Glucosylceramide synthase is an essential regulator of pathogenicity of Cryptococcus neoformans
Philipp C. Rittershaus, … , Chiara Luberto, Maurizio Del Poeta
Philipp C. Rittershaus, … , Chiara Luberto, Maurizio Del Poeta
Published June 1, 2006
Citation Information: J Clin Invest. 2006;116(6):1651-1659. https://doi.org/10.1172/JCI27890.
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Research Article Microbiology

Glucosylceramide synthase is an essential regulator of pathogenicity of Cryptococcus neoformans

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.

Authors

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

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Figure 2

C. neoformans GCS1 gene encodes for GCS.

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C. neoformans GCS1 gene encodes for GCS. (A) In vivo labeling of C. neo...
(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.