Quantitative Proteomic Profiling of Cryptococcus neoformans

Abstract

Cryptococcus neoformans is an opportunistic human fungal pathogen commonly associated with infection in immunocompromised individuals (e.g., patients with HIV/AIDS). Important virulence determinants include the production of a polysaccharide capsule, melanin, and extracellular enzymes, as well as the ability to grow at 37°C. C. neoformans controls a plethora of host defense and evasion mechanisms to survive during infection and to proliferate within the host, causing meningoencephalitis and death. Traditionally, characterization of C. neoformans under different environmental conditions and stresses has relied on genetic and phenotypic analyses, as well as biochemical assays. However, advances in mass spectrometry instrumentation, sample preparation protocols, and bioinformatic tools and databases promote comprehensive profiling of fungal cellular processes, secretion or protein release into the extracellular environment, and vesicle contents. Moreover, proteomics provides insight into regulatory mechanisms influencing signal transduction cascades and protein complexes or networks through profiling of post‐translational modifications and protein–protein interactions. Given the medical impact of C. neoformans infections and the recent emergence of antifungal‐resistant strains, defining proteins produced in response to unique environments provides an opportunity to uncover antivirulence strategies and alternative therapeutic options to combat infection. Here, we describe culturing and sample preparation of C. neoformans and outline protocols for comprehensively profiling changes in protein abundance within the cellular proteome and secretome. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Growth and sample preparation of Cryptococcus neoformans Basic Protocol 2: Protein extraction from supernatant Basic Protocol 3: Protein extraction from cell pellet Basic Protocol 4: Proteomic profiling and bioinformatics

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“…Cellular proteome samples were processed in quadruplicate per condition and subjected to a modified total proteome extraction protocol (Ball and Geddes-McAlister, 2019). Briefly, samples were washed twice with phosphate-buffered saline (PBS) and resuspended in 100 mM Tris-HCl (pH 8.5) containing a protease inhibitor cocktail tablet.…”

Proteome and secretome profiling of zinc availability in Cryptococcus neoformans identifies Wos2 as a subtle influencer of fungal virulence determinants

“…Sample preparation for mass spectrometry was performed as previously described [ 37 ]. Briefly, cell pellets were resuspended in 100 mM Tris-HCl (pH 8.5) and lysed using a probe sonicator (Thermo Fisher Scientific).…”

“…1 Overview of experimental design and mass spectrometry workflow. Cell pellets and supernatant from C. neoformans cultured in minimal medium (MM) MM + Zn or MM-Zn were subjected to protein extraction (e.g., sonication and detergent) and enzyme digestion (e.g., trypsin/Lys-C) followed by purification (C18 STAGE-tips) and measurement on a quadrupole orbitrap mass spectrometer [ 37 – 40 ]. Data analysis and visualization performed with MaxQuant and Perseus.…”

Dual perspective proteomics infectome profiling discoversSalmonellatype III secretion system effector functions in macrophages

“…Total proteome, secretome, and infectome profiling was performed as previously described with minor modifications [32, 33]. Cell pellets (bacterial and macrophage) were collected and washed with PBS, resuspended in 100 mM Tris-HCl, pH 8.5 with proteinase inhibitor capsule (PIC) and mechanically disrupted by probe sonication in an ice bath.…”