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Phospholipids trigger Cryptococcus neoformans capsular enlargement during interactions with amoebae and macrophages.

Chrisman CJ, Albuquerque P, Guimaraes AJ, Nieves E, Casadevall A - PLoS Pathog. (2011)

Bottom Line: The phenomenon required contact between fungal and protozoan cells but did not require amoeba viability.The observation that the incubation of C. neoformans with phospholipids led to the formation of giant cells provides the means to generate these enigmatic cells in vitro.The parallels apparent in the capsular response of C. neoformans to both amoebae and macrophages provide additional support for the notion that certain aspects of cryptococcal virulence emerged as a consequence of environmental interactions with other microorganisms such as protists.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT
A remarkable aspect of the interaction of Cryptococcus neoformans with mammalian hosts is a consistent increase in capsule volume. Given that many aspects of the interaction of C. neoformans with macrophages are also observed with amoebae, we hypothesized that the capsule enlargement phenomenon also had a protozoan parallel. Incubation of C. neoformans with Acanthamoeba castellanii resulted in C. neoformans capsular enlargement. The phenomenon required contact between fungal and protozoan cells but did not require amoeba viability. Analysis of amoebae extracts showed that the likely stimuli for capsule enlargement were protozoan polar lipids. Extracts from macrophages and mammalian serum also triggered cryptococcal capsular enlargement. C. neoformans capsule enlargement required expression of fungal phospholipase B, but not phospholipase C. Purified phospholipids, in particular, phosphatidylcholine, and derived molecules triggered capsular enlargement with the subsequent formation of giant cells. These results implicate phospholipids as a trigger for both C. neoformans capsule enlargement in vivo and exopolysaccharide production. The observation that the incubation of C. neoformans with phospholipids led to the formation of giant cells provides the means to generate these enigmatic cells in vitro. Protozoan- or mammalian-derived polar lipids could represent a danger signal for C. neoformans that triggers capsular enlargement as a non-specific defense mechanism against potential predatory cells. Hence, phospholipids are the first host-derived molecules identified to trigger capsular enlargement. The parallels apparent in the capsular response of C. neoformans to both amoebae and macrophages provide additional support for the notion that certain aspects of cryptococcal virulence emerged as a consequence of environmental interactions with other microorganisms such as protists.

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C. neoformans cell gigantism following treatment                            with phosphatidylcholine (PC).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.
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ppat-1002047-g008: C. neoformans cell gigantism following treatment with phosphatidylcholine (PC).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.

Mentions: Incubations of C. neoformans yeast with A. castellanii, macrophages, and their respective extracts were evaluated in longer incubation periods for the induction of cell gigantism. At days 2, 4, and 6, co-incubation with amoeba, macrophages, and the extracts all induced larger capsule volumes when compared to incubation in PBS or minimum medium alone (Figure 7A, p<0.05). After 8 days, we observed an increase in the cell body volume of these cells and a concurrent reduction of relative capsular volume, but the overall volume of the C. neoformans yeast did not display a statistically significant difference. Cells incubated with amoeba extract and with the intact amoeba cell have a distinct pattern, with a double-layered capsule and two regions of different density. These cells had diameters ranging from 15 to 20 µm, approximately the size of giant cells previously described as forming in vivo [7], [22] (Figure 7B). Analysis by indirect immunoflourescence of capsule-induced C. neoformans cells after 6 days revealed stronger binding than the capsules of C. neoformans cells in PBS, consistent with the capsule enlargement phenomenon previously described (Figure 7C). Additionally, India ink staining and immunofluorescence of C. neoformans cells exposed to PC manifested very large C. neoformans cells as early as 48 hours that were even larger than those observed after extended incubation with A. castellanii, macrophages, and their respective extracts (Figure 8A, 8B, and 8C). Those cells did not constitute the majority of the cells, but were notably larger than untreated cells (Insets in Figure 8A and in 8B and Figure 8D). The whole cells averaged from 20 to 30 µm and both cell body and capsule were significantly enlarged and their relative abundance appeared to increase after longer incubation periods (Figure 8C and 8D).


Phospholipids trigger Cryptococcus neoformans capsular enlargement during interactions with amoebae and macrophages.

Chrisman CJ, Albuquerque P, Guimaraes AJ, Nieves E, Casadevall A - PLoS Pathog. (2011)

C. neoformans cell gigantism following treatment                            with phosphatidylcholine (PC).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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102711&req=5

ppat-1002047-g008: C. neoformans cell gigantism following treatment with phosphatidylcholine (PC).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.
Mentions: Incubations of C. neoformans yeast with A. castellanii, macrophages, and their respective extracts were evaluated in longer incubation periods for the induction of cell gigantism. At days 2, 4, and 6, co-incubation with amoeba, macrophages, and the extracts all induced larger capsule volumes when compared to incubation in PBS or minimum medium alone (Figure 7A, p<0.05). After 8 days, we observed an increase in the cell body volume of these cells and a concurrent reduction of relative capsular volume, but the overall volume of the C. neoformans yeast did not display a statistically significant difference. Cells incubated with amoeba extract and with the intact amoeba cell have a distinct pattern, with a double-layered capsule and two regions of different density. These cells had diameters ranging from 15 to 20 µm, approximately the size of giant cells previously described as forming in vivo [7], [22] (Figure 7B). Analysis by indirect immunoflourescence of capsule-induced C. neoformans cells after 6 days revealed stronger binding than the capsules of C. neoformans cells in PBS, consistent with the capsule enlargement phenomenon previously described (Figure 7C). Additionally, India ink staining and immunofluorescence of C. neoformans cells exposed to PC manifested very large C. neoformans cells as early as 48 hours that were even larger than those observed after extended incubation with A. castellanii, macrophages, and their respective extracts (Figure 8A, 8B, and 8C). Those cells did not constitute the majority of the cells, but were notably larger than untreated cells (Insets in Figure 8A and in 8B and Figure 8D). The whole cells averaged from 20 to 30 µm and both cell body and capsule were significantly enlarged and their relative abundance appeared to increase after longer incubation periods (Figure 8C and 8D).

Bottom Line: The phenomenon required contact between fungal and protozoan cells but did not require amoeba viability.The observation that the incubation of C. neoformans with phospholipids led to the formation of giant cells provides the means to generate these enigmatic cells in vitro.The parallels apparent in the capsular response of C. neoformans to both amoebae and macrophages provide additional support for the notion that certain aspects of cryptococcal virulence emerged as a consequence of environmental interactions with other microorganisms such as protists.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT
A remarkable aspect of the interaction of Cryptococcus neoformans with mammalian hosts is a consistent increase in capsule volume. Given that many aspects of the interaction of C. neoformans with macrophages are also observed with amoebae, we hypothesized that the capsule enlargement phenomenon also had a protozoan parallel. Incubation of C. neoformans with Acanthamoeba castellanii resulted in C. neoformans capsular enlargement. The phenomenon required contact between fungal and protozoan cells but did not require amoeba viability. Analysis of amoebae extracts showed that the likely stimuli for capsule enlargement were protozoan polar lipids. Extracts from macrophages and mammalian serum also triggered cryptococcal capsular enlargement. C. neoformans capsule enlargement required expression of fungal phospholipase B, but not phospholipase C. Purified phospholipids, in particular, phosphatidylcholine, and derived molecules triggered capsular enlargement with the subsequent formation of giant cells. These results implicate phospholipids as a trigger for both C. neoformans capsule enlargement in vivo and exopolysaccharide production. The observation that the incubation of C. neoformans with phospholipids led to the formation of giant cells provides the means to generate these enigmatic cells in vitro. Protozoan- or mammalian-derived polar lipids could represent a danger signal for C. neoformans that triggers capsular enlargement as a non-specific defense mechanism against potential predatory cells. Hence, phospholipids are the first host-derived molecules identified to trigger capsular enlargement. The parallels apparent in the capsular response of C. neoformans to both amoebae and macrophages provide additional support for the notion that certain aspects of cryptococcal virulence emerged as a consequence of environmental interactions with other microorganisms such as protists.

Show MeSH
Related in: MedlinePlus