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 (NH4)(2)SO 4 fractionation, and hydrophobic-interaction, anion-exchange and gel-filtra tion chromatography. All three enzyme activities copurified as a single pro tein with an apparent molecular mass of 70-90 kDa by SDS/PAGE and 160-180 k Da by gel filtration. The ratio of the three activities remained constant a fter each purification step. The amino acid composition, as well as the seq uences of the N-terminus and of five internal peptide fragments were novel. The protein was an acidic glycoprotein containing N-linked carbohydrate mo ieties, with pi values of 5.5 and 3.5. The apparent V-max values for PLB an d LPL activities were 12.3 and 870 mu mol/min per mg of protein respectivel y; the corresponding K-m values were approx. 185.3 and 92.2 mu M. The enzym e was active only at acidic pH (pH optimum of 4.0 for PLB and 4.0-5.0 for L PL and LPTA). Enzyme activity did not require added cations, but was inhibi ted by Fe3+. 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 dipa lmitoyl phosphatidylcholine and dioleoyl phosphatidylcholine were the prefe rred substrates, This is the first complete description of the purification and properties of a phospholipase, which may be involved in virulence, fro m a pathogenic fungus.