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 extr acellular enzyme activities further by radiometric assays and P-31 nuclear magnetic resonance spectroscopy (NMR), All three enzymes were most active b etween 25 and 40 degrees C, Bovine lung surfactant and its major lipid comp onents, disaturated phosphatidylcholine and phosphatidylglycerol, were the optimal substrates for PLB, Lysophosphatidylcholine was the favoured substr ate for LPL and LPTA, PLB and LPL/LPTA were differentially affected by Trit on X-100, and palmitoyl carnitine was a potent inhibitor of the three phosp holipases, LPL and PLB activities were inhibited by dithiothreitol; N-ethyl maleimide inhibited LPL and LPTA activities. None of the enzymes was inhibi ted by N-bromosuccinimide or p-bromophenacyl bromide. Cellular disruption e xperiments indicated that >85% of the phospholipase activities were cell-as sociated, 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 vi ability of human neutrophils, This effect was attenuated by active supernat es. 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 p hospholipases and heat-stable substances secreted by C, neoformans at 37 de grees C could contribute to membrane degradation and virulence.