Species-Specific Differences in the Susceptibility of Fungi to the Antifungal Protein AFP Depend on C-3 Saturation of Glycosylceramides

AFP is an antimicrobial peptide (AMP) produced by the filamentous fungus Aspergillus giganteus and is a very potent inhibitor of fungal growth that does not affect the viability of bacteria, plant, or mammalian cells. It targets chitin synthesis and causes plasma membrane permeabilization in many human- and plant-pathogenic fungi, but its exact mode of action is not known. After adoption of the “damage-response framework of microbial pathogenesis” regarding the analysis of interactions between AMPs and microorganisms, we have recently proposed that the cytotoxic capacity of a given AMP depends not only on the presence/absence of its target(s) in the host and the AMP concentration applied but also on other variables, such as microbial survival strategies. We show here using the examples of three filamentous fungi (Aspergillus niger, Aspergillus fumigatus, and Fusarium graminearum) and two yeasts (Saccharomyces cerevisiae and Pichia pastoris) that the important parameters defining the AFP susceptibilities of these fungi are (i) the presence/absence of glycosylceramides, (ii) the presence/absence of Δ3(E) desaturation of the fatty acid chain therein, and (iii) the (dis)ability of these fungi to respond to AFP inhibitory effects with the fortification of their cell walls via increased chitin and β-(1,3)-glucan synthesis. These observations support the idea of the adoption of the damage-response framework to holistically understand the outcome of AFP inhibitory effects.TU Berlin, Open-Access-Mittel - 201

Updating genome annotation for the microbial cell factory Aspergillus niger using gene co-expression networks

A significant challenge in our understanding of biological systems is the high number of genes with unknown function in many genomes. The fungal genus Aspergillus contains important pathogens of humans, model organisms, and microbial cell factories. Aspergillus niger is used to produce organic acids, proteins, and is a promising source of new bioactive secondary metabolites. Out of the 14,165 open reading frames predicted in the A. niger genome only 2% have been experimentally verified and over 6,000 are hypothetical. Here, we show that gene co-expression network analysis can be used to overcome this limitation. A meta-analysis of 155 transcriptomics experiments generated co-expression networks for 9,579 genes (∼65%) of the A. niger genome. By populating this dataset with over 1,200 gene functional experiments from the genus Aspergillus and performing gene ontology enrichment, we could infer biological processes for 9,263 of A. niger genes, including 2,970 hypothetical genes. Experimental validation of selected co-expression sub-networks uncovered four transcription factors involved in secondary metabolite synthesis, which were used to activate production of multiple natural products. This study constitutes a significant step towards systems-level understanding of A. niger, and the datasets can be used to fuel discoveries of model systems, fungal pathogens, and biotechnology.DFG, 325093850, Open Access Publizieren 2017 - 2018 / Technische Universität BerlinEC/FP7/607332/EU/Quantitative Biology for Fungal Secondary Metabolite Producers/QuantFun

A Computational Modeling Approach Predicts Interaction of the Antifungal Protein AFP from Aspergillus giganteus with Fungal Membranes via Its γ-Core Motif

This work is licensed under a Creative Commons Attribution 4.0 International License.Fungal pathogens kill more people per year globally than malaria or tuberculosis and threaten international food security through crop destruction. New sophisticated strategies to inhibit fungal growth are thus urgently needed. Among the potential candidate molecules that strongly inhibit fungal spore germination are small cationic, cysteine-stabilized proteins of the AFP family secreted by a group of filamentous Ascomycetes. Its founding member, AFP from Aspergillus giganteus, is of particular interest since it selectively inhibits the growth of filamentous fungi without affecting the viability of mammalian, plant, or bacterial cells. AFPs are also characterized by their high efficacy and stability. Thus, AFP can serve as a lead compound for the development of novel antifungals. Notably, all members of the AFP family comprise a γ-core motif which is conserved in all antimicrobial proteins from pro- and eukaryotes and known to interfere with the integrity of cytoplasmic plasma membranes. In this study, we used classical molecular dynamics simulations combined with wet laboratory experiments and nuclear magnetic resonance (NMR) spectroscopy to characterize the structure and dynamical behavior of AFP isomers in solution and their interaction with fungal model membranes. We demonstrate that the γ-core motif of structurally conserved AFP is the key for its membrane interaction, thus verifying for the first time that the conserved γ-core motif of antimicrobial proteins is directly involved in protein-membrane interactions. Furthermore, molecular dynamic simulations suggested that AFP does not destroy the fungal membrane by pore formation but covers its surface in a well-defined manner, using a multistep mechanism to destroy the membranes integrity.NIH GM31749NIH GM103426Deutsche Forschungsgemeinschaft (Cluster of Excellence ‘Unifying Concepts in Catalysis’ and SFB1078

Production and characterization of antifungal peptides from the AFP family of Ascomycota

Die sehr variable und große Gruppe der antimikrobiellen Peptide, von der mehr als 10.000 Vertreter in der Datenbank „Collection of anti-microbial peptids“ CAMPR3 [1] gelistet sind, steht schon lange als potenzielle Basis für Wirkstoffe im Fokus der Forschung. Es gibt auf Grund steigender Zahlen von Pilzinfektionen und Resistenzen gegen antimykotische Wirkstoffe ein großes Bedürfnis diese Quelle zu erschließen. Die Mitglieder der AFP-Familie sind auf Grund ihrer Spezies-Spezifität, besonders auch für bekannte human- und pflanzenpathogene Pilze mit hohem Bedrohungspotential, ohne bekannte Nebenwirkungen von Interesse. In dieser Arbeit wurden daher Grundlagen zur Etablierung des antimykotischen Proteins AFP aus A. giganteus ausgearbeitet. Dazu gehören (i) verbesserte Methoden zur Herstellung und Charakterisierung, (ii) die Charakterisierung des Wirk-Mechanismus und (iii) in silico Analysen zur Identifizierung der Mitglieder der AFP-Familie. Die Etablierung der Bioreaktorkultivierung, mit standardisierter Ausbeute von 10 mg/L Kulturmedium des AFPs, bildet den Anfang der Arbeit. Vom nativen Produzenten A. giganteus werden mehrere AFP-Varianten sekretiert, welche schwer voreinander zu trennen sind. Aus NMR Versuchen ergab sich, dass durch die sequenzielle Prozession zusätzlich zur längeren AFP Variante „lf“-AFP eine kürzere Variante, das „sf“-AFP (-Alanin), gebildet wird. Die heterologe Expression in P. pastoris ist daher der geeignetste Ansatz für die AFP Produktion, die homologe Herstellung in A. giganteus ermöglicht die Funktion anderer AFP-Varianten aufzuklären. Im zweiten Teil der Arbeit wurde der Einfluss der C3-Sättigung von Glycosylceramiden auf die Wirksamkeit des AFPs untersucht. Die für Pilze eigenen Strukturmerkmale dieser Membranbestandteile können das spezifische Wirkspektrum des AFPs erklären. Es zeigte sich, dass die 2-Hydroxy Fettsäure N-Acyl-Δ3(E)-Desaturase für die Bildung der Doppelbindung zwischen dem dritten und vierten Kohlenstoffatom des Fettsäurerestes verantwortlich ist. Die Deletion dieses Enzyms führte zur Verringerung der Empfindlichkeit von A. niger oder F. graminearum gegen AFP. Die Expression des Enzyms in P. pastoris lässt einen AFP-sensitiven Stamm entstehen. Es konnte daher die Interaktion von AFP mit den Glycosylceramiden der pilzlichen Membran gezeigt werden. Im dritten Teil der Arbeit wurden Transkriptomdaten von A. niger, der ein anderes Mitglied der AFP-Familie, das AnAFP, sekretiert ausgewertet. Die Erstellung eines anafp-Ko-Expressionsnetzwerks ermöglichte die Zuordnung des AnAFP zu metabolischen Prozessen. Die in silico vorhergesagte Beteiligung des anafp wurde in Laborexperimenten überprüft, wodurch sie bestätigt werden konnte. Es fanden sich deutliche Hinweise für eine zellinterne Rolle des AnAFPs. Im letzten Teil der Arbeit konnten durch in silico Analysen über 50 AnAFP Orthologe in Ascomycota identifiziert werden. Hochkonserviert sind die Anzahl und Positionen der Cysteine sowie des „γ-cores“. Dabei handelt es sich um ein evolutionsbiologisch konserviertes dreidimensionales Strukturmotiv, welches Wechselwirkungen mit Membranen eingehtThe very variable and large group of antimicrobial peptides, of which more than 10,000 representatives are listed in the database "Collection of anti-microbial peptides" CAMPR3 [1], has long been in the focus of research as a potential basis for active substances. Due to the increasing number of fungal infections and resistance to antifungal agents, there is a great need to tap this source. The members of the AFP family are of interest due to their species-specificity, especially for known human and plant pathogenic fungi with high threat potential, without known side effects. Therefore, this work has provided the basis for establishing the antifungal protein AFP from A. giganteus. This includes (i) improved methods for production and characterization, (ii) characterization of the mechanism of action and (iii) in silico analyses to identify members of the AFP family. The establishment of bioreactor cultivation, with a standardized yield of 10 mg/L culture medium of AFP, is the beginning of the work. From the native producer A. giganteus several AFP variants are secreted, which are difficult to separate from each other. NMR experiments showed that the sequential procession produces a shorter variant, the "sf"-AFP (alanine), in addition to the longer AFP variant "lf"-AFP. Heterologous expression in P. pastoris is therefore the most suitable approach for AFP production. Homologous production in A. giganteus allows to elucidate the function of other AFP variants. In the second part of the work the influence of C3 saturation of glycosylceramides on the efficacy of AFP was investigated. The structural characteristics of these membrane components, which are specific to fungi, can explain the specific spectrum of activity of AFP. It was shown that the 2-hydroxy fatty acid N-acyl-Δ3(E)-desaturase is responsible for the formation of the double bond between the third and fourth carbon atom of the fatty acid residue. The deletion of this enzyme led to a reduction in the sensitivity of A. niger or F. graminearum to AFP. The expression of the enzyme in P. pastoris leads to the development of an AFP-sensitive strain. Therefore, the interaction of AFP with the glycosylceramides of the fungal membrane could be shown. In the third part of the work transcriptome data of A. niger, which secretes another member of the AFP family, AnAFP, were evaluated. The establishment of an anafp co-expression network enabled the assignment of AnAFP to metabolic processes. The in silico predicted involvement of anafp was verified in laboratory experiments, which confirmed it. There was clear evidence for a role of AnAFP in the cell. In the final part of the work, in silico analyses identified over 50 AnAFP orthologists in Ascomycota. Highly conserved are the number and positions of the cysteines and the "γ-core". This is an evolutionary-biologically conserved three-dimensional structural motif that interacts with membranes

A Transcriptome Meta-Analysis Proposes Novel Biological Roles for the Antifungal Protein AnAFP in Aspergillus niger.

Understanding the genetic, molecular and evolutionary basis of cysteine-stabilized antifungal proteins (AFPs) from fungi is important for understanding whether their function is mainly defensive or associated with fungal growth and development. In the current study, a transcriptome meta-analysis of the Aspergillus niger γ-core protein AnAFP was performed to explore co-expressed genes and pathways, based on independent expression profiling microarrays covering 155 distinct cultivation conditions. This analysis uncovered that anafp displays a highly coordinated temporal and spatial transcriptional profile which is concomitant with key nutritional and developmental processes. Its expression profile coincides with early starvation response and parallels with genes involved in nutrient mobilization and autophagy. Using fluorescence- and luciferase reporter strains we demonstrated that the anafp promoter is active in highly vacuolated compartments and foraging hyphal cells during carbon starvation with CreA and FlbA, but not BrlA, as most likely regulators of anafp. A co-expression network analysis supported by luciferase-based reporter assays uncovered that anafp expression is embedded in several cellular processes including allorecognition, osmotic and oxidative stress survival, development, secondary metabolism and autophagy, and predicted StuA and VelC as additional regulators. The transcriptomic resources available for A. niger provide unparalleled resources to investigate the function of proteins. Our work illustrates how transcriptomic meta-analyses can lead to hypotheses regarding protein function and predict a role for AnAFP during slow growth, allorecognition, asexual development and nutrient recycling of A. niger and propose that it interacts with the autophagic machinery to enable these processes

Analysis of <i>anafp</i> expression using a fluorescent reporter system.

<p>A) Luciferase expression under control of the <i>anafp</i> promoter was measured in a <i>ΔcreA</i> strain (NP6.4) and the corresponding wild type control strain (PK2.9) during 4 days of cultivation in complete medium using microtiter plates. Protein abundance was measured as luminescent counts per second normalized to culture optical density. Data of a representative experiment (n = 4) are shown. B) eYFP expression under control of the <i>anafp</i> promoter was measured in a <i>ΔcreA</i> strain (NP2.8, grey) and the corresponding wild type control strain (PK1.22, black) after 3 days of incubation on a complete medium agar. Expression levels were normalized to that of the control strain and are depicted as mean of three independent experiments performed as duplicate. *, p<0.03.</p

Expression levels of the <i>anafp</i> gene of <i>A</i>. <i>niger</i> strain N402 at different conditions.

<p>Transcription levels of the <i>anafp</i> gene were analyzed when exposed to different A) carbon sources in shaking flask cultures [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165755#pone.0165755.ref023" target="_blank">23</a>] and B) to carbon limitation in batch, retentostat [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165755#pone.0165755.ref027" target="_blank">27</a>] and chemostat bioreactor cultures [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165755#pone.0165755.ref028" target="_blank">28</a>]. <i>anafp</i> transcription levels are also depicted for batch cultivations in bioreactors with <i>ΔflbA</i> and <i>ΔbrlA</i> mutants, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165755#pone.0165755.ref033" target="_blank">33</a>]. Samples have been taken at four time points: t1–t4, exponential growth phase, 16 h, 60 h, 140 h post carbon depletion. In C) mRNA levels at different zones of a plated culture are depicted [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165755#pone.0165755.ref033" target="_blank">33</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165755#pone.0165755.ref034" target="_blank">34</a>]. Whereas zone 1 (z1) marks the center of the colony consisting, i.e. the oldest part of the culture, zone 5 (z5) marks the colony’s periphery. Zone 3 lies in between. Absolute values of transcription levels are depicted as arbitrary units of fluorescence intensity in a logarithmic scale and can be assessed using the legend. Color intensities were normalized to the highest absolute transcription level which is indicated by black color.</p

Membrane permeabilization induced by AnAFP in <i>A</i>. <i>niger</i>.

<p>The capability of AnAFP (100 μg/ml) to permeabilize the membranes of viable cells of <i>A</i>. <i>niger</i>, <i>A</i>. <i>nidulans</i> and <i>F</i>. <i>oxysporum</i> was monitored for 2 h of incubation. The fluorescence of the SYTOX Green dye was used as a measure of fungal cells with compromised membranes. Cells with intact membranes do not show a fluorescence signal. Data are averages of triplicate experiments.</p

Minimal inhibitory concentrations (MICs) of AnAFP and AFP on selected fungi.

<p>Minimal inhibitory concentrations (MICs) of AnAFP and AFP on selected fungi.</p

Fluorescence microscopic analysis of <i>anafp</i> promoter activity in reporter strain PK1.22.

<p>A) Shown is the fluorescence micrograph (left) and the fluorescence bright field overlay of strain PK1.22. CM agar sandwiched between two glass slides was inoculated with spores of strain PK1.22. After incubation at 30°C for two days in a humidified environment, the culture was analyzed using fluorescence microscopy. B) Shown are fluorescent (left column) and corresponding bright-field micrographs (right column) of culture samples taken from retentostat bioreactor cultivations of strain PK1.22 during exponential growth phase at day 0 with growth rate μ = 0.24 h^-1 and decreasing growth rates after the switch to the retentostat cultivation as indicated. The activity of the <i>anafp</i> promoter was followed by monitoring the green fluorescence signal of eYFP. Bar = 20 μm.</p