Abstract (Expand)

Filamentous fungi of the genus Aspergillus are of particular interest for biotechnological applications due to their natural capacity to secrete carbohydrate-active enzymes (CAZy) that target plant biomass. The presence of easily metabolizable sugars such as glucose, whose concentrations increase during plant biomass hydrolysis, results in the repression of CAZy-encoding genes in a process known as carbon catabolite repression (CCR), which is undesired for the purpose of large-scale enzyme production. To date, the C2H2 transcription factor CreA has been described as the major CC repressor in Aspergillus spp., although little is known about the role of posttranslational modifications in this process. In this work, phosphorylation sites were identified by mass spectrometry on Aspergillus nidulans CreA, and subsequently, the previously identified but uncharacterized site S262, the characterized site S319, and the newly identified sites S268 and T308 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was investigated. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 was not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. All sites were shown to be important for glycogen and trehalose metabolism. This study highlights the importance of CreA phosphorylation sites for the regulation of CCR. These sites are interesting targets for biotechnological strain engineering without the need to delete essential genes, which could result in undesired side effects.IMPORTANCE In filamentous fungi, the transcription factor CreA controls carbohydrate metabolism through the regulation of genes encoding enzymes required for the use of alternative carbon sources. In this work, phosphorylation sites were identified on Aspergillus nidulans CreA, and subsequently, the two newly identified sites S268 and T308, the previously identified but uncharacterized site S262, and the previously characterized site S319 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was characterized. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 is not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. This work characterized novel CreA phosphorylation sites under carbon catabolite-repressing conditions and showed that they are crucial for CreA protein turnover, control of carbohydrate utilization, and biotechnologically relevant enzyme production.

Authors: L. J. de Assis, L. P. Silva, O. Bayram, P. Dowling, Olaf Kniemeyer, Thomas Krüger, Axel Brakhage, Y. Chen, L. Dong, K. Tan, K. H. Wong, L. N. A. Ries, G. H. Goldman

Date Published: 5th Jan 2021

Journal: mBio

Abstract (Expand)

Mucormycosis is an emergent, fatal fungal infection of humans and warm-blooded animals caused by species of the order Mucorales. Immune cells of the innate immune system serve as the first line of defence against inhaled spores. Alveolar macrophages were challenged with the mucoralean fungus Lichtheimia corymbifera and subjected to biotinylation and streptavidin enrichment procedures followed by LC-MS/MS analyses. A total of 28 host proteins enriched for binding to macrophage-L. corymbifera interaction. Among those, the HSP70-family protein Hspa8 was found to be predominantly responsive to living and heat-killed spores of a virulent and an attenuated strain of L. corymbifera. Confocal scanning laser microscopy of infected macrophages revealed colocalization of Hspa8 with phagocytosed spores of L. corymbifera. The amount of detectable Hspa8 was dependent on the multiplicity of infection. Incubation of alveolar macrophages with an anti-Hspa8 antibody prior to infection reduced their capability to phagocytose spores of L. corymbifera. In contrast, anti-Hspa8 antibodies did not abrogate the phagocytosis of Aspergillus fumigatus conidia by macrophages. These results suggest an important contribution of the heat-shock family protein Hspa8 in the recognition of spores of the mucoralean fungus L. corymbifera by host alveolar macrophages and define a potential immunomodulatory therapeutic target.

Authors: M. I. A. Hassan, J. M. Kruse, Thomas Krüger, H. M. Dahse, Z. Cseresnyes, M. G. Blango, Hortense Slevogt, F. Horhold, V. Ast, R. Konig, Marc Thilo Figge, Olaf Kniemeyer, Axel Brakhage, Kerstin Voigt

Date Published: 26th Jun 2020

Journal: Environ Microbiol

Abstract (Expand)

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.

Authors: M. G. Blango, A. Pschibul, Flora Rivieccio, Thomas Krüger, M. Rafiq, L. J. Jia, T. Zheng, M. Goldmann, V. Voltersen, J. Li, Gianni Panagiotou, Olaf Kniemeyer, Axel Brakhage

Date Published: 1st May 2020

Journal: J Proteome Res

Abstract (Expand)

The capacity of Candida albicans to reversibly change its morphology between yeast and filamentous stages is crucial for its virulence. Formation of hyphae correlates with the upregulation of genes ALS3 and ECE1, which are involved in pathogenicity processes such as invasion, iron acquisition, and host cell damage. The global repressor Tup1 and its cofactor Nrg1 are considered to be the main antagonists of hyphal development in C. albicans However, our experiments revealed that Tup1, but not Nrg1, was required for full expression of ALS3 and ECE1 In contrast to NRG1, overexpression of TUP1 was found to inhibit neither filamentous growth nor transcription of ALS3 and ECE1 In addition, we identified the transcription factor Ahr1 as being required for full expression of both genes. A hyperactive version of Ahr1 bound directly to the promoters of ALS3 and ECE1 and induced their transcription even in the absence of environmental stimuli. This regulation worked even in the absence of the crucial hyphal growth regulators Cph1 and Efg1 but was dependent on the presence of Tup1. Overall, our results show that Ahr1 and Tup1 are key contributors in the complex regulation of virulence-associated genes in the different C. albicans morphologies.IMPORTANCE Candida albicans is a major human fungal pathogen and the leading cause of systemic Candida infections. In recent years, Als3 and Ece1 were identified as important factors for fungal virulence. Transcription of both corresponding genes is closely associated with hyphal growth. Here, we describe how Tup1, normally a global repressor of gene expression as well as of filamentation, and the transcription factor Ahr1 contribute to full expression of ALS3 and ECE1 in C. albicans hyphae. Both regulators are required for high mRNA amounts of the two genes to ensure functional relevant protein synthesis and localization. These observations identified a new aspect of regulation in the complex transcriptional control of virulence-associated genes in C. albicans.

Authors: S. Ruben, E. Garbe, Selene Mogavero, Daniela Albrecht-Eckardt, D. Hellwig, A. Hader, Thomas Krüger, K. Gerth, Ilse Jacobsen, O. Elshafee, S. Brunke, Kerstin Hünniger, Olaf Kniemeyer, Axel Brakhage, Joachim Morschhäuser, Bernhard Hube, Slavena Vylkova, Oliver Kurzai, R. Martin

Date Published: 28th Apr 2020

Journal: mBio

Abstract (Expand)

Polymorphonuclear granulocytes (PMNs) are indispensable for controlling life-threatening fungal infections. In addition to various effector mechanisms, PMNs also produce extracellular vesicles (EVs). Their contribution to antifungal defense has remained unexplored. We reveal that the clinically important human-pathogenic fungus Aspergillus fumigatus triggers PMNs to release a distinct set of antifungal EVs (afEVs). Proteome analyses indicated that afEVs are enriched in antimicrobial proteins. The cargo and the release kinetics of EVs are modulated by the fungal strain confronted. Tracking of afEVs indicated that they associated with fungal cells and even entered fungal hyphae, resulting in alterations in the morphology of the fungal cell wall and dose-dependent antifungal effects. To assess as a proof of concept whether the antimicrobial proteins found in afEVs might contribute to growth inhibition of hyphae when present in the fungal cytoplasm, two human proteins enriched in afEVs, cathepsin G and azurocidin, were heterologously expressed in fungal hyphae. This led to reduced fungal growth relative to that of a control strain producing the human retinol binding protein 7. In conclusion, extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. This finding offers an intriguing, previously overlooked mechanism of antifungal defense against A. fumigatus IMPORTANCE Invasive fungal infections caused by the mold Aspergillus fumigatus are a growing concern in the clinic due to the increasing use of immunosuppressive therapies and increasing antifungal drug resistance. These infections result in high rates of mortality, as treatment and diagnostic options remain limited. In healthy individuals, neutrophilic granulocytes are critical for elimination of A. fumigatus from the host; however, the exact extracellular mechanism of neutrophil-mediated antifungal activity remains unresolved. Here, we present a mode of antifungal defense employed by human neutrophils against A. fumigatus not previously described. We found that extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. In the end, antifungal extracellular vesicle biology provides a significant step forward in our understanding of A. fumigatus host pathogenesis and opens up novel diagnostic and therapeutic possibilities.

Authors: Iordana Shopova, I. Belyaev, Prasad Dasari, S. Jahreis, M. C. Stroe, Z. Cseresnyes, Ann-Kathrin Zimmermann, A. Medyukhina, Carl-Magnus Svensson, Thomas Krüger, V. Szeifert, S. Nietzsche, Theresia Conrad, M. G. Blango, Olaf Kniemeyer, M. von Lilienfeld-Toal, Peter Zipfel, E. Ligeti, Marc Thilo Figge, Axel Brakhage

Date Published: 14th Apr 2020

Journal: mBio

Abstract (Expand)

Th17 cells provide protection at barrier tissues but may also contribute to immune pathology. The relevance and induction mechanisms of pathologic Th17 responses in humans are poorly understood. Here, we identify the mucocutaneous pathobiont Candida albicans as the major direct inducer of human anti-fungal Th17 cells. Th17 cells directed against other fungi are induced by cross-reactivity to C. albicans. Intestinal inflammation expands total C. albicans and cross-reactive Th17 cells. Strikingly, Th17 cells cross-reactive to the airborne fungus Aspergillus fumigatus are selectively activated and expanded in patients with airway inflammation, especially during acute allergic bronchopulmonary aspergillosis. This indicates a direct link between protective intestinal Th17 responses against C. albicans and lung inflammation caused by airborne fungi. We identify heterologous immunity to a single, ubiquitous member of the microbiota as a central mechanism for systemic induction of human anti-fungal Th17 responses and as a potential risk factor for pulmonary inflammatory diseases.

Authors: P. Bacher, T. Hohnstein, E. Beerbaum, M. Rocker, M. G. Blango, S. Kaufmann, J. Rohmel, P. Eschenhagen, C. Grehn, K. Seidel, V. Rickerts, L. Lozza, U. Stervbo, M. Nienen, N. Babel, J. Milleck, M. Assenmacher, O. A. Cornely, M. Ziegler, H. Wisplinghoff, G. Heine, M. Worm, B. Siegmund, J. Maul, P. Creutz, C. Tabeling, C. Ruwwe-Glosenkamp, L. E. Sander, C. Knosalla, S. Brunke, Bernhard Hube, Olaf Kniemeyer, Axel Brakhage, C. Schwarz, A. Scheffold

Date Published: 7th Mar 2019

Journal: Cell

Abstract (Expand)

BACKGROUND: Omics data provide deep insights into overall biological processes of organisms. However, integration of data from different molecular levels such as transcriptomics and proteomics, still remains challenging. Analyzing lists of differentially abundant molecules from diverse molecular levels often results in a small overlap mainly due to different regulatory mechanisms, temporal scales, and/or inherent properties of measurement methods. Module-detecting algorithms identifying sets of closely related proteins from protein-protein interaction networks (PPINs) are promising approaches for a better data integration. RESULTS: Here, we made use of transcriptome, proteome and secretome data from the human pathogenic fungus Aspergillus fumigatus challenged with the antifungal drug caspofungin. Caspofungin targets the fungal cell wall which leads to a compensatory stress response. We analyzed the omics data using two different approaches: First, we applied a simple, classical approach by comparing lists of differentially expressed genes (DEGs), differentially synthesized proteins (DSyPs) and differentially secreted proteins (DSePs); second, we used a recently published module-detecting approach, ModuleDiscoverer, to identify regulatory modules from PPINs in conjunction with the experimental data. Our results demonstrate that regulatory modules show a notably higher overlap between the different molecular levels and time points than the classical approach. The additional structural information provided by regulatory modules allows for topological analyses. As a result, we detected a significant association of omics data with distinct biological processes such as regulation of kinase activity, transport mechanisms or amino acid metabolism. We also found a previously unreported increased production of the secondary metabolite fumagillin by A. fumigatus upon exposure to caspofungin. Furthermore, a topology-based analysis of potential key factors contributing to drug-caused side effects identified the highly conserved protein polyubiquitin as a central regulator. Interestingly, polyubiquitin UbiD neither belonged to the groups of DEGs, DSyPs nor DSePs but most likely strongly influenced their levels. CONCLUSION: Module-detecting approaches support the effective integration of multilevel omics data and provide a deep insight into complex biological relationships connecting these levels. They facilitate the identification of potential key players in the organism's stress response which cannot be detected by commonly used approaches comparing lists of differentially abundant molecules.

Authors: Theresia Conrad, Olaf Kniemeyer, S. G. Henkel, T. Kruger, D. J. Mattern, V. Valiante, Reinhard Guthke, Ilse Jacobsen, Axel Brakhage, S. Vlaic, Jörg Linde

Date Published: 20th Oct 2018

Journal: BMC Syst Biol

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