369910 Membrane Physical Properties and Its Differences in Virulent and Avirulent Strains of Cryptococcus
Cryptococcus is an environmental pathogen found in trees and bird excretions. Infections with Cryptococcus strains are initiated by inhaling fungal spores, followed by dissemination of fungal cells to the brain and the development of life threatening meningoencephalitis. Recent evidence suggests that the presence of rigid membrane microdomains consisting of saturated lipids and sterols (i.e. lipid rafts) might play a role in the pathogenicity of Cryptococcus. Certain virulence factors of Cryptococcus were shown to concentrate in lipid rafts. Also, mutations that affect the synthesis of the raft forming molecule glucosylceramide have been shown to reduce the virulence of the fungus, suggesting that the role of membrane physical properties in fungal pathogenicity deserves further attention. However, there is currently a lack of knowledge on whether there is a relationship between the membrane physical properties and the virulence of Cryptococcus. The present study focuses on characterizing the membrane physical properties of infectious and non-infectious strains of Cryptococcus aiming to clarify the role of membrane organization in pathogenicity.
The highly virulent Cryptococcus gattii R265 and the non-virulent C. gattii R272 strains were used in this study. Lipids were extracted from both strains and analyzed using thin layer chromatography and mass spectrometry. Small unilamellar vesicles (SUV) were synthesized using lipid extracts and their physical properties were investigated using fluorescent anisotropy and Forster resonance electron transfer (FRET) analyses. For a closer mimic of lipid rafts, detergent resistant membranes (DRM) were isolated by exposing cell lysates to cold Triton X-100 and floatation on a sucrose density gradient. The physical properties of DRMs were also examined using fluorescent anisotropy and FRET.
The lipid species were not different between the two strains of C. gattii. However, lipid extracts from the virulent C. gattii R265 showed higher anisotropy compared to the non-virulent C. gattii R272 suggesting the presence of a more ordered membrane. Despite the difference in anisotropy, no chain melting behavior was observed in FRET studies. The highly virulent R265 strain showed higher levels of a fluorescent lipid, which was likely dehydroergosterol. The composition of DRMs was similar between the two strains, mainly consisting of ergosterol, glucosylceramide and phospholipids. No significant difference was observed in the chain melting temperature of DRMs between the strains (35.0 ± 2.3 °C for C. gattii R265 vs. 38.8 ± 5.6 °C for C. gattii R272). This observation might suggest that the changes observed in the anisotropy of total lipid extracts are due to differences in the amount of unsaturated lipids. These studies link the biophysical properties of fungal membranes with their virulence for the first time and suggest that the biophysical properties of fungal membranes may play a role in their ability to cause disease.
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