Lipid Rafts in Cryptococcus neoformans Concentrate the Virulence Determinants Phospholipase B1 and Cu/Zn Superoxide Dismutase

Abstract

Lipid rafts have been identified in the membranes of mammalian cells, the yeast Saccharomyces cerevisiae, and the pathogenic fungus Candida albicans. Formed by a lateral association of sphingolipids and sterols, rafts concentrate proteins carrying a glycosylphosphatidylinositol (GPI) anchor. We report the isolation of membranes with the characteristics of rafts from the fungal pathogen Cryptococcus neoformans. These characteristics include insolubility in Triton X-100 (TX100) at 4°C, more-buoyant density within a sucrose gradient than the remaining membranes, and threefold enrichment with sterols. The virulence determinant phospholipase B1 (PLB1), a GPI-anchored protein, was highly concentrated in raft membranes and could be displaced from them by treatment with the sterol-sequestering agent methyl-␤-cyclodextrin (M␤CD). Phospholipase B enzyme activity was inhibited in the raft environment and increased 15-fold following disruption of rafts with TX100 at 37°C. Treatment of viable cryptococcal cells in suspension with M␤CD also released PLB1 protein and enzyme activity, consistent with localization of PLB1 in plasma membrane rafts prior to secretion. The antioxidant virulence factor Cu/Zn superoxide dismutase (SOD1) was concentrated six-to ninefold in raft membrane fractions compared with nonraft membranes, whereas the cell wall-associated virulence factor laccase was not detected in membranes. We hypothesize that raft membranes function to cluster certain virulence factors at the cell surface to allow efficient access to enzyme substrate and/or to provide rapid release to the external environment.

Cell Wall-linked Cryptococcal Phospholipase B1 Is a Source of Secreted Enzyme and a Determinant of Cell Wall Integrity

“…It facilitates invasion of host lung tissue (2), most likely by hydrolyzing phospholipids in mammalian cell membranes and pulmonary surfactant (3)(4)(5)(6) and is essential for hematogenous dissemination of infection (2). In the cryptococcal cell, Plb1 is localized in specialized membrane domains known as rafts (7). These domains contain phospholipid substrates, consistent with the putative role of Plb1 in cryptococcal membrane homeostasis and remodelling.…”

“…grubii serotype A (wild type), its PLB1 deletion mutant (⌬PLB1) and the reconstituted strain, ⌬PLB1 rec , were supplied by Dr. Gary Cox, Duke University. Subcellular Fractionation-Cryptococcal total and raft membranes were prepared as described (7). Briefly, cryptococcal cells, either grown on SAB agar for 72 h or in YNB/2% glucose for 24 h, were homogenized in MES-buffered saline (MBS; 25 mM MES, 150 mM NaCl, 2 mM EDTA, pH 6.5) containing protease inhibitors and 0.1% Triton X-100.…”

“…Antibodies-Antibodies to Plb1 were prepared as described (7,9). Antibodies to ␤-1,6-linked glucan were supplied by Frans Klis (Swammerdam Institute for Life Sciences, University of Amsterdam).…”

“…These domains contain phospholipid substrates, consistent with the putative role of Plb1 in cryptococcal membrane homeostasis and remodelling. It has been assumed that cell membranes are the source of secreted enzyme, based on studies of the Plb1 homologue in Saccharomyces cerevisiae (8), and our observation that incubation of cryptococci with the raft-disrupting agent, methyl ␤-cyclodextrin, releases Plb1 into the culture supernatant (7). We discovered recently that cryptococcal Plb1 is concentrated in the cell wall, but its function there is not known (9).…”

Silencing Prx1 and/or Prx5 sensitizes human esophageal cancer cells to ionizing radiation and increases apoptosis via intracellular ROS accumulation

“…Before IR, cell lysates were centrifuged at 12 000×g for 10 min at 4 °C and the supernatant was collected for measuring the activities of CAT, GPx, Cu/Zn-SOD and Mn-SOD, respectively by a Catalase Analysis Kit, a Cellular Glutathione Peroxidase Assay Kit, and Cu/Zn-SOD and Mn-SOD Assay Kit with WST-1 (Beytime Institute of Biotechnology, China). The measurements were performed according to the manufacturer's instructions as well as those described earlier [14][15][16] . Protein content was determined by using a BCA protein assay kit (Beytime Institute of Biotechnology, China).…”