Secretome profiling of Cryptococcus neoformans reveals regulation of a subset of virulence-associated proteins and potential biomarkers by protein kinase AReport as inadecuate

Secretome profiling of Cryptococcus neoformans reveals regulation of a subset of virulence-associated proteins and potential biomarkers by protein kinase A - Download this document for free, or read online. Document in PDF available to download.

BMC Microbiology

, 15:206

Microbe-host interactions and microbial pathogenicity


BackgroundThe 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.

MethodsThe 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.

ResultsWe 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.

ConclusionsOverall, 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.

KeywordsQuantitative proteomics Fungal pathogenesis Secretome PKA Virulence factors Biomarkers Multiple reaction monitoring AbbreviationsAIDSAcquired immune deficiency syndrome

BALBronchoalveolar lavage

cAMPcyclic-adenosine monophosphate

CIDCollision induced dissociation

CFUColony forming units


DMEMDulbecco’s modified eagle medium

FunSecKBFungal Secretome KnowledgeBase

GOGene ontology

GPCRG-protein coupled receptor


HCDHigher-energy collision dissociation

HIVHuman immunodeficiency virus

hpiHours post inoculation

HPLCHigh performance liquid chromatography

MM D-GMinimal media glucose-galactose

MRMMultiple reaction monitoring


PBSPhosphate buffered saline

PKAProtein kinase A

PMAPhorbol myristate acetate

qRT-PCRQuantitative real time – polymerase chain reaction

SIDStable isotope dilutions

STAGEStop and go extraction


YPDYeast peptone dextrose

Electronic supplementary materialThe online version of this article doi:10.1186-s12866-015-0532-3 contains supplementary material, which is available to authorized users.

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Author: Jennifer M. H. Geddes - Daniel Croll - Mélissa Caza - Nikolay Stoynov - Leonard J. Foster - James W. Kronstad


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