Lesley C. Wright

Extracellular phospholipase activity is a virulence factor for Cryptococcus neoformans

The human pathogenic fungus Cryptococcus neoformans secretes a phospholipase enzyme that demonstrates phospholipase B (PLB), lysophospholipase hydrolase and lysophospholipase transacylase activities. This enzyme has been postulated to be a cryptococcal virulence factor. We cloned a phospholipase‐encoding gene (PLB1) from C. neoformans and constructed plb1 mutants using targeted gene disruption. All three enzyme activities were markedly reduced in the mutants compared with the wild‐type parent. The plb1 strains did not have any defects in the known cryptococcal virulence phenotypes of growth at 37°C, capsule formation, laccase activity and urease activity. The plb1 strains were reconstituted using the wild‐type locus and this resulted in restoration of all extracellular PLB activities. In vivo testing demonstrated that the plb1 strain was significantly less virulent than the control strains in both the mouse inhalational model and the rabbit meningitis model. We also found that the plb1 strain exhibited a growth defect in a macrophage‐like cell line. These data demonstrate that secretory phospholipase is a virulence factor for C. neoformans.

Role of Extracellular Phospholipases and Mononuclear Phagocytes in Dissemination of Cryptococcosis in a Murine Model

ABSTRACT Secreted phospholipase B (PLB) activity promotes the survival and replication of Cryptococcus neoformans in macrophages in vitro. We therefore investigated the role of mononuclear phagocytes and cryptococcal PLB in the dissemination of infection in a mouse model, using C. neoformans var. grubii wild-type strain H99, a PLB1 deletion mutant (Δplb1), and a reconstituted strain (Δplb1rec). PLB facilitated the entry of endotracheally administered cryptococci into lung IM. PLB was also required for lymphatic spread from the lung to regional lymph nodes and for entry into the blood. Langhans-type giant cells containing budding cryptococci were seen free in the lymphatic sinuses of hilar nodes of H99- and Δplb1rec-infected mice, suggesting that they may have a role in the dissemination of cryptococcal infection. The transfer of infected lung macrophages to recipient mice by tail vein injections demonstrated that these cells can facilitate hematogenous dissemination of cryptococci to the brain, independent of cryptococcal PLB secretion. PLB activities of cryptococci isolated from lung macrophages or infected brains were not persistently increased. We conclude that mononuclear phagocytes are a vehicle for cryptococcal dissemination and that PLB activity is necessary for the initiation of interstitial pulmonary infections and for dissemination from the lung via the lymphatics and blood. PLB is not, however, essential for the establishment of neurological infections when cryptococci are presented within, or after passage through, mononuclear phagocytes.

Purification and characterization of secretory phospholipase B, lysophospholipase and lysophospholipase/transacylase from a virulent strain of the pathogenic fungus Cryptococcus neoformans.

Infection caused by the fungus Cryptococcus neoformans is potentially fatal. A highly active extracellular phospholipase, demonstrating phospholipase B (PLB), lysophospholipase (LPL) and lysophospholipase/transacylase (LPTA) activities, was purified to homogeneity from C. neoformans using (NH(4))(2)SO(4) fractionation, and hydrophobic-interaction, anion-exchange and gel-filtration chromatography. All three enzyme activities co-purified as a single protein with an apparent molecular mass of 70-90 kDa by SDS/PAGE and 160-180 kDa by gel filtration. The ratio of the three activities remained constant after each purification step. The amino acid composition, as well as the sequences of the N-terminus and of five internal peptide fragments were novel. The protein was an acidic glycoprotein containing N-linked carbohydrate moieties, with pI values of 5.5 and 3.5. The apparent V(max) values for PLB and LPL activities were 12.3 and 870 micromol/min per mg of protein respectively; the corresponding K(m) values were approx. 185.3 and 92.2 microM. The enzyme was active only at acidic pH (pH optimum of 4.0 for PLB and 4.0-5.0 for LPL and LPTA). Enzyme activity did not require added cations, but was inhibited by Fe(3+). LPL and LPTA activities were decreased by 0.1% (v/v) Triton X-100 to 50% of the control value. Palmitoylcarnitine (0.5 mM) inhibited PLB (97% inhibition) and LPL and LPTA activities (35% inhibition) competitively. All phospholipids except phosphatidic acid were degraded by PLB, but dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylcholine were the preferred substrates. This is the first complete description of the purification and properties of a phospholipase, which may be involved in virulence, from a pathogenic fungus.

Hexadecylphosphocholine (Miltefosine) Has Broad-Spectrum Fungicidal Activity and Is Efficacious in a Mouse Model of Cryptococcosis

ABSTRACT 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 μM (10.2 μg/ml) and was inactive against mammalian pancreatic phospholipase A2 (PLA2). At 250 μM, 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 μg/ml. The MICs of miltefosine against Candida tropicalis (n = 8) were 2 to 4 μg/ml, those against Aspergillus terreus and Candida parapsilosis were 8 μg/ml (MIC90), and those against Aspergillus flavus (n = 8) were 2 to 16 μg/ml. Miltefosine was fungicidal for C. neoformans, with rates of killing of 2 log units within 4 h at 7.0 μM (2.8 μg/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.

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-β-cyclodextrin (MβCD). 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 MβCD 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

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 β-1,6-linked glucan was released from H99 (wild-type strain) cell walls by β-1,3 glucanase, consistent with covalent attachment of Plb1 via β-1,6-linked glucans to β-1,3-linked glucan in the central scaffold of the wall. Naturally secreted Plb1 also contained β-1,6-linked glucan, confirming that it originated from the cell wall. Plb1 maintains cell wall integrity because a H99 deletion mutant, ΔPLB1, exhibited a morphological defect and was more susceptible than H99 to cell wall disruption by SDS and Congo red. Growth of ΔPLB1 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.

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.

Metabolites released by Cryptococcus neoformans var. neoformans and var. gattii differentially affect human neutrophil function

Differences in the ability of Cryptococcus neoformans var. neoformans (CNVN) and var. gattii (CNVG) to establish localized lesions in the lungs of healthy humans remain unexplained. In this study, CNVG infection in a rat model was characterized by early neutrophil invasion into lung tissue, but phagocytosis of cryptococci was not observed. The chemical composition of non-enzymic components secreted by one strain of each variety (heat-inactivated supernatants from CNVN and CNVG, termed vns and vgs, respectively) were compared, using magnetic resonance spectroscopy. Effects on human neutrophil viability and functions at both pH 5.5 and 7.0 were investigated, as the pH of cryptococcomas was found to be 5.4-5.6 in vivo. The supernatants were similar in composition, although metabolites in vns were generally present in higher concentrations. In addition, vgs contained two novel metabolites-acetoin and dihydroxyacetone. Polyphosphate was observed in cells from both varieties and may be a source of extracellular inorganic phosphate. Superoxide production in the presence of phorbol ester was enhanced by treatment with vns and decreased by vgs. At pH 5.5, vns caused high levels of necrosis in neutrophils, as well as increased adhesion/migration through A549 lung epithelial cell monolayers. Individual supernatant components such as polyols, acetoin, dihydroxyacetone, and gamma-aminobutyric acid exhibited both pro- and anti-inflammatory properties. Overall, we found that vgs was potentially less pro-inflammatory than vns. Inhibition of neutrophil function by products of CNVG may promote survival of extracellular organisms, and local multiplication to form cryptococcomas.

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 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.