Cryptococcus neoformans interactions with amoebae suggest an explanation for its virulence and intracellular pathogenic strategy in macrophages - PubMed (original) (raw)

Cryptococcus neoformans interactions with amoebae suggest an explanation for its virulence and intracellular pathogenic strategy in macrophages

J N Steenbergen et al. Proc Natl Acad Sci U S A. 2001.

Abstract

Cryptococcus neoformans (Cn) is a soil fungus that causes life-threatening meningitis in immunocompromised patients and is a facultative intracellular pathogen capable of replication inside macrophages. The mechanism by which environmental fungi acquire and maintain virulence for mammalian hosts is unknown. We hypothesized that the survival strategies for Cn after ingestion by macrophages and amoebae were similar. Microscopy, fungal and amoebae killing assays, and phagocytosis assays revealed that Cn is phagocytosed by and replicates in Acanthamoeba castellanii, which leads to death of amoebae. An acapsular strain of Cn did not survive when incubated with amoebae, but melanization protected these cells against killing by amoebae. A phospholipase mutant had a decreased replication rate in amoebae compared with isogenic strains. These observations suggest that cryptococcal characteristics that contribute to mammalian virulence also promote fungal survival in amoebae. Intracellular replication was accompanied by the accumulation of polysaccharide containing vesicles similar to those described in Cn-infected macrophages. The results suggest that the virulence of Cn for mammalian cells is a consequence of adaptations that have evolved for protection against environmental predators such as amoebae and provide an explanation for the broad host range of this pathogenic fungus.

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Figures

Figure 1

Figure 1

Properties of the infection of Ac by Cn. (A) Phagocytic index for five Cn strains incubated with Ac in PYG media. Error bars denote 1 SD. For each experiment, 100 Ac cells were counted in eight separate wells, and the number of measurements was eight. Similar results were obtained from an additional independent experiment. * indicates P < 0.001 for both Cap67 and F7 when compared with H99, 24067, or 3501. (B) Percent of infected amoebae with budding Cn cells. Error bars denote 1 SD. * denotes the absence of intact amoebae after this time. (C) Percent of Ac cells that do not exclude trypan blue after incubation with different strains of_Cn_. Error bars denote 1 SD. Similar results were obtained from an additional independent experiment. * indicates_P_ = 0.001 when 3501 and H99 were compared with amoeba alone at 24 h. ‡ indicates P < 0.001 at 48 h when 3501 and H99 were compared with amoebae alone.

Figure 2

Figure 2

Microscopy of Ac incubated with Cn in PYG media. Each field is representative of amoeba/Cn interactions at a given time. For both SEM figures, Cn cells are depicted in red and Ac cells are depicted in yellow: (A) SEM 30-min postincubation with_Cn_ 24067 and (B) SEM 90-min postincubation with Cn 24067. (C) TEM 24-h postincubation with Cn 3501. (D) TEM 1.5-h postincubation with Cn 24067. (E and F) Corresponding light microscopy (E) and immunofluorescent (F) pictures 2 h postincubation of Ac with FITC-labeled Cn 3501. (Magnifications: ×1,000.)

Figure 3

Figure 3

cfu counts of wild-type and mutant Cn strains after incubation with or without Ac in PBS. Bars represent average cfu at different times. Solid bars denote 0 h, white bars denote 24 h, and cross-hatched bars denote 48 h. (A and B) cfu counts of melanized and nonmelanized Cn cells incubated with or without_Ac_ in PBS. (A) Strain 3501 cells. (B) Strain Cap67 cells; ** denotes melanized Cap67 with amoebae has greater growth than Cap67 with amoebae (P < 0.001). (C) Comparison of the cfu counts of Cn between the parental strain, H99, the phospholipase-deficient strain, plb−, and the reconstituted mutant, rec1, strains after incubation with or without Ac in PBS. + indicates a P < 0.001 for plb− with amoebae versus H99 with amoebae. There is no significant difference between H99 and amoebae versus rec1 with amoebae. Error bars denote 1 SD. For each experiment, the number of repetitions was five, and similar results were obtained from two additional independent experiments.

Figure 4

Figure 4

Microscopy of Ac cells infected by Cn. Results are indicative of the cellular disruption induced by_Cn_ infection. (A and B) Corresponding bright-field (A) and immunofluorescent (B) microscopy photos. GXM-specific antibody, 2H1, was used for immunofluorescent staining. (C) TEM photograph showing the disruption of the amoebae phagocytic vacuole membrane (delta signs) and of small vacuole formation (arrows). (D) Immunogold-labeled TEM with 2H1 mAb. Small arrows show internal amoebae vacuoles containing Cn polysaccharide; * depicts the fungal capsule. (Magnifications:A and B, ×1,000; C and D, ×22,500.)

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