Biology subjects

Rivieccio, F.

Publications and source records attributed to Rivieccio, F..

4 recordsLinked to original sources

bioRxiv2022

Functional characterization of the RNA interference pathway in A. fumigatus reveals its potential for antifungal therapy

The RNA interference (RNAi) pathway has evolved numerous functionalities in eukaryotes, with many on display in Kingdom Fungi. RNAi can regulate gene expression, facilitate drug resistance, or even be altogether lost to improve growth potential in some fungal pathogens. In the WHO fungal priority pathogen, Aspergillus fumigatus, the RNAi system is known to be intact and functional. To extend our limited understanding of A. fumigatus RNAi, we first investigated the genetic variation in RNAi-associated genes in a collection of 217 environmental and 83 clinical genomes, where we found that RNAi components are conserved even in clinical strains. Using endogenously expressed inverted-repeat transgenes complementary to a conditionally essential gene (pabA) or a nonessential gene (pksP), we determined that a subset of the RNAi componentry is active in inverted-repeat transgene silencing in conidia and mycelium. Analysis of mRNA-seq data from RNAi double-knockout strains linked the A. fumigatus dicer-like enzymes (DclA/B) and RNA-dependent RNA polymerases (RrpA/B) to regulation of conidial ribosome biogenesis genes; however, surprisingly few endogenous small RNAs were identified in conidia that could explain this broad change. Although RNAi was not clearly linked to growth or stress response defects in the RNAi knockouts, serial passaging of RNAi knockout strains for six generations resulted in lineages with diminished spore production over time, indicating that loss of RNAi can exert a fitness cost on the fungus. Cumulatively, A. fumigatus RNAi appears to play an active role in defense against double-stranded RNA species alongside a previously unappreciated housekeeping function in regulation of conidial ribosomal biogenesis genes.

microbiology↗

bioRxiv2021

PLB-985 neutrophil-like cells release antifungal extracellular vesicles against the human-pathogenic fungus Aspergillus fumigatus

Fungal infections remain a major global concern. Emerging fungal pathogens and increasing rates of resistance mean that additional research efforts and resources must be allocated to advancing our understanding of fungal pathogenesis and developing new therapeutic interventions. Neutrophilic granulocytes are a major cell type involved in protection against the important fungal pathogen Aspergillus fumigatus, where they employ numerous defense mechanisms, including production of antimicrobial extracellular vesicles. A major draw-back to work with neutrophils is the lack of a suitable cell line system for the study of fungal pathogenesis. To address this problem, we assessed the feasibility of using differentiated PLB-985 neutrophil-like cells as an in vitro model to study A. fumigatus infection. We find that dimethylformamide-differentiated PLB-985 cells provide a useful recapitulation of many aspects of A. fumigatus interactions with primary human polymorphonuclear leukocytes. We show that differentiated PLB-985 cells phagocytose fungal conidia and acidify conidia-containing phagolysosomes similar to primary neutrophils, release neutrophil extracellular traps, and also produce antifungal extracellular vesicles in response to infection. In addition, we provide an improved method for the isolation of extracellular vesicles produced during infection by employing a size-exclusion chromatography-based approach. Advanced LC-MS/MS proteomics revealed an enrichment of extracellular vesicle marker proteins and a decrease of cytoplasmic proteins in extracellular vesicles isolated using this improved method. Ultimately, we find that differentiated PLB-985 cells can serve as a genetically tractable model to study many aspects of A. fumigatus pathogenesis. IMPORTANCEPolymorphonuclear leukocytes are an important defense against human fungal pathogens, yet our model systems to study this group of cells remains very limited in scope. In this study, we established that differentiated PLB-985 cells can serve as a model to recapitulate several important aspects of human polymorphonuclear leukocyte interactions with the important human fungal pathogen Aspergillus fumigatus. The proposed addition of a cultured neutrophil-like cell line to the experimental toolbox to study fungal pathogenesis will allow for a more mechanistic description of neutrophil antifungal biology. In addition, the easier handling of the cell line compared to primary human neutrophils allowed us to use PLB-985 cells to provide an improved method for isolation of neutrophil-derived extracellular vesicles using size-exclusion chromatography. Together, these results provide significant tools and a baseline knowledge for the future study of neutrophil-derived extracellular vesicles in the laboratory.

microbiology↗

bioRxiv2021

Dynamic optimization reveals alveolar epithelial cells as key mediators of host defense in invasive aspergillosis

Aspergillus fumigatus is an important human fungal pathogen and its conidia are constantly inhaled by humans. In immunocompromised individuals, conidia can grow out as hyphae that damage lung epithelium. The resulting invasive aspergillosis is associated with devastating mortality rates. Since infection is a race between the innate immune system and the outgrowth of A. fumigatus conidia, we use dynamic optimization to obtain insight into the recruitment and depletion of alveolar macrophages and neutrophils. Using this model, we obtain key insights into major determinants of infection outcome on host and pathogen side. On the pathogen side, we predict in silico and confirm in vitro that germination speed is a key virulence trait of fungal pathogens due to the vulnerability of conidia against host defense. On the host side, we find that epithelial cells play a so far underappreciated role in fungal clearance and are potent mediators of cytokine release which we confirm ex vivo. Further, our model affirms the importance of neutrophils in invasive aspergillosis and underlines that the role of macrophages remains elusive. We expect that our model will contribute to improvement of treatment protocols by focusing on the critical components of immune response to fungi but also fungal virulence.

systems biology↗

bioRxiv2020

The dynamic surface proteomes of allergenic fungal conidia

Fungal spores and hyphal fragments play an important role as allergens in respiratory diseases. In this study, we performed trypsin shaving and secretome analyses to identify the surface-exposed proteins and secreted/shed proteins of Aspergillus fumigatus conidia, respectively. We investigated the surface proteome under different conditions, including temperature variation and germination. We found that the surface proteome of resting A. fumigatus conidia is not static, but instead unexpectedly dynamic, as evidenced by drastically different surface proteomes under different growth conditions. Knockouts of two abundant A. fumigatus surface proteins, ScwA and CweA, were found to function only in fine-tuning the cell wall stress response, implying that the conidial surface is very robust against perturbations. We then compared the surface proteome of A. fumigatus to other allergy-inducing molds, including Alternaria alternata, Penicillium rubens, and Cladosporium herbarum, and performed comparative proteomics on resting and swollen conidia, as well as secreted proteins from germinating conidia. We detected 125 protein ortholog groups, including 80 with putative catalytic activity, in the extracellular region of all four molds, and 42 nonorthologous proteins produced solely by A. fumigatus. Ultimately, this study highlights the dynamic nature of the A. fumigatus conidial surface and provides targets for future diagnostics and immunotherapy.

microbiology↗