label-free-quantitative-proteomics-workflow-for-discovery-driven-host

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s ...

University of Guelph

The prevalence of fungal diseases is increasing on a global scale, ranging from acute to systemic infections caused by commensal or pathogenic microorganisms, often associated with the immune status of the host. Morbidity and mortality rates remain high and our ability to treat fungal infections is challenged by a limited arsenal of antifungal agents and the emergence of drug resistant pathogens. There is a high demand for new approaches to elucidate the fungal mechanisms of pathogenesis and the interplay between host and pathogen to discover novel treatment options. Moreover, the need for improved drug efficacy and reduced host toxicity requires the identification and characterization of antifungal biological targets and molecular mechanisms of action. Mass spectrometry (MS)-based proteomics is a rapidly advancing field capable of addressing these priorities by providing comprehensive information on the dynamics of cellular processes, modifications, and interactions. In this Review, we focus on applications of MS-based proteomics in a diverse array of fungal pathogens and host systems to define and distinguish the molecular details of fungal pathogenesis and host-fungal interactions. We also explore the emerging role of MS-based proteomics in the discovery and development of novel antifungal therapies and provide insight into the future of MS-based proteomics in fungal biology.

Mass Spectrometry-Based Proteomics of Fungal Pathogenesis, Host-Fungal Interactions, and Antifungal Development.

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.

Brianna Ball

Molecular and Cellular Biology Department

University of Guelph

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

Brianna Ball

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Molecular and Cellular Biology Department

University of Guelph

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

Molecular and Cellular Biology Department