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. 2006 Jun;116(6):1651-9.
doi: 10.1172/JCI27890.

Glucosylceramide synthase is an essential regulator of pathogenicity of Cryptococcus neoformans

Philipp C Rittershaus  1 , Talar B Kechichian, Jeremy C Allegood, Alfred H Merrill Jr, Mirko Hennig, Chiara Luberto, Maurizio Del Poeta
Affiliations

Glucosylceramide synthase is an essential regulator of pathogenicity of Cryptococcus neoformans

Philipp C Rittershaus et al. J Clin Invest. 2006 Jun.

Erratum in

  • J Clin Invest. 2007 May;117(5):1450

Abstract

The pathogenic fungus Cryptococcus neoformans infects humans upon inhalation and causes the most common fungal meningoencephalitis in immunocompromised subjects worldwide. In the host, C. neoformans is found both intracellularly and extracellularly, but how these two components contribute to the development of the disease is largely unknown. Here we show that the glycosphingolipid glucosylceramide (GlcCer), which is present in C. neoformans, was essential for fungal growth in host extracellular environments, such as in alveolar spaces and in the bloodstream, which are characterized by a neutral/alkaline pH, but not in the host intracellular environment, such as in the phagolysosome of macrophages, which is characteristically acidic. Indeed, a C. neoformans mutant strain lacking GlcCer did not grow in vitro at a neutral/alkaline pH, yet it had no growth defect at an acidic pH. The mechanism by which GlcCer regulates alkali tolerance was by allowing the transition of C. neoformans through the cell cycle. This study establishes C. neoformans GlcCer as a key virulence factor of cryptococcal pathogenicity, with important implications for future development of new antifungal strategies.

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Figures

Figure 1
Figure 1. Expression of GCS1 genes in S. cerevisiae.
(A) In vivo labeling in S. cerevisiae with [3H]-DHS. Expression of cryptococcal and human GCS1 produced GlcCer (boxed region) in galactose (+), but not in glucose (–). DHS, dihydrosphingosine; S1P, sphingosine-1-phosphate. Inositol phosphoryl ceramide (IPC), mannose-IPC (MIPC), and mannose-(inositol phosphoryl)2 ceramide (MIP2C) are complex sphingolipids. (B) In vitro assay in S. cerevisiae using NBD-C6-ceramides. Proteins extracted from yeast cells expressing pYES2-HuGCS1 and incubated with NBD-ceramides produced NBD-GlcCer under induction by galactose. NBD-GlcCer production was not observed in yeast cells expressing pYES2-CnGCS1 or pYES2 empty vector.
Figure 2
Figure 2. C. neoformans GCS1 gene encodes for GCS.
(A) In vivo labeling of C. neoformans WT, Δgcs1, and Δgcs1 + GCS1 using [3H]-DHS. The formation of GlcCer (boxed area) was examined by analysis of the extracted lipids onto a TLC. The soy GlcCer standard was visualized by iodine stain. (B) Analysis of purified GlcCer from the cultured strains using HPTLC. The lipids containing the sugar residues were visualized by staining with orcinol in 70% sulfuric acid, and the putative GlcCer is indicated. Plates were also stained with iodine to ensure equal lipid loading among the lanes (arrowhead). (C) Electrospray tandem mass spectrometric analysis of the glycosphingolipid analyzed on the HPTLC. The regions of the chromatogram indicated in B were extracted and analyzed by mass spectrometry as described in Methods. The indicated peaks are consistent with the [M+H]+ ions for monoexosylceramide with a methyl-sphingadienine backbone and a hydroxy-C18:1 fatty acid (the major species, m/z 756.7), a non–hydroxy-C18:0 fatty acid (m/z 740.7), and a hydroxy-C16:0 fatty acid (m/z 728.7). (D and E) 1H-1H–double quantum filtered correlation spectroscopy (D) and 1H-13C heteronuclear single quantum correlation (E) NMR spectra of the monohexosylceramide WT extracted from the HPTLC. The solid connectivities in D among the 7 nonexchangeable hexose protons and their 1H and 13C chemical shift characteristics (E) show that glucose was attached to the ceramide backbone, defining this glycosphingolipid as GlcCer. Dashed connectivities in D identify the exchangeable 6-OH in 100% dimethyl-sulfoxide-d6.
Figure 3
Figure 3. Survival studies of C. neoformans strains in a murine animal model.
(A) CBA/J mice infected intranasally with C. neoformans cells. The average survival of mice infected with the C. neoformans WT and Δgcs1 + GCS1 strains was 24.6 ± 3.9 days and 27.3 ± 4.8 days, respectively. All mice infected with Δgcs1 mutant strain survived after 90 days of infection (P < 0.0001). (B) CBA/J mice infected intravenously with C. neoformans cells. The average survival of mice infected with the C. neoformans WT and Δgcs1 + GCS1 strains was 6.3 ± 0.51 days and 6.2 ± 0.46 days, respectively, whereas the average survival of mice infected with C. neoformans Δgcs1 strain was 15 days (P < 0.01).
Figure 4
Figure 4. Histopathology of 2 different lungs obtained from CBA/J mice infected intranasally with the C. neoformans Δgsc1 strain.
(A and B) Movat stain. (C and D) Verhoeff–van Gieson stain. Boxed areas in A and C are magnified in B and D. In A, white arrowhead indicates C. neoformans cells stained alcian blue, white arrow indicates necrotic tissue, black arrowheads indicate macrophages, black arrows indicate lymphocyte infiltration with fibroblasts and fibrotic tissue, and green arrows indicate normal lung tissue. In B, yeast cells (white arrowhead) were found within necrotic tissue. Also, many yeast cells appear as “ghosts” or degenerated cells (yellow arrowheads) within macrophages. In C, black arrowhead indicates collagen stained red in the peripheral of a nodule containing necrotic tissue, yeast cells, macrophages, and lymphocytes. In D, note the collagen deposition (red stain) between lymphocytes and fibrotic tissue. A giant cell in gray (black arrowheads) was loaded with C. neoformans in the internal side of the nodule, surrounded by granulocytes and lymphocytes. Scale bars: 500 μm (A and C); 50 μm (B and D).
Figure 5
Figure 5. GlcCer is required for growth of C. neoformans cells in 5% CO2 at pH 7.4. C. neoformans WT, Δgcs1, and Δgcs1 + GCS1 strains were grown in DMEM medium at 37°C in 5% CO2 at pH 7.4 (A) and pH 4.0 (B).
*P < 0.001, Δgcs1 versus WT. (C) Succession of biological events characterizing a yeast cell cycle.
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