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.
Santangelo
Rosemary T
RT
Department of Biological Sciences, University of Western Sydney, Nepean, Kingswood.|Centre for Infectious Diseases and Microbiology, Westmead Hospital, Westmead.
Nouri-Sorkhabi
M Hossein
MH
Department of Biochemistry, University of Sydney, Sydney New South Wales, Australia.
Sorrell
Tania C
TC
Centre for Infectious Diseases and Microbiology, Westmead Hospital, Westmead.
Cagney
Michelle
M
Department of Infectious Diseases, University of Sydney, Westmead Hospital, Westmead.|Centre for Infectious Diseases and Microbiology, Westmead Hospital, Westmead.
Chen
Sharon C A
SCA
Centre for Infectious Diseases and Microbiology, Westmead Hospital, Westmead.
Kuchel
Philip W
PW
Department of Biochemistry, University of Sydney, Sydney New South Wales, Australia.
Wright
Lesley C
LC
Centre for Infectious Diseases and Microbiology, Westmead Hospital, Westmead.
D002470:Cell Survival; D003455:Cryptococcus neoformans / Q000187:drug effects / Q000201:enzymology*; D004229:Dithiothreitol / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008245:Lysophospholipase / Q000378:metabolism; D000432:Methanol / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology; D017830:Octoxynol / Q000494:pharmacology; D010172:Palmitoylcarnitine / Q000494:pharmacology; D010740:Phospholipases / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature
D010172:Palmitoylcarnitine; D017830:Octoxynol; D010740:Phospholipases; D008245:Lysophospholipase; D004229:Dithiothreitol; D000432:Methanol