PMC

This effect was apparent only when the experiment was carried out in PBS and no effect was observed when the yeast cells were suspended in minimal medium (MM), possibly due to glucose repression.

C. neoformans is proposed to release enzymes that damage the cell membrane of amoebae and macrophages thus releasing phospholipids, possibly in combination with proteins. Those phospholipids are then cleaved by PLB, releasing their polar heads that are in turn sensed by C. neoformans cells, triggering capsule enlargement and the formation of giant cells.

A) Deconvolved immunofluorescence of H99 cells treated with 5 mM PC for 48 hours and labeled with Uvitex 2B (blue) and DTAF-18B7 (green). Inset represents untreated cells under the same magnification. B) Differential interference contrast of Panel A. C) India ink preparation of H99 cells treated with 5 mM PC for 96 hours. (D) India ink preparation of untreated H99 cells at the same magnification. Scale bars  = 5 µm.

Activity assays were performed to test the effects of GPC, GPE, or PC on the capsule of C. neoformans. A) H99 and B) 24067 fungal cells were incubated alone in minimal medium (MM), or with 10 µM of GPC or GPE or 5 mM of PC for 48 hours. Each assay represents a minimum of 50 C. neoformans cells. C) H99, plb1,1 and plb1 ^REC1 cells were incubated either alone in MM or with 10 µM of GPC or GPE or 5 mM of PC for 48 hours. The numbers shown represents the average of a minimum of 50 C. neoformans cells. (*) indicates P<0.01 for capsule enlargement for each condition when compared to control incubation alone in MM.

A) C. neoformans strain 24067 (Cn) was incubated alone, with intact A. castellanii (Ac), and with the three fractions (upper, interface, and lower) that resulted from lipid extraction of A. castellanii. The statistically significant increase in capsule volume was only observed with intact amoeba and with the upper phase polar fraction. Each condition represents a minimum of 50 C. neoformans cells and experiments were done in triplicate. P<0.05. B) C. neoformans strain H99 (Cn) was incubated in the same manner as 24067, with the same conditions as stated above. The effects for strain 24067 were proportionately greater than for strain H99. C) Capsule-inducing activity of A. castellanii extracts declines as a function of time. The half-life of activity decay at room temperature was 1.385 d.

A) Initial effects involve capsule enlargement but continued incubation results in cells with giant cell bodies. B) Uvitex 2B preparations demonstrate an increased double-layered capsule of C. neoformans over time when co-incubated with A. castellanii cells and extracts. An increased cell body size, along with a decreased capsule size, was observed after an 8 day incubation period. C) Immunofluorescence demonstrates that C. neoformans cells are more intensely labeled with capsular antibodies following treatment with A. castellanii cells and extracts. Scale bars  = 10 µm.

A) The C. neoformans (Cn) cells were incubated alone in PBS, with live and heat-killed A. castellanii (Ac), live and heat-killed J774.14 macrophage-like cells (Mac), and polystyrene beads. Live amoebae and macrophages were more effective than heat-killed cells in triggering capsular enlargement. In contrast, there was no enlargement in the presence of the polystyrene beads, when compared to incubation in PBS alone. Each assay was performed independently on 3 separate days and the numbers shown represent the average of a minimum of 50 C. neoformans cells. B) Capsule enlargement activity of A. castellanii extracts as a function of temperature. Activity assays were done at 4°C, room temperature (RT), 28°C, and 37°C. This experiment was done two times on separate days and each condition represents a minimum of 50 cells. C) Representative C. neoformans cells suspended in India ink showing enlargement of the polysaccharide capsule after incubation with A. castellani (40x). (*) indicates P<0.001 for capsule enlargement for each condition when compared to control incubation alone in PBS.

A) C. neoformans strains H99, plb1, and plb1 ^REC1 were used in an activity assay with intact A. castellanii and the upper phase polar fraction. The wild type and reconstituted strains showed significant increases in capsule volume in the presence of the intact organism and the polar lipid fraction. The plb1 mutant, however, did not show a statistically significant increase in either condition. The numbers shown represent the average of a minimum of 50 C. neoformans cells. The experiment was done independently on 3 separate days and the results were reproducible. B) The Fetal Calf Serum (FCS) upper phase fraction elicits capsular enlargement, but is not dependent on PLB. FCS was subjected to lipid extraction and separated into fractions. The fractions were tested in the activity assay with the C. neoformans strains H99, plb1, and plb1 ^REC1. The active component separated into the polar fraction, similar to results seen with intact A. castellanii; however, the effect was not dependent on PLB activity as the enlargement of the capsule was observed in the PLB-deficient strain. (*) indicates P<0.05 for capsule enlargement for each condition when compared to control incubation alone in PBS for each strain.

A) Activity of upper phase amoebae extracts (AE) on C. neoformans strain H99 after extract treatment with heat, Proteinase K (Ptn K), and glucanases. Neither heat nor glucanase treatments had a significant effect on capsule enlargement activity. In contrast, Proteinase K + heat treatment elicited cells with larger capsule volumes. This experiment was done two times on separate days and the results were reproducible. The numbers shown represent the average of a minimum of 50 C. neoformans cells. B) Thin liquid chromatography shows an increase in the mobility of the phospholipid bands upon treatment of amoebae extracts with Proteinase K, and fractionation of the free lipid compounds to the lower phase fraction. Lane 1: Phospholipid markers: (PE) phosphatidylethanolamine; (PS) phosphatidylserine; (PI) phosphatidylinositol; and (PC) phosphatidylcholine; Lane 2: Untreated extracts; Lane 3: Untreated extract + Proteinase K (before treatment); Lane 4: Proteinase K-treated extract; Lane 5: Lower phase fraction after treatment with Proteinase K; Lane 6: Upper phase fraction after treatment with Proteinase K. C) and D) Demonstration that the active substance in the amoeba extract (AE) shifts from the upper phase to the lower phase after digestion with Proteinase K (Ptn K). Panels C and D show results from overnight and 48 h, respectively. Chloroform extraction of the upper phase after Proteinase K digestion (Cn + AE upper phase) enhances the activity of the upper phase to promote cell body size increase, while the capsule inducing substance transfers to the lower phase (Cn + AE lower phase). (*) indicates P<0.05 for capsule enlargement for each condition when compared to control incubation in PBS. In Panel C, the (**) indicates P<0.05 for a decrease in capsule relative to incubation in PBS, while in Panel D (**), indicates P<0.05 for a decrease in cell body volume relative to incubation in PBS.