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.

Cellular Tracking and Gene Profiling of Fusarium graminearum during Maize Stalk Rot Disease Development Elucidates Its Strategies in Confronting Phosphorus Limitation in the Host Apoplast.

The ascomycete fungus Fusarium graminearum causes stalk rot in maize. We tracked this pathogen's growth in wound-inoculated maize stalks using a fluorescence-labeled fungal isolate and observed that invasive hyphae grew intercellularly up to 24 h post inoculation, grew intra- and inter-cellularly between 36-48 h, and fully occupied invaded cells after 72 h. Using laser microdissection and microarray analysis, we profiled changes in global gene expression during pathogen growth inside pith tissues of maize stalk from 12 h to six days after inoculation and documented transcriptomic patterns that provide further insights into the infection process. Expression changes in transcripts encoding various plant cell wall degrading enzymes appeared to correlate with inter- and intracellular hyphal growth. Genes associated with 36 secondary metabolite biosynthesis clusters were expressed. Expression of several F. graminearum genes potentially involved in mobilization of the storage lipid triacylglycerol and phosphorus-free lipid biosynthesis were induced during early infection time points, and deletion of these genes caused reduction of virulence in maize stalk. Furthermore, we demonstrated that the F. graminearum betaine lipid synthase 1 (BTA1) gene was necessary and sufficient for production of phosphorus-free membrane lipids, and that deletion of BTA1 interfered with F. graminearum's ability to advance intercellularly. We conclude that F. graminearum produces phosphorus-free membrane lipids to adapt to a phosphate-limited extracellular microenvironment during early stages of its invasion of maize stalk.

In planta stage-specific fungal gene profiling elucidates the molecular strategies of Fusarium graminearum growing inside wheat coleoptiles.

The ascomycete Fusarium graminearum is a destructive fungal pathogen of wheat (Triticum aestivum). To better understand how this pathogen proliferates within the host plant, we tracked pathogen growth inside wheat coleoptiles and then examined pathogen gene expression inside wheat coleoptiles at 16, 40, and 64 h after inoculation (HAI) using laser capture microdissection and microarray analysis. We identified 344 genes that were preferentially expressed during invasive growth in planta. Gene expression profiles for 134 putative plant cell wall-degrading enzyme genes suggest that there was limited cell wall degradation at 16 HAI and extensive degradation at 64 HAI. Expression profiles for genes encoding reactive oxygen species (ROS)-related enzymes suggest that F. graminearum primarily scavenges extracellular ROS before a later burst of extracellular ROS is produced by F. graminearum enzymes. Expression patterns of genes involved in primary metabolic pathways suggest that F. graminearum relies on the glyoxylate cycle at an early stage of plant infection. A secondary metabolite biosynthesis gene cluster was specifically induced at 64 HAI and was required for virulence. Our results indicate that F. graminearum initiates infection of coleoptiles using covert penetration strategies and switches to overt cellular destruction of tissues at an advanced stage of infection.

Aspergillus fumigatus hijacks human p11 to redirect fungal-containing phagosomes to non-degradative pathway.

The decision whether endosomes enter the degradative or recycling pathway in mammalian cells is of fundamental importance for pathogen killing, and its malfunctioning has pathological consequences. We discovered that human p11 is a critical factor for this decision. The HscA protein present on the conidial surface of the human-pathogenic fungus Aspergillus fumigatus anchors p11 on conidia-containing phagosomes (PSs), excludes the PS maturation mediator Rab7, and triggers binding of exocytosis mediators Rab11 and Sec15. This reprogramming redirects PSs to the non-degradative pathway, allowing A. fumigatus to escape cells by outgrowth and expulsion as well as transfer of conidia between cells. The clinical relevance is supported by the identification of a single nucleotide polymorphism in the non-coding region of the S100A10 (p11) gene that affects mRNA and protein expression in response to A. fumigatus and is associated with protection against invasive pulmonary aspergillosis. These findings reveal the role of p11 in mediating fungal PS evasion.

Antigen specificity and cross-reactivity drive functionally diverse anti-Aspergillus fumigatus T cell responses in cystic fibrosis.

BACKGROUNDThe fungus Aspergillus fumigatus causes a variety of clinical phenotypes in patients with cystic fibrosis (pwCF). Th cells orchestrate immune responses against fungi, but the types of A. fumigatus-specific Th cells in pwCF and their contribution to protective immunity or inflammation remain poorly characterized.METHODSWe used antigen-reactive T cell enrichment (ARTE) to investigate fungus-reactive Th cells in peripheral blood of pwCF and healthy controls.RESULTSWe show that clonally expanded, high-avidity A. fumigatus-specific effector Th cells, which were absent in healthy donors, developed in pwCF. Individual patients were characterized by distinct Th1-, Th2-, or Th17-dominated responses that remained stable over several years. These different Th subsets target different A. fumigatus proteins, indicating that differential antigen uptake and presentation directs Th cell subset development. Patients with allergic bronchopulmonary aspergillosis (ABPA) are characterized by high frequencies of Th2 cells that cross-recognize various filamentous fungi.CONCLUSIONOur data highlight the development of heterogenous Th responses targeting different protein fractions of a single fungal pathogen and identify the development of multispecies cross-reactive Th2 cells as a potential risk factor for ABPA.FUNDINGGerman Research Foundation (DFG), under Germany's Excellence Strategy (EXC 2167-390884018 "Precision Medicine in Chronic Inflammation" and EXC 2051-390713860 "Balance of the Microverse"); Oskar Helene Heim Stiftung; Christiane Herzog Stiftung; Mukoviszidose Institut gGmb; German Cystic Fibrosis Association Mukoviszidose e.V; German Federal Ministry of Education and Science (BMBF) InfectControl 2020 Projects AnDiPath (BMBF 03ZZ0838A+B).

Biotinylated Surfome Profiling Identifies Potential Biomarkers for Diagnosis and Therapy of Aspergillus fumigatus Infection.

Aspergillus fumigatus is one of the most common airborne molds capable of causing mycoses and allergies in humans. During infection, fungal surface proteins mediate the first contact with the human immune system to evade immune responses or to induce hypersensitivity. Several methods have been established for surface proteomics (surfomics). Biotinylation coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of peptides is a particularly efficient method to identify the surface-exposed regions of proteins that potentially mediate interaction with the host. After biotinylation of surface proteins during spore germination, we detected 231 different biotinylated surface proteins (including several well-known proteins such as RodA, CcpA, and DppV; allergens; and heat shock proteins [HSPs]), as well as some previously undescribed surface proteins. The dynamic change of the surface proteome was illustrated by detection of a relatively high number of proteins exclusively at one developmental stage. Using immunofluorescence microscopy, we confirmed the surface localization of several HSPs of the HSP70 family, which may have moonlighting functions. Collectively, by comparing our data with data representative of previously published A. fumigatus surface proteomes, our study generated a comprehensive data set corresponding to the A. fumigatus surfome and uncovered the surface-exposed regions of many proteins on the surface of conidia or hyphae. These surface-exposed regions are candidates for direct interaction with host cells and may represent antigenic epitopes that either induce protective immune responses or mediate immune evasion. Thus, our data sets provided and compiled here represent reasonable immunotherapy and diagnostic targets for future investigations.IMPORTANCEAspergillus fumigatus is the most important airborne human-pathogenic mold, capable of causing both life-threatening invasive pulmonary aspergillosis in immunocompromised patients and allergy-inducing infections in individuals with atopic allergy. Despite its obvious medical relevance, timely diagnosis and efficient antifungal treatment of A. fumigatus infection remain major challenges. Proteins on the surface of conidia (asexually produced spores) and mycelium directly mediate host-pathogen interaction and also may serve as targets for diagnosis and immunotherapy. However, the similarity of protein sequences between A. fumigatus and other organisms, sometimes even including the human host, makes selection of targets for immunological-based studies difficult. Here, using surface protein biotinylation coupled with LC-MS/MS analysis, we identified hundreds of A. fumigatus surface proteins with exposed regions, further defining putative targets for possible diagnostic and immunotherapeutic design.

A linear nonribosomal octapeptide from Fusarium graminearum facilitates cell-to-cell invasion of wheat.

Fusarium graminearum is a destructive wheat pathogen. No fully resistant cultivars are available. Knowledge concerning the molecular weapons of F. graminearum to achieve infection remains limited. Here, we report that deletion of the putative secondary metabolite biosynthesis gene cluster fg3_54 compromises the pathogen's ability to infect wheat through cell-to-cell penetration. Ectopic expression of fgm4, a pathway-specific bANK-like regulatory gene, activates the transcription of the fg3_54 cluster in vitro. We identify a linear, C- terminally reduced and D-amino acid residue-rich octapeptide, fusaoctaxin A, as the product of the two nonribosomal peptide synthetases encoded by fg3_54. Chemically-synthesized fusaoctaxin A restores cell-to-cell invasiveness in fg3_54-deleted F. graminearum, and enables colonization of wheat coleoptiles by two Fusarium strains that lack the fg3_54 homolog and are nonpathogenic to wheat. In conclusion, our results identify fusaoctaxin A as a virulence factor required for cell-to-cell invasion of wheat by F. graminearum.

Wheat Coleoptile Inoculation by Fusarium graminearum for Large-scale Phenotypic Analysis.

The ascomycete fungus Fusarium graminearum is a destructive fungal pathogen of wheat, barley and maize. Although reverse genetics and homologous recombination gene deletion methods have generated thousands of gene deletion mutants of F. graminearum, evaluating virulence of these fungal mutants is still a rate-limiting step. Here we present a protocol for inoculation of wheat coleoptiles with conidial suspensions for large-scale phenotypic analysis, and describe how it can also be used to assess fungal infectious growth and symptom developmentat a cellular scale. The inoculation method described in this protocol provides highly reproducible results in wheat coleoptile infection by F. graminearum.