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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.

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

Small and medium sized food businesses have been slow to adopt food safety management systems (FSMSs) such as good manufacturing practices and Hazard Analysis Critical Control Point (HACCP). This study identifies factors influencing workers in their implementation of food safety practices in small and medium meat processing establishments in Ontario, Canada. A qualitative approach was used to explore in-plant factors that influence the implementation of FSMSs. Thirteen in-depth interviews in five meat plants and two focus group interviews were conducted. These generated 219 pages of verbatim transcripts which were analysed using NVivo 7 software. Main themes identified in the data related to production systems, organisational characteristics and employee characteristics. A socio-psychological model based on the theory of planned behaviour is proposed to describe how these themes and underlying sub-themes relate to FSMS implementation. Addressing the various factors that influence production workers is expected to enhance FSMS implementation and increase food safety.

The landscape of infectious fungal agents includes previously unidentified or rare pathogens with the potential to cause unprecedented casualties in biodiversity, food security, and human health. The influences of human activity, including the crisis of climate change, along with globalized transport, are underlying factors shaping fungal adaptation to increased temperature and expanded geographical regions. Furthermore, the emergence of novel antifungal-resistant strains linked to excessive use of antifungals (in the clinic) and fungicides (in the field) offers an additional challenge to protect major crop staples and control dangerous fungal outbreaks. Hence, the alarming frequency of fungal infections in medical and agricultural settings requires effective research to understand the virulent nature of fungal pathogens and improve the outcome of infection in susceptible hosts. Mycology-driven research has benefited from a contemporary and unified approach of omics technology, deepening the biological, biochemical, and biophysical understanding of these emerging fungal pathogens. Here, we review the current state-of-the-art multi-omics technologies, explore the power of data integration strategies, and highlight discovery-based revelations of globally important and taxonomically diverse fungal pathogens. This information provides new insight for emerging pathogens through an in-depth understanding of well-characterized fungi and provides alternative therapeutic strategies defined through novel findings of virulence, adaptation, and resistance.

Escherichia coli represents a classical intestinal gram-negative commensal. Despite this commensalism, different E . coli strains can mediate disparate immunogenic properties in a given host. Symbiotic E . coli strains such as E . coli Nissle 1917 (EcN) are attributed beneficial properties, e.g., promotion of intestinal homeostasis. Therefore, we aimed to identify molecular features derived from symbiotic bacteria that might help to develop innovative therapeutic alternatives for the treatment of intestinal immune disorders. This study was performed using the dextran sodium sulphate (DSS)-induced colitis mouse model, which is routinely used to evaluate potential therapeutics for the treatment of Inflammatory Bowel Diseases (IBDs). We focused on the analysis of flagellin structures of different E . coli strains. EcN flagellin was found to harbor a substantially longer hypervariable region (HVR) compared to other commensal E . coli strains, and this longer HVR mediated symbiotic properties through stronger activation of Toll-like receptor (TLR)5, thereby resulting in interleukin (IL)-22–mediated protection of mice against DSS-induced colitis. Furthermore, using bone-marrow–chimeric mice (BMCM), CD11c+ cells of the colonic lamina propria (LP) were identified as the main mediators of these flagellin-induced symbiotic effects. We propose flagellin from symbiotic E . coli strains as a potential therapeutic to restore intestinal immune homeostasis, e.g., for the treatment of IBD patients.

Post-translational modifications (PTMs) change the structure and function of proteins and regulate a diverse array of biological processes. Fungal pathogens rely on PTMs to modulate protein production and activity during infection, manipulate the host response, and ultimately, promote fungal survival. Given the high mortality rates of fungal infections on a global scale, along with the emergence of antifungal-resistant species, identifying new treatment options is critical. In this review, we focus on the role of PTMs (e.g., phosphorylation, acetylation, ubiquitination, glycosylation, and methylation) among the highly prevalent and medically relevant fungal pathogens, Candida spp., Aspergillus spp., and Cryptococcus spp. We explore the role of PTMs in fungal stress response and host adaptation, the use of PTMs to manipulate host cells and the immune system upon fungal invasion, and the importance of PTMs in conferring antifungal resistance. We also provide a critical view on the current knowledgebase, pose questions key to our understanding of the intricate roles of PTMs within fungal pathogens, and provide research opportunities to uncover new therapeutic strategies.