Biochemical and Functional Characterisation of Secreted Phospholipase Activities from Cryptococcus Neoformans in their Naturally Occurring State

Abstract

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 31 P nuclear magnetic resonance spectroscopy (NMR). All three enzymes were most active between 25 and 40OC. 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°C could contribute to membrane degradation and virulence.

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

“…4 show that raft-associated PLB activity increased approximately 15-fold following raft disruption. This was not due to stimulation of enzyme activity, since the detergent concentration in the assay was Ͻ0.1%, a concentration at which we have shown previously causes no stimulation of PLB activity (8,40). In contrast, only a fivefold increase in PLB activity was obtained following disruption of nonraft membranes.…”

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

“…Analysis of four cryptococcal strains with various levels of phospholipase activity revealed a correlation between phospholipase activity and virulence in BALB\c mice [22]. We also showed that phospholipid components of lung surfactant and cellular membranes were targets for C. neoformans phospholipases in their natural state [24]. Vidotto et al subsequently suggested that phospholipase production by C. neoformans may be linked to capsule size [25].…”

“…The respective roles of extracellular PLB, LPL and LPTA activities in human cryptococcal infection may vary in specific microenvironments within the host. We have previously shown that the LPL and LPTA activities of the native enzyme can reacylate lysophospholipids and reverse damage caused to neutrophils by other components secreted by cryptococci [24]. The ability to degrade (PLB) membrane phospholipids may promote invasion of lung tissue, whereas the ability to repair (LPL\LPTA) the phospholipids may enable cryptococci to parasitize phagocytes without eliciting an immune response.…”

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

“…The secreted enzyme, phospholipase B (Plb1), 2 is an established cryptococcal virulence determinant (1). 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).…”