title: Secretome profiling of Cryptococcus neoformans reveals regulation of a subset of virulence-associated proteins and potential biomarkers by protein kinase A.
abstract: BACKGROUND
  The pathogenic yeast Cryptococcus neoformans causes life-threatening meningoencephalitis in individuals suffering from HIV/AIDS. The cyclic-AMP/protein kinase A (PKA) signal transduction pathway regulates the production of extracellular virulence factors in C. neoformans, but the influence of the pathway on the secretome has not been investigated. In this study, we performed quantitative proteomics using galactose-inducible and glucose-repressible expression of the PKA1 gene encoding the catalytic subunit of PKA to identify regulated proteins in the secretome.
  
  
  METHODS
  The proteins in the supernatants of cultures of C. neoformans were precipitated and identified using liquid chromatography-coupled tandem mass spectrometry. We also employed multiple reaction monitoring in a targeted approach to identify fungal proteins in samples from macrophages after phagocytosis of C. neoformans cells, as well as from the blood and bronchoalveolar fluid of infected mice.
  
  
  RESULTS
  We identified 61 secreted proteins and found that changes in PKA1 expression influenced the extracellular abundance of five proteins, including the Cig1 and Aph1 proteins with known roles in virulence. We also observed a change in the secretome profile upon induction of Pka1 from proteins primarily involved in catabolic and metabolic processes to an expanded set that included proteins for translational regulation and the response to stress. We further characterized the secretome data using enrichment analysis and by predicting conventional versus non-conventional secretion. Targeted proteomics of the Pka1-regulated proteins allowed us to identify the secreted proteins in lysates of phagocytic cells containing C. neoformans, and in samples from infected mice. This analysis also revealed that modulation of PKA1 expression influences the intracellular survival of cryptococcal cells upon phagocytosis.
  
  
  CONCLUSIONS
  Overall, we found that the cAMP/PKA pathway regulates specific components of the secretome including proteins that affect the virulence of C. neoformans. The detection of secreted cryptococcal proteins from infected phagocytic cells and tissue samples suggests their potential utility as biomarkers of infection. The proteomics data are available via ProteomeXchange with identifiers PXD002731 and PASS00736.
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada. jenngedd13@gmail.com.|Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada. jenngedd13@gmail.com.
authors.forename: Jennifer M H
authors.initials: JM
authors.lastname: Geddes
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada. daniel.croll@usys.ethz.ch.
authors.forename: Daniel
authors.initials: D
authors.lastname: Croll
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada. mcaza@msl.ubc.ca.
authors.forename: Mélissa
authors.initials: M
authors.lastname: Caza
authors.affiliation: Centre for High-Throughput Biology, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada. nstoynov@mail.ubc.ca.
authors.forename: Nikolay
authors.initials: N
authors.lastname: Stoynov
authors.affiliation: Centre for High-Throughput Biology, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada. foster@chibi.ubc.ca.
authors.forename: Leonard J
authors.initials: LJ
authors.lastname: Foster
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada. kronstad@msl.ubc.ca.|Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada. kronstad@msl.ubc.ca.
authors.forename: James W
authors.initials: JW
authors.lastname: Kronstad
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000032:analysis*; D011232:Chemical Precipitation; D002853:Chromatography, Liquid; D003455:Cryptococcus neoformans / Q000737:chemistry*; D003470:Culture Media / Q000737:chemistry; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005260:Female; D005656:Fungal Proteins / Q000378:metabolism*; D008264:Macrophages / Q000382:microbiology; D008807:Mice, Inbred BALB C; D020543:Proteome / Q000032:analysis*; D053719:Tandem Mass Spectrometry; D037521:Virulence Factors / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D003470:Culture Media; D005656:Fungal Proteins; D020543:Proteome; D037521:Virulence Factors; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases