Cryptococcal phospholipase B antigen is not detected in serum of patients infected with Cryptococcus neoformans using a sandwich enzyme-linked immunosorbent assay.

Extracellular phospholipase B (PLB) is a virulence determinant of Cryptococcus neoformans and Cryptococcus gattii. In this study, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) for PLB antigen with a detection limit of 3.9 ng mL(-1). PLB was detected in culture supernatants of C. neoformans and C. gattii. PLB, however, was not detected in sera of seven human patients and 10 feline patients with active cryptococcosis. Furthermore, none of five rats with extensive pulmonary C. gattii infection had a positive ELISA test result. In conclusion, cryptococcal PLB could not be detected in serum using a PLB antigen-based ELISA. Despite its sensitivity, this ELISA is of limited diagnostic value. Exploration of further extracellular molecules suitable for serodiagnosis of active cryptococcal infection is warranted.

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.

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.