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.
Background Fungal infections impact over 25% of the global population. For the opportunistic fungal pathogen, Cryptococcus neoformans, infection leads to cryptococcosis. In the presence of the host, disease is enabled by elaboration of sophisticated virulence determinants, including polysaccharide capsule, melanin, thermotolerance, and extracellular enzymes. Conversely, the host protects itself from fungal invasion by regulating and sequestering transition metals (e.g., iron, zinc, copper) important for microbial growth and survival. Results Here, we explore the intricate relationship between zinc availability and fungal virulence via mass spectrometry-based quantitative proteomics. We observe a core proteome along with a distinct zinc-regulated protein-level signature demonstrating a shift away from transport and ion binding under zinc-replete conditions towards transcription and metal acquisition under zinc-limited conditions. In addition, we revealed a novel connection among zinc availability, thermotolerance, as well as capsule and melanin production through the detection of a Wos2 ortholog in the secretome under replete conditions. Conclusions Overall, we provide new biological insight into cellular remodeling at the protein level of C. neoformans under regulated zinc conditions and uncover a novel connection between zinc homeostasis and fungal virulence determinants.
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and chromogenic media are widely used in clinical microbiology laboratories to facilitate the rapid selection and identification of pathogens. The aim of this study was to evaluate whether usage of chromogenic media limits the diagnostic performance of MALDI-TOF MS for microbial identification. A total of 386 microorganisms collected and analyzed at five laboratories were included. Isolates were cultured on relevant chromogenic media and non-selective agar plates in parallel and identified using the Bruker MALDI-TOF MS. Among the tested isolates, no misidentification was recorded and there was no medium-related difference in the identification level. However, score values were overall slightly but significantly lower for isolates grown on chromogenic media. In conclusion, the use of chromogenic culture media tested here had no relevant impact on MALDI-TOF MS performance for diagnostic purposes.
Based on newly discovered characters on the male genitalia, external morphology and an accumulation of ecological data, we revise the single member of the genus Hemiquedius. Two new species, H. infinitus Brunke & Smetana, sp. n. and H. castoris Brunke & Smetana, sp. n., from eastern North America are described, and H. ferox (LeConte), restricted to peninsular Florida, is re-described. Hemiquedius castoris is strongly associated with the microhabitats provided by nest materials of the North American beaver and muskrat. A key to the three species of Hemiquedius is provided and diagnostic characters are illustrated. We also review the beetles known to be obligate associates of beavers and muskrats, and discuss the potential role of these keystone vertebrates in beetle evolution and distribution. Based on nest-associated beetles and their closest living relatives, beaver and muskrat lodges may extend distributions northward by moderating winters, promote sympatric speciation and act as refugia against extinction of lineages on a broader timescale. Further research into these potential impacts by ecologists and evolutionary biologists is encouraged.
Fungal pathogens are critically important threats to global health with over 300 million people affected by serious fungal diseases worldwide. Fungal pathogens have evolved sophisticated strategies, including the secretion of virulence factors to interfere with host cell functions and to perturb immune responses. Our ‘infectome’ analysis identifies previously undescribed proteins involved in fungal virulence and host immune response, representing an opportunity to elucidate molecular mechanisms of host‐pathogen interplay during disease. Using state‐of‐the‐art mass spectrometry‐based proteomics we profile the total proteome and secretome of Cryptococcus neoformans wild‐type (H99) under in vitro growth conditions. We also define the infectome of C. neoformans and BALB/c macrophages in single runs using high resolution mass spectrometry on a Quadrupole Orbitrap instrument. The in vitro and infectome datasets were integrated using Perseus and candidate fungal proteins of interest were prioritized based on predicted secretory roles, novelty, and abundance profiles. Deletion strains were constructed by double‐joint PCR and characterized by phenotypic screening and cell death assays. Virulence‐associated candidates will be evaluated in a murine infection model and further characterized by immunofluorescence and interactome analyses. Our preliminary results identify interactions between the host and pathogen critical for disease. Comprehensive profiling of infection from both host and pathogen perspectives unveils new anti‐virulence strategies to combat fungal infection.
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