Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2013

Composition of the Cryptococcus neoformans secretome and post-mitotic cell separation are affected by the lipid transfer protein Sec14 (#178)

Sophia Lev 1 , Ben Crossett 2 , Methee Chayakulkeeree 3 , Desmarini Desmarini 1 , Christabel F Wilson 1 , Peter Williamson 4 , Tania C Sorrell 1 5 , Julianne T Djordjevic 1
  1. Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute and Sydney Medical School, University of Sydney at Westmead Hospital, Westmead, NSW, Australia
  2. School of Molecular Bioscience, University of Sydney, Sydney, NSW, Australia
  3. Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand
  4. Laboratory of Clinical Infectious Diseases , National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland , USA
  5. Sydney Emerging Infections and Biosecurity Institute, University of Sydney at Westmead Hospital, Westmead, NSW, Australia

The opportunistic fungal pathogen, Cryptococcus neoformans, secretes a diverse array of proteins that contribute to its virulence within the mammalian host. We established that the phosphatidylinositol/phosphatidylcholine transfer protein Sec14 regulates secretion of the C. neoformans invasin, phospholipase B1 (Plb1), and is essential for virulence. To identify other secreted proteins regulated by CnSec14 that potentially contribute to the virulence of C. neoformans, we analyzed the secretomes of WT (strain H99) and a SEC14 deletion mutant (Δsec14) using mass spectrometry. We identified 124 proteins in WT secretions; 32 contained a signal peptide, implying that they are canonically secreted via the ER/Golgi. The abundance of 25 proteins was reduced in Δsec14 secretions, 11 of which were either, canonically secreted enzymes or cell wall associated proteins/enzymes. In addition to Plb1, secretion of other virulence-related proteins, including acid phosphatase (Aph1) and laccase, was reduced in Δsec14 mutant. Fluorescent labelling of Aph1 and Plb1 demonstrated that these proteins are transported to the cell periphery via endosome-dependent and independent routes respectively, with both proteins enriched in bud necks. Phenotypic analysis of Δsec14 revealed cell growth and post-mitotic cell separation defects: Δsec14 cells were enlarged and formed multicellular aggregates containing nucleated cells connected by septa. Defective septum dissolution in Δsec14 correlates with reduced secretion of cell wall modifying enzymes. In summary we report the most comprehensive C. neoformans var grubii secretome to date and have demonstrated the importance of Sec14 in cell separation and secretion of proteins via the canonical route, particularly those involved in cell wall homeostasis.