Cryptococcus neoformans is an environmental fungal pathogen and the most common cause of brain infection in immunocompromised individuals. Cryptococcus infections afflict a large population, especially among HIV/AIDS patients, resulting in an estimated 625000 deaths per year. Recent studies from our group have shown that changes in the chemical structure of the membrane-localized lipid, glucosylceramide (GlcCer), are directly linked with cryptococcal virulence. While the virulent C. neoformans strain synthesizes GlcCer with a double bond in carbon position 8 and a methyl group in carbon position 9, genetically modified strains that lack the methyl group (strain Δsmt1) and the double bond (strain Δsld8) in their GlcCer structure become avirulent in the mouse model. It has been suggested that changes in GlcCer structure might affect membrane biophysical properties and contribute to loss of virulence. In an attempt to investigate this potential mechanism, the present study focuses on the role of GlcCer chemical structure in regulating the biophysical properties of the cell membrane in C. neoformans.
Three strains were used in this study: the highly virulent C. neoformans wild-type strain H99 (WT), strain Δsmt1, and strain Δsld8. GlcCer was purified from all strains using column chromatography and characterized using thin layer chromatography and liquid chromatography-mass spectrometry. Purified lipids were formulated into vesicles either alone or in combination with ergosterol and the partially unsaturated lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to generate a simplistic membrane model. The thermotropic behavior of lipid vesicles was investigated by monitoring the fluorescence anisotropy of the fluorescent probe, diphenylhexatriene, in the temperature range of 16 °C to 60 °C. The tendency of lipids to phase segregate and form ordered domains was examined using Forster resonance energy transfer (FRET). Gas chromatography-mass spectrometry (GC-MS) was used to analyze ergosterol levels in the cell membrane of each strain.
The thermotropic behavior of purified GlcCers was dependent on their structure. The temperature at which purified lipids transitioned from ordered to disordered phase (Tm) was higher in GlcCer purified from the mutant strains compared to the WT (Tm-WT= 34.7 ± 0.0 °C, Tm-Δsmt1= 40.7 ± 1.1 °C, Tm-Δsld8= 43.2 ± 1.1 °C), suggesting an increased ability of GlcCer from mutants to form ordered domains at physiological temperatures. Similar trends were observed in membrane models, in which the more saturated GlcCer structures showed higher lipid order. GlcCer structures purified from all strains formed ordered domains as evidenced by FRET studies. GC-MS analysis revealed that the mutant strains contained up to 4 times higher amount of ergosterol, a sterol known to promote ordered lipid domain formation. Taken together, these data suggest that mutations in GlcCer chemical structure are likely to increase lipid order and domain formation in the cell membrane and disrupt normal cell behavior. These results provide information on the role of lipid chemical structure on cell membrane physical properties and are of importance in the fields of fungal pathogenesis and membrane biophysics.