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

ABSTRACT: 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 (NH(4))(2)SO(4) fractionation, and hydrophobic-interaction, anion-exchange and gel-filtration chromatography. All three enzyme activities co-purified as a single protein with an apparent molecular mass of 70-90 kDa by SDS/PAGE and 160-180 kDa by gel filtration. The ratio of the three activities remained constant after each purification step. The amino acid composition, as well as the sequences of the N-terminus and of five internal peptide fragments were novel. The protein was an acidic glycoprotein containing N-linked carbohydrate moieties, with pI values of 5.5 and 3.5. The apparent V(max) values for PLB and LPL activities were 12.3 and 870 micromol/min per mg of protein respectively; the corresponding K(m) values were approx. 185.3 and 92.2 microM. The enzyme was active only at acidic pH (pH optimum of 4.0 for PLB and 4.0-5.0 for LPL and LPTA). Enzyme activity did not require added cations, but was inhibited by Fe(3+). 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 dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylcholine were the preferred substrates. This is the first complete description of the purification and properties of a phospholipase, which may be involved in virulence, from a pathogenic fungus.

SUBMITTER: Chen SC

PROVIDER: S-EPMC1220975 | biostudies-other | 2000 Apr

REPOSITORIES: biostudies-other

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Project description:The Cryptococcus complex consists of at least seven evolutionary divergent lineages and causes ?200,000 fatal human infections each year worldwide. The dominant lineage is Cryptococcus neoformans which consists of three haploid clades VNI, VNII, and VNB, their haploid hybrids, and various diploids derived from intra- and inter-clade mating events. In this study, we analyzed the mitogenomes of 184 strains of C. neoformans. Our analyses revealed that all 184 mitogenomes contained the same 15 protein-coding genes in the same gene order. However, their mitogenome sizes varied between 24,740 and 31,327 bp, primarily due to differences in the number and size of mitochondrial introns. Twelve nucleotide sites within five mitochondrial genes were found to contain introns in at least one of the 184 strains, ranging from 2 to 7 introns within each mitogenome. The concatenated mitochondrial exon sequences of the 15 protein-coding genes and two rRNA genes showed that VNI, VNII, and VNB strains were separated into distinct clades or sub-clades, largely consistent with results based on nuclear genome SNPs. However, several novel findings were observed. First, one strain of the VNB clade contained mitogenome exon sequences identical to the main VNI mitogenome type but was distant to other VNB mitogenomes. Second, hybrids among clades VNI, VNII, and VNB identified based on their nuclear genome SNPs contained mitogenomes from different clades, with evidence of their mitogenomes inherited from either the MAT a or the MAT ? parents. Third, the eight diploid VNB (C. neoformans) × VNIV (C. deneoformans) hybrids contained recombinant mitogenomes. Fourth, analyses of intron distribution and the paired exon-intron phylogenies for each of the 12 exon-intron pairs suggested frequent gains and losses of mitochondrial introns during the evolution of C. neoformans. The combined mitogenome exon-based phylogeny and intron distributions suggested that clades VNI, VNII and VNB could be further divided into sub-clades. Together, our results revealed a dynamic evolution of mitochondrial genomes in this important human fungal pathogen.

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

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