Changes in glucosylceramide structure affect virulence and membrane biophysical properties of Cryptococcus neoformans

Abstract

Fungal glucosylceramide (GlcCer) is a plasma membrane sphingolipid in which the sphingosine backbone is unsaturated in carbon position 8 (C8) and methylated in carbon position 9 (C9). Studies in the fungal pathogen, Cryptococcus neoformans, have shown that loss of GlcCer synthase activity results in complete loss of virulence in the mouse model. However, whether the loss of virulence is due to the lack of the enzyme or to the loss of the sphingolipid is not known. In this study, we used genetic engineering to alter the chemical structure of fungal GlcCer and studied its effect on fungal growth and pathogenicity. Here we show that unsaturation in C8 and methylation in C9 is required for virulence in the mouse model without affecting fungal growth in vitro or common virulence factors. However, changes in GlcCer structure led to a dramatic susceptibility to membrane stressors resulting in increased cell membrane permeability and rendering the fungal mutant unable to grow within host macrophages. Biophysical studies using synthetic vesicles containing GlcCer revealed that the saturated and unmethylated sphingolipid formed vesicles with higher lipid order that were more likely to phase separate into ordered domains. Taken together, these studies show for the first time that a specific structure of GlcCer is a major regulator of membrane permeability required for fungal pathogenicity.

The putative flippase Apt1 is required for intracellular membrane architecture and biosynthesis of polysaccharide and lipids in Cryptococcus neoformans

“…C. neoformans GlcCer was also characterized as a key virulence regulator of cryptococcal pathogenicity [56]. Importantly, the pathogenic role of GlcCer required unsaturation in C8 and methylation inC9 [57]. In our study, 9-methylated, 8-unsaturated GlcCer was reduced in the aptlΔ mutant.…”

Blood vessel occlusion byCryptococcus neoformansis a mechanism for haemorrhagic dissemination of infection

“…In order to investigate whether cryptococcal cell size or rigidity may affect the frequency of trapping and the extent of blood vessel vasodilation, we used mutant cryptococci with altered physical properties. Recently, the biophysical properties of several ceramide pathway mutants have been described (24) in which the accumulation of saturated GluCer ( Δsld8 ) was suggested to increase the rigidity of cryptococcal membranes and therefore reduce their ability to traverse smaller blood vessels. In contrast, mutants in Δgcs1 and Δsmt1 have reduced amounts of the more rigid ceramide lipids or differences in lipid packing respectively.…”

“…The C. neoformans variety grubii strain KN99, its GFP-expressing derivative KN99:GFP and mCherry-expressing derivative KN99:mCherry were used in this study (39). We used GFP expressing Δplb1-H99, Δlac1-H99 or parental H99-GFP (27) and Δgsc, Δsmt, Δsld8 and parental strain (24). Cultures were grown in 2□ml of yeast extract peptone dextrose (YPD) (all reagents are from Sigma-Aldrich, Poole, UK unless otherwise stated) inoculated from YPD agar plates and grown for 18□hours at 28□°C, rotating horizontally at 20□rpm.…”

Sterol-response pathways mediate alkaline survival in diverse fungi

“…These processes include the production of glycosphingolipids (GSLs) that associate with proteins in the outer leaflet of fungal plasma membranes to form lipid rafts and maintain membrane fluidity and organization (5–7). Recent studies have demonstrated that mutations resulting in reduced or absent GSLs render yeasts such as Kluyveromyces lactis , Neurospora crassa, and C. neoformans unable to grow in alkaline environments (8–11). The connection between membrane composition and the ability for fungal cells to grow in alkaline environments has been associated with defects in cytokinesis, altered activity of plasma membrane proton pumps, as well as an altered lipid profile (10).…”