IDENTIFICATION OF EXTRACELLULAR PHOSPHOLIPASE-B, LYSOPHOSPHOLIPASE, AND ACYLTRANSFERASE PRODUCED BY CRYPTOCOCCUS-NEOFORMANS

We recently identified phospholipase activity as a potential virulence factor of Cryptococcus neoformans. We have now defined the nature of the phospholipase activity produced by a clinical isolate of C. neofor mans var. neoformans, under native conditions, by H-1 and P-31 nuclear magnetic resonance (NMR) spectroscopy and thin-layer chromatography ( TLC) of radiolabelled substrates. Glycerophosphocholine was identified by NMR spectroscopy as the sole phospholipid degradation product of t he reaction between substrate phosphatidylcholine (PC) and cryptococca l culture supernatants indicating the presence of phospholipase B (PLB ). No lysophosphatidylcholine (lyse-PC) or products indicative of phos pholipase C, phospholipase D, or other lipase activity were identified . Use of PC and lyse-PC containing radiolabelled acyl chains and separ ation of products by TLC confirmed the PLB and lysophospholipase (LPL) activities. Lysophospholipase transacylase (LPTA) activity was identi fied by the formation of radioactive PC from lyse-PC. Extracellular en zyme production was maximal after 6 to 10 h in fresh medium. Assay con ditions were optimized for pH, linearity with time, enzyme concen trat ion, and saturation by substrates to allow comparison with phospholipa ses from other organisms. LPL activity was 10- to 20-fold greater than PLB activity, with mean (a standard deviation) specific activities of 34.9 +/- 7.9 and 3.18 +/- 0.2 mu mol of substrate hydrolyzed per min per mg of protein, respectively. The response of PLB to increasing sub strate concentrations was bimodal, whereas inhibition of LPL and LPTA activities occurred at concentrations of substrate lyse-PC greater tha n 200 mu M. Enzyme activities were stable at acid pH (3.8), with pH op tima of 3.5 to 4.5. Activities were unchanged in the presence of exoge nous serine protease inhibitors, divalent cations, and EDTA. We conclu de that C. neoformans produces highly active extracellular PLB, LPL, a nd LPTA under native conditions.