title: Cell wall-linked cryptococcal phospholipase B1 is a source of secreted enzyme and a determinant of cell wall integrity.
abstract: Phospholipase B (Plb1) is secreted by pathogenic fungi and is a proven virulence determinant in Cryptococcus neoformans. Cell-associated Plb1 is presumptively involved in fungal membrane biogenesis and remodelling. We have also identified it in cryptococcal cell walls. Motif scanning programs predict that Plb1 is attached to cryptococcal membranes via a glycosylphosphatidylinositol (GPI) anchor, which could regulate Plb1 export and secretion. A functional GPI anchor was identified in cell-associated Plb1 by (G)PI-specific phospholipase C (PLC)-induced release of Plb1 from strain H99 membrane rafts and inhibition of GPI anchor synthesis by YW3548, which prevented Plb1 secretion and transport to membranes and cell walls. Plb1 containing beta-1,6-linked glucan was released from H99 (wild-type strain) cell walls by beta-1,3 glucanase, consistent with covalent attachment of Plb1 via beta-1,6-linked glucans to beta-1,3-linked glucan in the central scaffold of the wall. Naturally secreted Plb1 also contained beta-1,6-linked glucan, confirming that it originated from the cell wall. Plb1 maintains cell wall integrity because a H99 deletion mutant, DeltaPLB1, exhibited a morphological defect and was more susceptible than H99 to cell wall disruption by SDS and Congo red. Growth of DeltaPLB1 was unaffected by caffeine, excluding an effect of Plb1 on cell wall biogenesis-related signaling pathways. Environmental (heat) stress caused Plb1 accumulation in cell walls, with loss from membranes and reduced secretion, further supporting the importance of Plb1 in cell wall integrity. This is the first demonstration that Plb1 contributes to fungal survival by maintaining cell wall integrity and that the cell wall is a source of secreted enzyme.
authors.affiliation: Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, University of Sydney at Westmead Hospital, NSW, Australia.
authors.forename: A Rosemary
authors.initials: AR
authors.lastname: Siafakas
authors.forename: Tania C
authors.initials: TC
authors.lastname: Sorrell
authors.forename: Lesley C
authors.initials: LC
authors.lastname: Wright
authors.forename: Christabel
authors.initials: C
authors.lastname: Wilson
authors.forename: Michelle
authors.initials: M
authors.lastname: Larsen
authors.forename: Ross
authors.initials: R
authors.lastname: Boadle
authors.forename: Peter R
authors.initials: PR
authors.lastname: Williamson
authors.forename: Julianne T
authors.initials: JT
authors.lastname: Djordjevic
mesh_terms: D020816:Amino Acid Motifs; D001692:Biological Transport; D002473:Cell Wall / Q000378:metabolism*; D003455:Cryptococcus neoformans / Q000201:enzymology*; D005656:Fungal Proteins; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D008245:Lysophospholipase / Q000737:chemistry / Q000502:physiology*; D021962:Membrane Microdomains / Q000378:metabolism; D008954:Models, Biological; D013347:Subcellular Fractions; D013482:Superoxide Dismutase / Q000378:metabolism; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D013482:Superoxide Dismutase; D008245:Lysophospholipase