N-linked glycosylation sites affect secretion of cryptococcal phospholipase B1, irrespective of glycosylphosphatidylinositol anchoring.

Secreted phospholipase B enzymes (PLB1) with high levels of N-linked glycosylation are proven fungal virulence determinants. We demonstrated that removal of N-linked glycans from secreted cryptococcal PLB1 leads to loss of enzyme activity. To determine if individual N-glycan attachment sites affect secretion of active enzyme, we altered three along the entire length of the protein, by site-directed mutagenesis, namely Asn56, Asn430 and Asn550 to Ala, in wild-type PLB1 (full length) and a glycosylphosphatidylinositol (GPI) anchorless version (PLB1(GPI-)) that is hypersecreted due to lack of membrane association. Alteration of Asn56 and Asn550 in both PLB1 and PLB1(GPI-) abolished enzyme secretion while alteration of Asn430 reduced secretion by 60%, following expression in Saccharomyces cerevisiae. Reduced secretion coincided with reduced enzyme in membranes and cell walls confirming a reduction in the rate of PLB1 transport to the cell surface. Deglycosylation of cryptococcal PLB1 increased its susceptibility to proteolysis suggesting that the absence of full glycosylation status leads to degradation of unstable PLB1, resulting in reduced traffic through the secretory pathway. We conclude that individual N-linked glycans are required for optimal transport of PLB1 to the cell surface and optimal secretion of both PLB1 and PLB1(GPI-).

Correlation of antifungal activity with fungal phospholipase inhibition using a series of bisquaternary ammonium salts.

A series of bisquaternary ammonium salts with a 12-carbon spacer between the positive charges were synthesized, and their antifungal activity has been investigated. Compounds with butyl, pentyl, and isopentyl headgroups were the most potent antifungal agents with MICs in the range of 2.2-5.5 microM against both Cryptococcus neoformans and Candida albicans. The antifungal activity of these compounds correlated with their inhibition of cryptococcal phospholipase B1 (PLB1), a newly identified virulence factor. This indicates that the mode of action of these compounds may be inhibition of the fungal PLB1 enzyme, further validating this enzyme as a target for the development of novel antifungal therapies.

Secretion of cryptococcal phospholipase B1 (PLB1) is regulated by a glycosylphosphatidylinositol (GPI) anchor.

The secreted, multifunctional enzyme PLB1 (phospholipase B1 protein encoded by the PLB1 gene) is a virulence determinant of the pathogenic fungus Cryptococcus neoformans, but the mechanism of its secretion is unknown. The cryptococcal PLB1 gene encodes putative, N-terminal LP (leader peptide) and C-terminal GPI (glycosylphosphatidylinositol) anchor attachment motifs, suggesting that PLB1 is GPI-anchored before secretion. To investigate the role of these motifs in PLB1 secretion, four cDNA constructs were created encoding the full-length construct (PLB1) and three truncated versions without the LP and/or the GPI anchor attachment motifs [(LP-)PLB1 (PLB1 expressed without the LP consensus motif), (LP-)PLB1(GPI-) (PLB1 expressed without the LP and GPI consensus motifs) and PLB1(GPI-) (PLB1 expressed without the GPI anchor attachment motif) respectively]. The constructs were ligated into pYES2, and galactose-induced expression was achieved in Saccharomyces cerevisiae. The LP was essential for secretion of the PLB1 protein and its three activities (PLB, lysophospholipase and lysophospholipase transacylase). Deletion of the GPI motif to create PLB1(GPI-) resulted in a redistribution of activity from the cell wall and membranes to the secreted and cytosolic fractions, with 36-54% of the total activity being secreted as compared with <5% for PLB1. PLB1 produced the maximum cell-associated activity (>2-fold more than that for PLB1(GPI-)), with 75-86% of this in the cell-wall fraction, 6-19% in the membrane fraction and 3-7% in the cytosolic fraction. Cell-wall localization was confirmed by release of activity with beta-glucanase in both S. cerevisiae recombinants and wild-type C. neoformans. The dominant location of PLB1 in the cell wall via GPI anchoring may permit immediate release of the enzyme in response to changing environmental conditions and may represent part of a novel mechanism for regulating the secretion of a fungal virulence determinant.

Inhibition of tetraphenylphosphonium-induced NMR-visible lipid accumulation in human breast cells by chlorpromazine.

The effects of chlorpromazine (CPZ) on the lipid accumulation induced by the cationic lipophilic compound tetraphenylphosphonium chloride (TPP) were examined using proton nuclear magnetic resonance spectroscopy (NMR), lipid extraction and thin layer chromatography (TLC), and electron microscopy (EM). Chlorpromazine at concentrations of 12 or 25 microM significantly reduced the NMR-visible lipid accumulation induced by a 48-h treatment with 6.25 microM TPP in the human breast cell line, HBL-100, without affecting cell viability. TPP caused threefold increases in whole-cell triglyceride levels that were attenuated by the addition of CPZ. Electron micrographs of TPP-treated HBL-100 cells showed that the destruction of mitochondria was accompanied by the accumulation of lipid droplets and myelinoid bodies. The addition of CPZ to TPP-treated cells reduced the occurrence of lipid droplets but not of mitochondrial destruction. Treatment with CPZ, in the presence or absence of TPP, resulted in large cytoplasmic inclusions indicating the inhibition of lysosomal metabolism. The induction and attenuation of NMR-visible lipids in conjunction with concomitant changes in both intracellular lipid droplets and whole-cell triglyceride levels provides evidence that NMR-visible lipids arise from cytoplasmic neutral lipid droplets. The observation that CPZ, a known inhibitor of lysosomal and cytosolic lipid metabolism, attenuates the formation of neutral triglycerides indicates that lysosomal processing may be a major step in the accumulation of NMR-visible lipid in breast cell lines.

Cryptococcal phospholipases: a novel lysophospholipase discovered in the pathogenic fungus Cryptococcus gattii.

The pathogenic fungus Cryptococcus neoformans produces an extracellular PLB1 (phospholipase B1), shown previously to be a virulence factor. A novel phospholipase (LPL1) with only LPL (lysophospholipase) and LPTA (transacylase) activities has now been characterized in C. gattii, and found to be a 66-kDa glycoprotein (by SDS/PAGE), with a native molecular mass of 670 kDa. The pI was 6.3, and it was active at high temperatures (to 70 degrees C), as well as at both acidic and neutral pH values. It was stimulated by calcium and palmitoyl carnitine at pH 7.0, but not at pH 5.0, and palmitoyl lysophosphatidylcholine was the preferred substrate. Sequencing indicated that LPL1 is a novel cryptococcal lysophospholipase, and not the gene product of CnLYSO1 or PLB1. A protein with only LPL and LPTA activities was subsequently isolated from two strains of C. neoformans var. grubii. A PLB1 enzyme was isolated from both C. gattii and a highly virulent strain of C. neoformans var. grubii (H99). In both cases, all three enzyme activities (PLB, LPL and LPTA) were present in one 95-120 kDa glycoprotein (by SDS/PAGE) with pI 3.9-4.3. Characterization of PLB1 from C. gattii showed that it differed from that of C. neoformans in its larger native mass (275 kDa), high PLB activity relative to LPL and LPTA, and preference for saturated lipid substrates. Differences in the properties between the secreted phospholipases of the two cryptococcal species could contribute to phenotypic differences that determine their respective environmental niches and different clinical manifestations.

Cloning of CnLYSO1, a novel extracellular lysophospholipase of the pathogenic fungus Cryptococcus neoformans.

We cloned a novel lysophospholipase (CnLYSO1) from Cryptococcus neoformans var. grubii by PCR amplification and a cDNA library screen. The open reading frame (ORF) of 1278 nucleotides coded for a predicted 426-amino-acid protein (CnLyso1p) with two highly conserved GXSXG lipase-specific catalytic motifs and a molecular weight of 48.3 kDa. CnLyso1p exhibited 14% and 21% identity to Arabidopsis thaliana and human lysophospholipases, respectively. Immunoprecipitation and Western blot analyses indicated that CnLyso1p was secreted as a high molecular weight protein of 97-140 kDa. CnLYSO1 expressed in a phospholipase B-null mutant of Saccharomyces cerevisiae demonstrated lysophospholipase and lysophospholipase transacylase activities at pH 4.0. Targeted disruption of CnLYSO1 did not affect growth, melanin or capsule production by C. neoformans. Secreted lysophospholipase and transacylase activities (pH 4.0) were 50% of wild type and CnLyso1p was undetectable on Western blots. Phospholipase B activity was reduced at pH 7.0 (P<0.006) and at pH 4.0 (P=NS). The amount of secreted Plb1p (the gene product of PLB1) was also reduced. Deletion of PLB1 abolished all three secreted activities at pH 4.0 and 7.0. These results are best explained by post-translational interaction, most likely the formation of a functional complex between the independently regulated gene products, CnLyso1p and CnPlb1p.

Strain-dependent effects of environmental signals on the production of extracellular phospholipase by Cryptococcus neoformans.

Extracellular phospholipase (PL) activities comprising phospholipase B, lysophospholipase and lysophospholipase transacylase have been identified in culture supernatants of Cryptococcus neoformans and contribute to virulence. We found that PL production was optimal after fungal growth at 30 degrees C and secretion at 37 degrees C for all six C. neoformans isolates studied (four C. neoformans var. neoformans and two C. neoformans var. gattii). No increase in PL activity was found in one strain, NU-2, in low iron or tissue culture media, conditions where upregulation of other virulence factors has been reported. The most virulent strains in an intravenous mouse model of infection were best able to produce PL at growth and secretion temperatures of 37 degrees C, in tissue culture media and under assay conditions of pH 7.0.

Biochemical and functional characterisation of secreted phospholipase activities from Cryptococcus neoformans in their naturally occurring state.

A recent study demonstrated that phospholipase B (PLB), lysophospholipase (LPL) and lysophopholipase transacylase (LPTA) are secreted by Cryptococcus neoformans var. neoformans and showed that the amount of enzyme production correlated with virulence in mice. The present study characterised the extracellular enzyme activities further by radiometric assays and 31P nuclear magnetic resonance spectroscopy (NMR). All three enzymes were most active between 25 and 40 degrees C. Bovine lung surfactant and its major lipid components, disaturated phosphatidylcholine and phosphatidylglycerol, were the optimal substrates for PLB. Lysophosphatidylcholine was the favoured substrate for LPL and LPTA. PLB and LPL/LPTA were differentially affected by Triton X-100, and palmitoyl carnitine was a potent inhibitor of the three phospholipases. LPL and PLB activities were inhibited by dithiothreitol; N-ethylmaleimide inhibited LPL and LPTA activities. None of the enzymes was inhibited by N-bromosuccinimide or p-bromophenacyl bromide. Cellular disruption experiments indicated that >85% of the phospholipase activities were cell-associated, with LPL and LPTA being more easily released than PLB. At pH 5.5 and 7.0, the heat-inactivated secreted enzyme preparations decreased the viability of human neutrophils. This effect was attenuated by active supernates. The relative activities of the PLB, LPL and LPTA in the environment of neutrophils are likely to determine the fate of these cells in vivo. Both phospholipases and heat-stable substances secreted by C. neoformans at 37 degrees C could contribute to membrane degradation and virulence.

Cryptococcal lipid metabolism: phospholipase B1 is implicated in transcellular metabolism of macrophage-derived lipids.

Cryptococci survive and replicate within macrophages and can use exogenous arachidonic acid for the production of eicosanoids. Phospholipase B1 (PLB1) has a putative, but uninvestigated, role in these processes. We have shown that uptake and esterification of radiolabeled arachidonic, palmitic, and oleic acids by the Cryptococcus neoformans var. grubii H99 wild-type strain and its PLB1 deletion mutant strain (the Deltaplb1 strain) are independent of PLB1, except under hyperosmolar stress. Similarly, PLB1 was required for metabolism of 1-palmitoyl lysophosphatidylcholine (LysoPC), which is toxic to eukaryotic cell membranes, under hyperosmolar conditions. During both logarithmic and stationary phases of growth, the physiologically relevant phospholipids, dipalmitoyl phosphatidylcholine (DPPC) and dioleoyl phosphatidylcholine, were taken up and metabolized via PLB1. Exogenous DPPC did not enhance growth in the presence of glucose as a carbon source but could support it for at least 24 h in glucose-free medium. Detoxification of LysoPC by reacylation occurred in both the H99 wild-type and the Deltaplb1 strains in the presence of glucose, but PLB1 was required when LysoPC was the sole carbon source. This indicates that both energy-independent (via PLB1) and energy-dependent transacylation pathways are active in cryptococci. Phospholipase A(1) activity was identified by PLB1-independent degradation of 1-palmitoyl-2-arachidonoyl phosphatidylcholine, but the arachidonoyl LysoPC formed was not detoxified by reacylation. Using the human macrophage-like cell line THP-1, we demonstrated the PLB1-dependent incorporation of macrophage-derived arachidonic acid into cryptococcal lipids during cryptococcus-phagocyte interaction. This pool of arachidonate can be sequestered for eicosanoid production by the fungus and/or suppression of host phagocytic activity, thus diminishing the immune response.

Synthesis, antifungal and antimicrobial activity of alkylphospholipids.

The antifungal, antibacterial and haemolytic activity of a series of alkylphosphocholines (e.g., miltefosine) and alkylglycerophosphocholines (e.g., edelfosine) has been investigated. These compound classes exhibit significant antifungal and moderate antibacterial activities. Several new alkylphosphocholine derivatives with amide or ester bonds in the alkyl chain have been synthesised. These compounds show much lower haemolytic activity than miltefosine. Alkylphosphocholines and alkylglycerophosphocholines show significant promise as novel orally available antifungal and antibacterial therapeutics.

Synthesis, antifungal and haemolytic activity of a series of bis(pyridinium)alkanes.

A series of bis(pyridinium)alkanes have been prepared and their antifungal activity, haemolytic activity and ability to inhibit fungal phospholipase B1 have been investigated, together with those of the commercially available antiseptics octenidine and dequalinium. Removal of the amino substituents from the pyridinium rings resulted in a significant decrease in antifungal activity. However, shortening or removing the alkyl chains attached to the amino groups had little effect on antifungal activity and significantly reduced haemolytic activity. Only octenidine was a strong inhibitor of fungal phospholipase B1.

Role of conserved active site residues in catalysis by phospholipase B1 from Cryptococcus neoformans.

Phospholipase B1 (PLB1), secreted by the pathogenic yeast Cryptococcus neoformans, has an established role in virulence. Although the mechanism of its phospholipase B, lysophospholipase, and lysophospholipase transacylase activities is unknown, it possesses lipase, subtilisin protease aspartate, and phospholipase motifs containing putative catalytic residues S146, D392, and R108, respectively, conserved in fungal PLBs and essential for human cytosolic phospholipase A2 (cPLA2) catalysis. To determine the role of these residues in PLB1 catalysis, each was substituted with alanine, and the mutant cDNAs were expressed in Saccharomyces cerevisiae. The mutant PLB1s were deficient in all three enzymatic activities. As the active site structure of PLB1 is unknown, a homology model was developed, based on the X-ray structure of the cPLA2 catalytic domain. This shows that the two proteins share a closely related fold, with the three catalytic residues located in identical positions as part of a single active site, with S146 and D392 forming a catalytic dyad. The model suggests that PLB1 lacks the "lid" region which occludes the cPLA2 active site and provides a mechanism of interfacial activation. In silico substrate docking studies with cPLA2 reveal the binding mode of the lipid headgroup, confirming the catalytic dyad mechanism for the cleavage of the sn-2 ester bond within one of two separate binding tracts for the lipid acyl chains. Residues specific for binding arachidonic and palmitic acids, preferred substrates for cPLA2 and PLB1, respectively, are identified. These results provide an explanation for differences in substrate specificity between lipases sharing the cPLA2 catalytic domain fold and for the differential effect of inhibitors on PLB1 enzymatic activities.

Cell wall-linked cryptococcal phospholipase B1 is a source of secreted enzyme and a determinant of cell wall integrity.

Phospholipase B (Plb1) is secreted by pathogenic fungi and is a proven virulence determinant in Cryptococcus neoformans. Cell-associated Plb1 is presumptively involved in fungal membrane biogenesis and remodelling. We have also identified it in cryptococcal cell walls. Motif scanning programs predict that Plb1 is attached to cryptococcal membranes via a glycosylphosphatidylinositol (GPI) anchor, which could regulate Plb1 export and secretion. A functional GPI anchor was identified in cell-associated Plb1 by (G)PI-specific phospholipase C (PLC)-induced release of Plb1 from strain H99 membrane rafts and inhibition of GPI anchor synthesis by YW3548, which prevented Plb1 secretion and transport to membranes and cell walls. Plb1 containing beta-1,6-linked glucan was released from H99 (wild-type strain) cell walls by beta-1,3 glucanase, consistent with covalent attachment of Plb1 via beta-1,6-linked glucans to beta-1,3-linked glucan in the central scaffold of the wall. Naturally secreted Plb1 also contained beta-1,6-linked glucan, confirming that it originated from the cell wall. Plb1 maintains cell wall integrity because a H99 deletion mutant, DeltaPLB1, exhibited a morphological defect and was more susceptible than H99 to cell wall disruption by SDS and Congo red. Growth of DeltaPLB1 was unaffected by caffeine, excluding an effect of Plb1 on cell wall biogenesis-related signaling pathways. Environmental (heat) stress caused Plb1 accumulation in cell walls, with loss from membranes and reduced secretion, further supporting the importance of Plb1 in cell wall integrity. This is the first demonstration that Plb1 contributes to fungal survival by maintaining cell wall integrity and that the cell wall is a source of secreted enzyme.

In vitro activities of miltefosine and two novel antifungal biscationic salts against a panel of 77 dermatophytes.

The susceptibilities of 77 dermatophytes to miltefosine (MI), 1,12-bis(4-pentylpyridinium)dodecane (PYR), 1,12-bis(tributylammonium)dodecane (AM), itraconazole (ITC), terbinafine (TRB), and butenafine (BTF) were compared. Geometric mean MICs of TRB, BTF, ITC, MI, PYR, and AM were 0.039, 0.059, 1.718, 0.671, 6.006, and 4.771 microg/ml, respectively. MI was more active than ITC (P < 0.001).

Application of proton nuclear magnetic resonance spectroscopy to the study of Cryptococcus and cryptococcosis.

Proton nuclear magnetic resonance spectroscopy is a nondestructive technique that identifies chemicals in solution and in living cells. It has been used in cryptococcal research to identify the primary structure of capsular glucuronoxylomannans, link cellular apoptosis susceptibility (CAS) genes to positioning of residues on the mannose backbone of glucuronoxylomannan, and verify that the cryptococcal virulence determinant, phospholipase B, is elaborated in vivo. Promising clinical applications include speciation (Cryptococcus neoformans and Cryptococcus gattii), with preliminary evidence that varieties neoformans and grubii can also be distinguished, non-invasive diagnosis of cerebral cryptococcomas, and, in cases of meningitis, monitoring therapeutic response by analysis of cerebrospinal fluid.

Lipid rafts in Cryptococcus neoformans concentrate the virulence determinants phospholipase B1 and Cu/Zn superoxide dismutase.

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 degrees 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-beta-cyclodextrin (MbetaCD). Phospholipase B enzyme activity was inhibited in the raft environment and increased 15-fold following disruption of rafts with TX100 at 37 degrees C. Treatment of viable cryptococcal cells in suspension with MbetaCD 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.

Hexadecylphosphocholine (miltefosine) has broad-spectrum fungicidal activity and is efficacious in a mouse model of cryptococcosis.

The alkyl phosphocholine drug miltefosine is structurally similar to natural substrates of the fungal virulence determinant phospholipase B1 (PLB1), which is a potential drug target. We determined the MICs of miltefosine against key fungal pathogens, correlated antifungal activity with inhibition of the PLB1 activities (PLB, lysophospholipase [LPL], and lysophospholipase-transacylase [LPTA]), and investigated its efficacy in a mouse model of disseminated cryptococcosis. Miltefosine inhibited secreted cryptococcal LPTA activity by 35% at the subhemolytic concentration of 25 microM (10.2 microg/ml) and was inactive against mammalian pancreatic phospholipase A2 (PLA2). At 250 microM, cytosolic PLB, LPL, and LPTA activities were inhibited by 25%, 51%, and 77%, respectively. The MICs at which 90% of isolates were inhibited (MIC90s) against Candida albicans, Candida glabrata, Candida krusei, Cryptococcus neoformans, Cryptococcus gattii, Aspergillus fumigatus, Fusarium solani, Scedosporium prolificans, and Scedosporium apiospermum were 2 to 4 microg/ml. The MICs of miltefosine against Candida tropicalis (n = 8) were 2 to 4 microg/ml, those against Aspergillus terreus and Candida parapsilosis were 8 microg/ml (MIC90), and those against Aspergillus flavus (n = 8) were 2 to 16 microg/ml. Miltefosine was fungicidal for C. neoformans, with rates of killing of 2 log units within 4 h at 7.0 microM (2.8 microg/ml). Miltefosine given orally to mice on days 1 to 5 after intravenous infection with C. neoformans delayed the development of illness and mortality and significantly reduced the brain cryptococcal burden. We conclude that miltefosine has broad-spectrum antifungal activity and is active in vivo in a mouse model of disseminated cryptococcosis. The relatively small inhibitory effect on PLB1 enzyme activities at concentrations exceeding the MIC by 2 to 20 times suggests that PLB1 inhibition is not the only mechanism of the antifungal effect.

Detergent-resistant membrane fractions contribute to the total 1H NMR-visible lipid signal in cells.

Leukocytes and other cells show an enhanced intensity of mobile lipid in their 1H NMR spectra under a variety of conditions. Such conditions include stimulation, which has recently been shown to involve detergent-resistant, plasma membrane domains (DRMs) often called lipid rafts. As there is much speculation surrounding the origin of cellular NMR-visible lipid, we analysed subcellular fractions, including DRMs, by NMR spectroscopy. We demonstrated that DRMs isolated by density gradient centrifugation from lymphoid (CEM-T4, stimulated Jurkat cells), and monocytoid (THP-1) cells produced NMR-visible, lipid signals. Large scale subfractionation of THP-1 cells determined that while cytoplasmic lipid droplets constituted much of the total NMR-visible lipid, the contribution of DRMs was significant. Qualitative and quantitative lipid analyses revealed that DRMs and lipid droplets differed in their lipid composition. DRMs were enriched in cholesterol and ganglioside GM1, and contained relatively unsaturated fatty acids compared with the lipid droplets. Both lipid droplets and DRMs contained neutral lipids (triacylgycerols, cholesterol ester, fatty acids in THP-1 cells) that could, in addition to phospholipids, contribute to the NMR-visible lipid. The lipid droplets also exhibited different protein profiles and contained 500-fold less protein than DRMs, confirming that DRMs and droplets were fractionated as separate entities. The NMR-visible lipid in DRMs is therefore unlikely to be a contaminant from lipid droplets. We propose a micropartitioning of the NMR-visible mobile lipid of whole cells between intracellular lipid droplets, where most of this lipid resides, and detergent-resistant plasma membrane domains.