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Defining the humoral immune response to infectious agents is important for gaining insights into infectious diseases and the response of the immune system. It can further aid development of serodiagnostic tests, discovery of vaccine antigen candidates, and immuno-epidemiological research. During the last three decades, serological proteome analyses (SERPAs) have played a significant role in characterizing the antibody response of humans or animals to fungal pathogens. SERPA combines 2D-gel electrophoresis with Western blotting. The introduction of multiplexing approaches by means of fluorescent dyes has greatly improved the reliability of the 2D technique and has boosted also the qualitative capabilities of the SERPA approach. In this chapter, we detail a SERPA protocol using fungal extracellular proteins from a fungal culture, here as an example the mold Aspergillus fumigatus.

Defining the humoral immune response to infectious agents is important for gaining insights into infectious diseases and the response of the immune system. It can further aid development of serodiagnostic tests, discovery of vaccine antigen candidates, and immuno-epidemiological research. During the last three decades, serological proteome analyses (SERPAs) have played a significant role in characterizing the antibody response of humans or animals to fungal pathogens. SERPA combines 2D-gel electrophoresis with Western blotting. The introduction of multiplexing approaches by means of fluorescent dyes has greatly improved the reliability of the 2D technique and has boosted also the qualitative capabilities of the SERPA approach. In this chapter, we detail a SERPA protocol using fungal extracellular proteins from a fungal culture, here as an example the mold Aspergillus fumigatus.

Aspergillus fumigatus is a human pathogen and the principal etiologic agent of invasive and chronic aspergillosis leading to several hundreds of thousands of deaths every year. Very few antifungals are available to treat infections caused by A. fumigatus, and resistance is developing to those we have. Our understanding of the molecular mechanisms that drive pathogenicity and drug resistance have been hampered by the lack of large mutant collections, which limits our ability to perform functional genomics analysis. Here we present a high-throughput gene knockout method that combines a highly reproducible fusion PCR method to enable generation of gene replacement cassettes with a multiwell format transformation procedure. This process can be used to generate 96 null mutants within 5 days by a single person at a cost of less than £18 ($24) per mutant and is being employed in our laboratory to generate a barcoded genome-wide knockout library in A. fumigatus. © 2019 The Authors.

Natural product discovery efforts have focused primarily on microbial biosynthetic gene clusters (BGCs) containing large multimodular polyketide synthases and nonribosomal peptide synthetases; however, sequencing of fungal genomes has revealed a vast number of BGCs containing smaller NRPS-like genes of unknown biosynthetic function. Using comparative metabolomics, we show that a BGC in the human pathogen Aspergillus fumigatus named fsq, which contains an NRPS-like gene lacking a condensation domain, produces several new isoquinoline alkaloids known as the fumisoquins. These compounds derive from carbon-carbon bond formation between two amino acid-derived moieties followed by a sequence that is directly analogous to isoquinoline alkaloid biosynthesis in plants. Fumisoquin biosynthesis requires the N-methyltransferase FsqC and the FAD-dependent oxidase FsqB, which represent functional analogs of coclaurine N-methyltransferase and berberine bridge enzyme in plants. Our results show that BGCs containing incomplete NRPS modules may reveal new biosynthetic paradigms and suggest that plant-like isoquinoline biosynthesis occurs in diverse fungi.

Systemic infections with the opportunistic mold Aspergillus fumigatus are a great threat to immunocompromised patients such as transplant recipients. Immunological research on A. fumigatus involves the measurement of phagocytosis of fungal conidia (spores) by human phagocytes. Here, we present a fast and flexible way to analyze phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC) labeling of conidia prior to co-incubation with human leukocytes and an anti-FITC counterstaining step postincubation to allow the discrimination of internalized and adherent conidia. In contrast to many other protocols, this method can be combined with further surface marker analyses. We sought to determine phagocytosis rates of A. fumigatus conidia in different stages and after several incubation times using this method. Moreover, we provide an example of application by comparing phagocytosis of A. fumigatus mutants to the wild type. © 2018 International Society for Advancement of Cytometry.

Summary Aspergillus fumigatus is an opportunistic human pathogenic fungus causing life-threatening infections in immunocompromised patients. Adaptation to different habitats and also virulence of the fungus depends on signal perception and transduction by modules such as the cyclic AMP-dependent protein kinase A (PKA) pathway. Here, by transcriptome analysis, 632 differentially regulated genes of this important signaling cascade were identified, including 23 putative transcriptional regulators. The highest upregulated transcription factor gene was located in a previously unknown secondary metabolite gene cluster, which we named fmp, encoding an incomplete non-ribosomal peptide synthetase, FmpE. Overexpression of the regulatory gene fmpR using the TetOn system led to the specific expression of the other six genes of the fmp cluster. Metabolic profiling of wild type and fmpR overexpressing strain by HPLC-DAD and HPLC-HRESI-MS and structure elucidation by NMR led to identification of 5-benzyl-1H-pyrrole-2-carboxylic acid, which we named fumipyrrole. Fumipyrrole was not described as natural product yet. Chemical synthesis of fumipyrrole confirmed its structure. Interestingly, deletion of fmpR or fmpE led to reduced growth and sporulation of the mutant strains. Although fmp cluster genes were transcribed in infected mouse lungs, deletion of fmpR resulted in wild-type virulence in a murine infection model.

Aspergillus fumigatus faces abrupt changes in oxygen concentrations at the site of infection. An increasing number of studies has demonstrated that elevated production of intracellular reactive oxygen species (ROS) under low oxygen conditions plays a regulatory role in modulating cellular responses for adaptation to hypoxia. To learn more about this process in A. fumigatus, intracellular ROS production during hypoxia has been determined. The results confirm increased amounts of intracellular ROS in A. fumigatus exposed to decreased oxygen levels. Moreover, nuclear accumulation of the major oxidative stress regulator AfYap1 is observed after low oxygen cultivation. For further analysis, iodoTMT labeling of redox-sensitive cysteine residues is applied to identify proteins that are reversibly oxidized. This analysis reveals that proteins with important roles in maintaining redox balance and protein folding, such as the thioredoxin Asp f 29 and the disulfide-isomerase PdiA, undergo substantial thiol modification under hypoxia. The data also show that the mitochondrial respiratory complex IV assembly protein Coa6 is significantly oxidized by hypoxic ROS. Deletion of the corresponding gene results in a complete absence of hypoxic growth, indicating the importance of complex IV during adaptation of A. fumigatus to oxygen-limiting conditions.

Fungi have the capability to produce a tremendous number of so-called secondary metabolites, which possess a multitude of functions, e.g., communication signals during coexistence with other microorganisms, virulence factors during pathogenic interactions with plants and animals, and in medical applications. Therefore, research on this topic has intensified significantly during the past 10 years and thus knowledge of regulatory mechanisms and the understanding of the role of secondary metabolites have drastically increased. This review aims to depict the complexity of all the regulatory elements involved in controlling the expression of secondary metabolite gene clusters, ranging from epigenetic control and signal transduction pathways to global and specific transcriptional regulators. Furthermore, we give a short overview on the role of secondary metabolites, focusing on the interaction with other microorganisms in the environment as well as on pathogenic relationships.

Aspergillus fumigatus is the most important airborne fungal pathogen, causing severe infections with invasive growth in immunocompromised patients. The fungal cell wall prevents the cell from lysing and protects the fungus against environmental stress conditions. Because it is absent in humans and because of its essentiality, the fungal cell wall is a promising target for antifungal drugs. Nowadays, compounds acting on the cell wall, i.e. echinocandin derivatives, are used to treat A. fumigatus infections. However, studies demonstrating the clinical effectiveness of echinocandins in comparison with antifungals currently recommended for first-line treatment of invasive aspergillosis are still lacking. Therefore, it is important to elucidate cell wall biosynthesis pathways and their signal transduction cascades, which potentially compensate the inhibition caused by cell wall- perturbing compounds. Like in other fungi, the central core of the cell wall integrity (CWI) signalling pathway in A. fumigatus is composed of three mitogen activated protein kinases. Deletion of these genes resulted in severely enhanced sensitivity of the mutants against cell wall-disturbing compounds and in drastic alterations of the fungal morphology. Additionally, several cross-talk interactions between the CWI pathways and other signalling pathways are emerging, raising the question about their role in the cell wall compensatory mechanisms. In this review we focused on recent advances in understanding the CWI signalling pathway in A. fumigatus and its role during drug stress response and virulence.

The opportunistic human pathogenic fungus Aspergillus fumigatus produces at least two types of melanin, namely pyomelanin and dihydroxynaphthalene (DHN) melanin. Pyomelanin is produced during tyrosine catabolism via accumulation of homogentisic acid. Although pyomelanin protects the fungus against reactive oxygen species (ROS) and acts as a defense compound in response to cell wall stress, mutants deficient for pyomelanin biosynthesis do not differ in virulence when tested in a murine infection model for invasive pulmonary aspergillosis. DHN melanin is responsible for the characteristic gray-greenish color of A. fumigatus conidia. Mutants lacking a functional polyketide synthase PksP, the enzyme responsible for the initial step in DHN-melanin formation, i.e., the synthesis of naphthopyrone, produce white spores and are attenuated in virulence. The activity of PksP was found to be essential not only for inhibition of apoptosis of phagocytes by interfering with the host PI3K/Akt signaling cascade but also for effective inhibition of acidification of conidia-containing phagolysosomes. These features allow A. fumigatus to survive in phagocytes and thereby to escape from human immune effector cells and to become a successful pathogen.