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J. Biol. Chem., Vol. 282, Issue 52, 37508-37514, December 28, 2007

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Cell Wall-linked Cryptococcal Phospholipase B1 Is a Source of Secreted Enzyme and a Determinant of Cell Wall Integrity^*

A. Rosemary Siafakas, Tania C. Sorrell, Lesley C. Wright, Christabel Wilson, Michelle Larsen, Ross Boadle, Peter R. Williamson^¶, and Julianne T. Djordjevic¹

From the Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, University of Sydney at Westmead Hospital, New South Wales 2145, Australia, The Electron Microscope Laboratory, ICPMR and Westmead Millennium Institute, Westmead Hospital, New South Wales 2145, Australia, and the ^¶Section of Infectious Diseases, Department of Medicine, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612

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 β-1,6-linked glucan was released from H99 (wild-type strain) cell walls by β-1,3 glucanase, consistent with covalent attachment of Plb1 via β-1,6-linked glucans to β-1,3-linked glucan in the central scaffold of the wall. Naturally secreted Plb1 also contained β-1,6-linked glucan, confirming that it originated from the cell wall. Plb1 maintains cell wall integrity because a H99 deletion mutant, PLB1, exhibited a morphological defect and was more susceptible than H99 to cell wall disruption by SDS and Congo red. Growth of PLB1 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.

Received for publication, September 21, 2007 , and in revised form, October 17, 2007.

^* This work was supported by National Health and Medical Research Council of Australia Project Grant 352354. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, University of Sydney at Westmead Hospital, Level 3 ICPMR Bldg., NSW 2145, Australia. Tel.: 61-2-9845-7367; Fax: 61-2-9891-5317; E-mail: julie_djordjevic{at}wmi.usyd.edu.au.

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