J2 conidia after 24 h of incubation with amoebae illustrating conidial, yeast, and hyphal forms of H. capsulatum J2. (H) Micrograph of J2 conidia after 24 h of incubation in PBS. Magnification, ×15,000 (A), ×12,000 (B and C), and ×200 (F, G, and H).

FIG. 9.

Hematoxylin and eosin staining of lung tissue from mice 14 days after infection with H. capsulatum CIB 1980 grown in PYG media without (A) or with (B) amoebae. Magnification, ×250. The inset in panel B shows the tissue stained with Gomori's methenamine silver. The arrowheads indicate H. capsulatum yeast cells.