TY - JOUR A1 - Gupta, Shishir K. A1 - Srivastava, Mugdha A1 - Osmanoglu, Özge A1 - Xu, Zhuofei A1 - Brakhage, Axel A. A1 - Dandekar, Thomas T1 - Aspergillus fumigatus versus genus Aspergillus: conservation, adaptive evolution and specific virulence genes JF - Microorganisms N2 - Aspergillus is an important fungal genus containing economically important species, as well as pathogenic species of animals and plants. Using eighteen fungal species of the genus Aspergillus, we conducted a comprehensive investigation of conserved genes and their evolution. This also allows us to investigate the selection pressure driving the adaptive evolution in the pathogenic species A. fumigatus. Among single-copy orthologs (SCOs) for A. fumigatus and the closely related species A. fischeri, we identified 122 versus 50 positively selected genes (PSGs), respectively. Moreover, twenty conserved genes of unknown function were established to be positively selected and thus important for adaption. A. fumigatus PSGs interacting with human host proteins show over-representation of adaptive, symbiosis-related, immunomodulatory and virulence-related pathways, such as the TGF-β pathway, insulin receptor signaling, IL1 pathway and interfering with phagosomal GTPase signaling. Additionally, among the virulence factor coding genes, secretory and membrane protein-coding genes in multi-copy gene families, 212 genes underwent positive selection and also suggest increased adaptation, such as fungal immune evasion mechanisms (aspf2), siderophore biosynthesis (sidD), fumarylalanine production (sidE), stress tolerance (atfA) and thermotolerance (sodA). These genes presumably contribute to host adaptation strategies. Genes for the biosynthesis of gliotoxin are shared among all the close relatives of A. fumigatus as an ancient defense mechanism. Positive selection plays a crucial role in the adaptive evolution of A. fumigatus. The genome-wide profile of PSGs provides valuable targets for further research on the mechanisms of immune evasion, antimycotic targeting and understanding fundamental virulence processes. KW - molecular evolution KW - phylogenetic analysis KW - adaptation KW - recombination KW - positive selection KW - human pathogenic fungi KW - genus Aspergillus KW - Aspergillus fumigatus Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-246318 SN - 2076-2607 VL - 9 IS - 10 ER - TY - JOUR A1 - Ruf, Dominik A1 - Brantl, Victor A1 - Wagener, Johannes T1 - Mitochondrial Fragmentation in \(Aspergillus\) \(fumigatus\) as Early Marker of Granulocyte Killing Activity JF - Frontiers in Cellular and Infection Microbiology N2 - The host's defense against invasive mold infections relies on diverse antimicrobial activities of innate immune cells. However, studying these mechanisms in vitro is complicated by the filamentous nature of such pathogens that typically form long, branched, multinucleated and compartmentalized hyphae. Here we describe a novel method that allows for the visualization and quantification of the antifungal killing activity exerted by human granulocytes against hyphae of the opportunistic pathogen Aspergillus fumigatus. The approach relies on the distinct impact of fungal cell death on the morphology of mitochondria that were visualized with green fluorescent protein (GFP). We show that oxidative stress induces complete fragmentation of the tubular mitochondrial network which correlates with cell death of affected hyphae. Live cell microscopy revealed a similar and non-reversible disruption of the mitochondrial morphology followed by fading of fluorescence in Aspergillus hyphae that were killed by human granulocytes. Quantitative microscopic analysis of fixed samples was subsequently used to estimate the antifungal activity. By utilizing this assay, we demonstrate that lipopolysaccharides as well as human serum significantly increase the killing efficacy of the granulocytes. Our results demonstrate that evaluation of the mitochondrial morphology can be utilized to assess the fungicidal activity of granulocytes against A. fumigatus hyphae. KW - Aspergillus fumigatus KW - killing KW - assay KW - PMNs KW - granulocytes KW - mitochondria KW - mitochondrial morphology KW - fungicidal activity Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227133 VL - 8 IS - 128 ER - TY - JOUR A1 - Schmidt, Stefanie A1 - Ebner, Friederike A1 - Rosen, Kerstin A1 - Kniemeyer, Olaf A1 - Brakhage, Axel A. A1 - Löffler, Jürgen A1 - Seif, Michelle A1 - Springer, Jan A1 - Schlosser, Josephine A1 - Scharek‐Tedin, Lydia A1 - Scheffold, Alexander A1 - Bacher, Petra A1 - Kühl, Anja A. A1 - Rösler, Uwe A1 - Hartmann, Susanne T1 - The domestic pig as human‐relevant large animal model to study adaptive antifungal immune responses against airborne Aspergillus fumigatus JF - European Journal of Immunology N2 - Pulmonary mucosal immune response is critical for preventing opportunistic Aspergillus fumigatus infections. Although fungus‐specific CD4\(^{+}\) T cells in blood are described to reflect the actual host–pathogen interaction status, little is known about Aspergillus‐specific pulmonary T‐cell responses. Here, we exploit the domestic pig as human‐relevant large animal model and introduce antigen‐specific T‐cell enrichment in pigs to address Aspergillus‐specific T cells in the lung compared to peripheral blood. In healthy, environmentally Aspergillus‐exposed pigs, the fungus‐specific T cells are detectable in blood in similar frequencies as observed in healthy humans and exhibit a Th1 phenotype. Exposing pigs to 10\(^{6}\) cfu/m\(^{3}\) conidia induces a long‐lasting accumulation of Aspergillus‐specific Th1 cells locally in the lung and also systemically. Temporary immunosuppression during Aspergillus‐exposure showed a drastic reduction in the lung‐infiltrating antifungal T‐cell responses more than 2 weeks after abrogation of the suppressive treatment. This was reflected in blood, but to a much lesser extent. In conclusion, by using the human‐relevant large animal model the pig, this study highlights that the blood clearly reflects the mucosal fungal‐specific T‐cell reactivity in environmentally exposed as well as experimentally exposed healthy pigs. But, immunosuppression significantly impacts the mucosal site in contrast to the initial systemic immune response. KW - fungal aerosolization KW - porcine large animal model KW - pulmonary immune response KW - T cells KW - Aspergillus fumigatus Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-216085 VL - 50 IS - 11 SP - 1712 EP - 1728 ER - TY - JOUR A1 - Czakai, Kristin A1 - Leonhardt, Ines A1 - Dix, Andreas A1 - Bonin, Michael A1 - Linde, Joerg A1 - Einsele, Hermann A1 - Kurzai, Oliver A1 - Loeffler, Jürgen T1 - Krüppel-like Factor 4 modulates interleukin-6 release in human dendritic cells after in vitro stimulation with Aspergillus fumigatus and Candida albicans JF - Scientific Reports N2 - Invasive fungal infections are associated with high mortality rates and are mostly caused by the opportunistic fungi Aspergillus fumigatus and Candida albicans. Immune responses against these fungi are still not fully understood. Dendritic cells (DCs) are crucial players in initiating innate and adaptive immune responses against fungal infections. The immunomodulatory effects of fungi were compared to the bacterial stimulus LPS to determine key players in the immune response to fungal infections. A genome wide study of the gene regulation of human monocyte-derived dendritic cells (DCs) confronted with A. fumigatus, C. albicans or LPS was performed and Krüppel-like factor 4 (KLF4) was identified as the only transcription factor that was down-regulated in DCs by both fungi but induced by stimulation with LPS. Downstream analysis demonstrated the influence of KLF4 on the interleukine-6 expression in human DCs. Furthermore, KLF4 regulation was shown to be dependent on pattern recognition receptor ligation. Therefore KLF4 was identified as a controlling element in the IL-6 immune response with a unique expression pattern comparing fungal and LPS stimulation. KW - gene regulation in immune cells KW - fungal host response KW - Aspergillus fumigatus KW - Candida albicans Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-181185 VL - 6 ER - TY - JOUR A1 - White, P. Lewis A1 - Wiederhold, Nathan P. A1 - Loeffler, Juergen A1 - Najvar, Laura K. A1 - Melchers, Willem A1 - Herrera, Monica A1 - Bretagne, Stephane A1 - Wickes, Brian A1 - Kirkpatrick, William R. A1 - Barnes, Rosemary A. A1 - Donnelly, J. Peter A1 - Patterson, Thomas F. T1 - Comparison of nonculture blood-based tests for diagnosing invasive aspergillosis in an animal model JF - Journal of Clinical Microbiology N2 - The European Aspergillus PCR Initiative (EAPCRI) has provided recommendations for the PCR testing of whole blood (WB) and serum/plasma. It is important to test these recommended protocols on nonsimulated "in vivo" specimens before full clinical evaluation. The testing of an animal model of invasive aspergillosis (IA) overcomes the low incidence of disease and provides experimental design and control that is not possible in the clinical setting. Inadequate performance of the recommended protocols at this stage would require reassessment of methods before clinical trials are performed and utility assessed. The manuscript describes the performance of EAPCRI protocols in an animal model of invasive aspergillosis. Blood samples taken from a guinea pig model of IA were used for WB and serum PCR. Galactomannan and beta-D-glucan detection were evaluated, with particular focus on the timing of positivity and on the interpretation of combination testing. The overall sensitivities for WB PCR, serum PCR, galactomannan, and beta-D-glucan were 73%, 65%, 68%, and 46%, respectively. The corresponding specificities were 92%, 79%, 80%, and 100%, respectively. PCR provided the earliest indicator of IA, and increasing galactomannan and beta-D-glucan values were indicators of disease progression. The combination of WB PCR with galactomannan and beta-D-glucan proved optimal (area under the curve AUC], 0.95), and IA was confidently diagnosed or excluded. The EAPRCI-recommended PCR protocols provide performance comparable to commercial antigen tests, and clinical trials are warranted. By combining multiple tests, IA can be excluded or confirmed, highlighting the need for a combined diagnostic strategy. However, this approach must be balanced against the practicality and cost of using multiple tests. KW - high-risk hematology KW - Guinea pig model KW - real-time PCR KW - fungal disease KW - galactomannan KW - pulmonary aspergillosis KW - amphotericin B KW - Aspergillus fumigatus KW - beta-D-glucan Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-189674 VL - 54 IS - 4 ER - TY - JOUR A1 - Ziegler, Sabrina A1 - Weiss, Esther A1 - Schmitt, Anna-Lena A1 - Schlegel, Jan A1 - Burgert, Anne A1 - Terpitz, Ulrich A1 - Sauer, Markus A1 - Moretta, Lorenzo A1 - Sivori, Simona A1 - Leonhardt, Ines A1 - Kurzai, Oliver A1 - Einsele, Hermann A1 - Loeffler, Juergen T1 - CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells JF - Scientific Reports N2 - Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response. KW - pattern recognition receptors KW - fungal infection KW - Aspergillus fumigatus KW - natural killer cells Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170637 VL - 7 IS - 6138 ER - TY - JOUR A1 - Hellmann, Anna-Maria A1 - Lother, Jasmin A1 - Wurster, Sebastian A1 - Lutz, Manfred B. A1 - Schmitt, Anna Lena A1 - Morton, Charles Oliver A1 - Eyrich, Matthias A1 - Czakai, Kristin A1 - Einsele, Hermann A1 - Loeffler, Juergen T1 - Human and Murine Innate Immune Cell Populations Display Common and Distinct Response Patterns during Their In Vitro Interaction with the Pathogenic Mold Aspergillus fumigatus JF - Frontiers in Immunology N2 - Aspergillus fumigatus is the main cause of invasive fungal infections occurring almost exclusively in immunocompromised patients. An improved understanding of the initial innate immune response is key to the development of better diagnostic tools and new treatment options. Mice are commonly used to study immune defense mechanisms during the infection of the mammalian host with A. fumigatus. However, little is known about functional differences between the human and murine immune response against this fungal pathogen. Thus, we performed a comparative functional analysis of human and murine dendritic cells (DCs), macrophages, and polymorphonuclear cells (PMNs) using standardized and reproducible working conditions, laboratory protocols, and readout assays. A. fumigatus did not provoke identical responses in murine and human immune cells but rather initiated relatively specific responses. While human DCs showed a significantly stronger upregulation of their maturation markers and major histocompatibility complex molecules and phagocytosed A. fumigatus more efficiently compared to their murine counterparts, murine PMNs and macrophages exhibited a significantly stronger release of reactive oxygen species after exposure to A. fumigatus. For all studied cell types, human and murine samples differed in their cytokine response to conidia or germ tubes of A. fumigatus. Furthermore, Dectin-1 showed inverse expression patterns on human and murine DCs after fungal stimulation. These specific differences should be carefully considered and highlight potential limitations in the transferability of murine host–pathogen interaction studies. KW - murine model KW - humans KW - Aspergillus fumigatus KW - innate immune response KW - fungal infection Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-169926 VL - 8 IS - 1716 ER - TY - JOUR A1 - Marischen, Lothar A1 - Englert, Anne A1 - Schmitt, Anna-Lena A1 - Einsele, Hermann A1 - Loeffler, Juergen T1 - Human NK cells adapt their immune response towards increasing multiplicities of infection of Aspergillus fumigatus JF - BMC Immunology N2 - Background: The saprophytic fungus Aspergillus fumigatus reproduces by generation of conidia, which are spread by airflow throughout nature. Since humans are inhaling certain amounts of spores every day, the (innate) immune system is constantly challenged. Even though macrophages and neutrophils carry the main burden, also NK cells are regarded to contribute to the antifungal immune response. While NK cells reveal a low frequency, expression and release of immunomodulatory molecules seem to be a natural way of their involvement. Results: In this study we show, that NK cells secrete chemokines such as CCL3/MIP-1α, CCL4/MIP-1β and CCL5/RANTES early on after stimulation with Aspergillus fumigatus and, in addition, adjust the concentration of chemokines released to the multiplicity of infection of Aspergillus fumigatus. Conclusions: These results further corroborate the relevance of NK cells within the antifungal immune response, which is regarded to be more and more important in the development and outcome of invasive aspergillosis in immunocompromised patients after hematopoietic stem cell transplantation. Additionally, the correlation between the multiplicity of infection and the expression and release of chemokines shown here may be useful in further studies for the quantification and/or surveillance of the NK cell involvement in antifungal immune responses. KW - Aspergillus fumigatus KW - aspergillosis KW - NK cells KW - chemokines KW - CCL4 KW - multiplicity of infection KW - MIP-1β Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176331 VL - 19 IS - 39 ER - TY - JOUR A1 - Remmele, Christian W. A1 - Luther, Christian H. A1 - Balkenhol, Johannes A1 - Dandekar, Thomas A1 - Müller, Tobias A1 - Dittrich, Marcus T. T1 - Integrated inference and evaluation of host-fungi interaction networks JF - Frontiers in Microbiology N2 - Fungal microorganisms frequently lead to life-threatening infections. Within this group of pathogens, the commensal Candida albicans and the filamentous fungus Aspergillus fumigatus are by far the most important causes of invasive mycoses in Europe. A key capability for host invasion and immune response evasion are specific molecular interactions between the fungal pathogen and its human host. Experimentally validated knowledge about these crucial interactions is rare in literature and even specialized host pathogen databases mainly focus on bacterial and viral interactions whereas information on fungi is still sparse. To establish large-scale host fungi interaction networks on a systems biology scale, we develop an extended inference approach based on protein orthology and data on gene functions. Using human and yeast intraspecies networks as template, we derive a large network of pathogen host interactions (PHI). Rigorous filtering and refinement steps based on cellular localization and pathogenicity information of predicted interactors yield a primary scaffold of fungi human and fungi mouse interaction networks. Specific enrichment of known pathogenicity-relevant genes indicates the biological relevance of the predicted PHI. A detailed inspection of functionally relevant subnetworks reveals novel host fungal interaction candidates such as the Candida virulence factor PLB1 and the anti-fungal host protein APP. Our results demonstrate the applicability of interolog-based prediction methods for host fungi interactions and underline the importance of filtering and refinement steps to attain biologically more relevant interactions. This integrated network framework can serve as a basis for future analyses of high-throughput host fungi transcriptome and proteome data. KW - candida genome database KW - computational prediction KW - potential role KW - network inference KW - bioinformatics and computational biology KW - protein interaction database KW - Aspergillus fumigatus KW - cell wall KW - functional modules KW - alzheimers disease KW - molecular cloning KW - Candida albicans KW - pathogen-host interaction (PHI) KW - protein-protein interaction KW - pathogenicity KW - interolog Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148278 VL - 6 IS - 764 ER - TY - JOUR A1 - Irmer, Henriette A1 - Tarazona, Sonia A1 - Sasse, Christoph A1 - Olbermann, Patrick A1 - Loeffler, Jürgen A1 - Krappmann, Sven A1 - Conesa, Ana A1 - Braus, Gerhard H. T1 - RNAseq analysis of Aspergillus fumigatus in blood reveals a just wait and see resting stage behavior JF - BMC Genomics N2 - Background: Invasive aspergillosis is started after germination of Aspergillus fumigatus conidia that are inhaled by susceptible individuals. Fungal hyphae can grow in the lung through the epithelial tissue and disseminate hematogenously to invade into other organs. Low fungaemia indicates that fungal elements do not reside in the bloodstream for long. Results: We analyzed whether blood represents a hostile environment to which the physiology of A. fumigatus has to adapt. An in vitro model of A. fumigatus infection was established by incubating mycelium in blood. Our model allowed to discern the changes of the gene expression profile of A. fumigatus at various stages of the infection. The majority of described virulence factors that are connected to pulmonary infections appeared not to be activated during the blood phase. Three active processes were identified that presumably help the fungus to survive the blood environment in an advanced phase of the infection: iron homeostasis, secondary metabolism, and the formation of detoxifying enzymes. Conclusions: We propose that A. fumigatus is hardly able to propagate in blood. After an early stage of sensing the environment, virtually all uptake mechanisms and energy-consuming metabolic pathways are shut-down. The fungus appears to adapt by trans-differentiation into a resting mycelial stage. This might reflect the harsh conditions in blood where A. fumigatus cannot take up sufficient nutrients to establish self-defense mechanisms combined with significant growth. KW - Saccharomyces cerevisiae KW - cerebral aspergillosis KW - gene expression KW - Aspergillus fumigatus KW - iron homeostasis KW - invasive pulmonary aspergillosis KW - Candida albicans KW - cell wall KW - lysine biosynthesis KW - human pathogen KW - murine model KW - virulence KW - mRNA-Seq KW - transcriptome KW - human pathogenic fungi KW - secondary metabolite gene cluster KW - detoxification Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-151390 VL - 16 IS - 640 ER - TY - JOUR A1 - Amich, Jorge A1 - Krappmann, Sven T1 - Deciphering metabolic traits of the fungal pathogen Aspergillus fumigatus: redundancy vs. essentiality JF - Frontiers in Microbiology N2 - Incidence rates of infections caused by environmental opportunistic fungi have risen over recent decades. Aspergillus species have emerged as serious threat for the immunecompromised, and detailed knowledge about virulence-determining traits is crucial for drug target identification. As a prime saprobe, A. fumigatus has evolved to efficiently adapt to various stresses and to sustain nutritional supply by osmotrophy, which is characterized by extracellular substrate digestion followed by efficient uptake of breakdown products that are then fed into the fungal primary metabolism. These intrinsic metabolic features are believed to be related with its virulence ability. The plethora of genes that encode underlying effectors has hampered their in-depth analysis with respect to pathogenesis. Recent developments in Aspergillus molecular biology allow conditional gene expression or comprehensive targeting of gene families to cope with redundancy. Furthermore, identification of essential genes that are intrinsically connected to virulence opens accurate perspectives for novel targets in antifungal therapy. KW - Aspergillus fumigatus KW - aspergillosis KW - virulence KW - conditional promoter replacement KW - nutrients KW - gene family targeting Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-123669 VL - 3 ER -