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74 Publications visible to you, out of a total of 74
Triple RNA-Seq Reveals Synergy in a Human Virus-Fungus Co-infection Model.

Abstract (Expand)

High-throughput RNA sequencing (RNA-seq) is routinely applied to study diverse biological processes; however, when performed separately on interacting organisms, systemic noise intrinsic to RNA extraction, … library preparation, and sequencing hampers the identification of cross-species interaction nodes. Here, we develop triple RNA-seq to simultaneously detect transcriptomes of monocyte-derived dendritic cells (moDCs) infected with the frequently co-occurring pulmonary pathogens Aspergillus fumigatus and human cytomegalovirus (CMV). Comparing expression patterns after co-infection with those after single infections, our data reveal synergistic effects and mutual interferences between host responses to the two pathogens. For example, CMV attenuates the fungus-mediated activation of pro-inflammatory cytokines through NF-kappaB (nuclear factor kappaB) and NFAT (nuclear factor of activated T cells) cascades, while A. fumigatus impairs viral clearance by counteracting viral nucleic acid-induced activation of type I interferon signaling. Together, the analytical power of triple RNA-seq proposes molecular hubs in the differential moDC response to fungal/viral single infection or co-infection that contribute to our understanding of the etiology and, potentially, clearance of post-transplant infections.

Authors: B. Seelbinder, J. Wallstabe, L. Marischen, E. Weiss, S. Wurster, L. Page, C. Loffler, L. Bussemer, A. L. Schmitt, T. Wolf, J. Linde, L. Cicin-Sain, J. Becker, U. Kalinke, J. Vogel, G. Panagiotou, H. Einsele, A. J. Westermann, S. Schauble, J. Loeffler

Date Published: 17th Nov 2020

Publication Type: Not specified

Survival Strategies of Pathogenic Candida Species in Human Blood Show Independent and Specific Adaptations.

Abstract (Expand)

Only four species, Candida albicans, C. glabrata, C. parapsilosis, and C. tropicalis, together account for about 90% of all Candida bloodstream infections and are among the most common causes of invasive … fungal infections of humans. However, virulence potential varies among these species, and the phylogenetic tree reveals that their pathogenicity may have emerged several times independently during evolution. We therefore tested these four species in a human whole-blood infection model to determine, via comprehensive dual-species RNA-sequencing analyses, which fungal infection strategies are conserved and which are recent evolutionary developments. The ex vivo infection progressed from initial immune cell interactions to nearly complete killing of all fungal cells. During the course of infection, we characterized important parameters of pathogen-host interactions, such as fungal survival, types of interacting immune cells, and cytokine release. On the transcriptional level, we obtained a predominantly uniform and species-independent human response governed by a strong upregulation of proinflammatory processes, which was downregulated at later time points after most of the fungal cells were killed. In stark contrast, we observed that the different fungal species pursued predominantly individual strategies and showed significantly different global transcriptome patterns. Among other findings, our functional analyses revealed that the fungal species relied on different metabolic pathways and virulence factors to survive the host-imposed stress. These data show that adaptation of Candida species as a response to the host is not a phylogenetic trait, but rather has likely evolved independently as a prerequisite to cause human infections.IMPORTANCE To ensure their survival, pathogens have to adapt immediately to new environments in their hosts, for example, during the transition from the gut to the bloodstream. Here, we investigated the basis of this adaptation in a group of fungal species which are among the most common causes of hospital-acquired infections, the Candida species. On the basis of a human whole-blood infection model, we studied which genes and processes are active over the course of an infection in both the host and four different Candida pathogens. Remarkably, we found that, while the human host response during the early phase of infection is predominantly uniform, the pathogens pursue largely individual strategies and each one regulates genes involved in largely disparate processes in the blood. Our results reveal that C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis all have developed individual strategies for survival in the host. This indicates that their pathogenicity in humans has evolved several times independently and that genes which are central for survival in the host for one species may be irrelevant in another.

Authors: P. Kammer, S. McNamara, T. Wolf, T. Conrad, S. Allert, F. Gerwien, K. Hunniger, O. Kurzai, R. Guthke, B. Hube, J. Linde, S. Brunke

Date Published: 6th Oct 2020

Publication Type: Not specified

Reconstituting NK Cells After Allogeneic Stem Cell Transplantation Show Impaired Response to the Fungal Pathogen Aspergillus fumigatus.

Abstract (Expand)

Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells … with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1alpha, MIP-1beta, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56(bright)CD16(dim) cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1alpha, MIP-1beta, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.

Authors: E. Weiss, J. Schlegel, U. Terpitz, M. Weber, J. Linde, A. L. Schmitt, K. Hunniger, L. Marischen, F. Gamon, J. Bauer, C. Loffler, O. Kurzai, C. O. Morton, M. Sauer, H. Einsele, J. Loeffler

Date Published: 5th Oct 2020

Publication Type: Not specified

Treatment with etanercept and low monocyte concentration contribute to the risk of invasive aspergillosis in patients post allogeneic stem cell transplantation.

Abstract (Expand)

Invasive aspergillosis (IA) is a life-threatening complication among allogeneic hematopoietic stem cell transplant (alloSCT) recipients. Despite well known risk factors and different available assays, … diagnosis of invasive aspergillosis remains challenging. 103 clinical variables from patients with hematological malignancies and subsequent alloSCT were collected. Associations between collected variables and patients with (n = 36) and without IA (n = 36) were investigated by applying univariate and multivariable logistic regression. The predictive power of the final model was tested in an independent patient cohort (23 IA cases and 25 control patients). Findings were investigated further by in vitro studies, which analysed the effect of etanercept on A. fumigatus-stimulated macrophages at the gene expression and cytokine secretion. Additionally, the release of C-X-C motif chemokine ligand 10 (CXCL10) in patient sera was studied. Low monocyte concentration (p = 4.8 x 10(-06)), severe GvHD of the gut (grade 2-4) (p = 1.08 x 10(-02)) and etanercept treatment of GvHD (p = 3.5 x 10(-03)) were significantly associated with IA. Our studies showed that etanercept lowers CXCL10 concentrations in vitro and ex vivo and down-regulates genes involved in immune responses and TNF-alpha signaling. Our study offers clinicians new information regarding risk factors for IA including low monocyte counts and administration of etanercept. After necessary validation, such information may be used for decision making regarding antifungal prophylaxis or closely monitoring patients at risk.

Authors: T. Zoran, M. Weber, J. Springer, P. L. White, J. Bauer, A. Schober, C. Loffler, B. Seelbinder, K. Hunniger, O. Kurzai, A. Scherag, S. Schauble, C. O. Morton, H. Einsele, J. Linde, J. Loffler

Date Published: 21st Nov 2019

Publication Type: Not specified

Module-detection approaches for the integration of multilevel omics data highlight the comprehensive response of Aspergillus fumigatus to caspofungin.

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: T. Conrad, O. Kniemeyer, S. G. Henkel, T. Kruger, D. J. Mattern, V. Valiante, R. Guthke, I. D. Jacobsen, A. A. Brakhage, S. Vlaic, J. Linde

Date Published: 20th Oct 2018

Publication Type: Not specified

Comparative Study on Alternative Splicing in Human Fungal Pathogens Suggests Its Involvement During Host Invasion.

Abstract (Expand)

Alternative splicing (AS) is an important regulatory mechanism in eukaryotes but only little is known about its impact in fungi. Human fungal pathogens are of high clinical interest causing recurrent … or life-threatening infections. AS can be well-investigated genome-wide and quantitatively with the powerful technology of RNA-Seq. Here, we systematically studied AS in human fungal pathogens based on RNA-Seq data. To do so, we investigated its effect in seven fungi during conditions simulating ex vivo infection processes and during in vitro stress. Genes undergoing AS are species-specific and act independently from differentially expressed genes pointing to an independent mechanism to change abundance and functionality. Candida species stand out with a low number of introns with higher and more varying lengths and more alternative splice sites. Moreover, we identified a functional difference between response to host and other stress conditions: During stress, AS affects more genes and is involved in diverse regulatory functions. In contrast, during response-to-host conditions, genes undergoing AS have membrane functionalities and might be involved in the interaction with the host. We assume that AS plays a crucial regulatory role in pathogenic fungi and is important in both response to host and stress conditions.

Authors: P. Sieber, K. Voigt, P. Kammer, S. Brunke, S. Schuster, J. Linde

Date Published: 19th Oct 2018

Publication Type: Not specified

Two's company: studying interspecies relationships with dual RNA-seq.

Abstract (Expand)

Organisms do not exist isolated from each other, but constantly interact. Cells can sense the presence of interaction partners by a range of receptors and, via complex regulatory networks, specifically … react by changing the expression of many of their genes. Technological advances in next-generation sequencing over the recent years now allow us to apply RNA sequencing to two species at the same time (dual RNA-seq), and thus to directly study the gene expression of two interacting species without the need to physically separate cells or RNA. In this review, we give an overview over the latest studies in interspecies interactions made possible by dual RNA-seq, ranging from pathogenic to symbiotic relationships. We summarize state-of-the-art experimental techniques, bioinformatic data analysis and data interpretation, while also highlighting potential problems and pitfalls starting from the selection of meaningful time points and number of reads to matters of rRNA depletion. A short outlook on new trends in the field of dual RNA-seq concludes this review, looking at sequencing of non-coding RNAs during host-pathogen interactions and the prediction of molecular interspecies interactions networks.

Authors: T. Wolf, P. Kammer, S. Brunke, J. Linde

Date Published: 29th Sep 2017

Publication Type: Not specified

LASSIM-A network inference toolbox for genome-wide mechanistic modeling.

Abstract (Expand)

Recent technological advancements have made time-resolved, quantitative, multi-omics data available for many model systems, which could be integrated for systems pharmacokinetic use. Here, we present … large-scale simulation modeling (LASSIM), which is a novel mathematical tool for performing large-scale inference using mechanistically defined ordinary differential equations (ODE) for gene regulatory networks (GRNs). LASSIM integrates structural knowledge about regulatory interactions and non-linear equations with multiple steady state and dynamic response expression datasets. The rationale behind LASSIM is that biological GRNs can be simplified using a limited subset of core genes that are assumed to regulate all other gene transcription events in the network. The LASSIM method is implemented as a general-purpose toolbox using the PyGMO Python package to make the most of multicore computers and high performance clusters, and is available at https://gitlab.com/Gustafsson-lab/lassim. As a method, LASSIM works in two steps, where it first infers a non-linear ODE system of the pre-specified core gene expression. Second, LASSIM in parallel optimizes the parameters that model the regulation of peripheral genes by core system genes. We showed the usefulness of this method by applying LASSIM to infer a large-scale non-linear model of naive Th2 cell differentiation, made possible by integrating Th2 specific bindings, time-series together with six public and six novel siRNA-mediated knock-down experiments. ChIP-seq showed significant overlap for all tested transcription factors. Next, we performed novel time-series measurements of total T-cells during differentiation towards Th2 and verified that our LASSIM model could monitor those data significantly better than comparable models that used the same Th2 bindings. In summary, the LASSIM toolbox opens the door to a new type of model-based data analysis that combines the strengths of reliable mechanistic models with truly systems-level data. We demonstrate the power of this approach by inferring a mechanistically motivated, genome-wide model of the Th2 transcription regulatory system, which plays an important role in several immune related diseases.

Authors: R. Magnusson, G. P. Mariotti, M. Kopsen, W. Lovfors, D. R. Gawel, R. Jornsten, J. Linde, T. E. M. Nordling, E. Nyman, S. Schulze, C. E. Nestor, H. Zhang, G. Cedersund, M. Benson, A. Tjarnberg, M. Gustafsson

Date Published: 24th Jun 2017

Publication Type: Not specified

Specific and Novel microRNAs Are Regulated as Response to Fungal Infection in Human Dendritic Cells.

Abstract (Expand)

Within the last two decades, the incidence of invasive fungal infections has been significantly increased. They are characterized by high mortality rates and are often caused by Candida albicans and … Aspergillus fumigatus. The increasing number of infections underlines the necessity for additional anti-fungal therapies, which require extended knowledge of gene regulations during fungal infection. MicroRNAs are regulators of important cellular processes, including the immune response. By analyzing their regulation and impact on target genes, novel therapeutic and diagnostic approaches may be developed. Here, we examine the role of microRNAs in human dendritic cells during fungal infection. Dendritic cells represent the bridge between the innate and the adaptive immune systems. Therefore, analysis of gene regulation of dendritic cells is of particular significance. By applying next-generation sequencing of small RNAs, we quantify microRNA expression in monocyte-derived dendritic cells after 6 and 12 h of infection with C. albicans and A. fumigatus as well as treatment with lipopolysaccharides (LPS). We identified 26 microRNAs that are differentially regulated after infection by the fungi or LPS. Three and five of them are specific for fungal infections after 6 and 12 h, respectively. We further validated interactions of miR-132-5p and miR-212-5p with immunological relevant target genes, such as FKBP1B, KLF4, and SPN, on both RNA and protein level. Our results indicate that these microRNAs fine-tune the expression of immune-related target genes during fungal infection. Beyond that, we identified previously undiscovered microRNAs. We validated three novel microRNAs via qRT-PCR. A comparison with known microRNAs revealed possible relations with the miR-378 family and miR-1260a/b for two of them, while the third one features a unique sequence with no resemblance to known microRNAs. In summary, this study analyzes the effect of known microRNAs in dendritic cells during fungal infections and proposes novel microRNAs that could be experimentally verified.

Authors: A. Dix, K. Czakai, I. Leonhardt, K. Schaferhoff, M. Bonin, R. Guthke, H. Einsele, O. Kurzai, J. Loffler, J. Linde

Date Published: 11th Mar 2017

Publication Type: Not specified

Smelling the difference: Transcriptome, proteome and volatilome changes after mating

Abstract (Expand)

Mushrooms, such as Schizophyllum commune, have a specific odor. Whether this is linked to mating, prerequisite for mushroom formation, or also found in monokaryotic, unmated strains, was investigated … with a comprehensive study on the transcriptome and proteome of this model organism. Mating interactions were investigated using a complete, cytosolic proteome map for unmated, monokaryotic, as well as for mated, dikaryotic mycelia. The regulations of the proteome were compared to transcriptional changes upon mating and to changes in smell by volatilome studies. We could show a good overlap between proteome and transcriptome data, but extensive posttranslational regulation was identified for more than 80% of transcripts. This suggests down-stream regulation upon interaction of mating partners and formation of the dikaryon that is competent to form fruiting bodies. The volatilome was shown to respond to mating by a broader spectrum of volatiles and increased emission of the mushroom smell molecules 3-octanone and 1-octen-3-ol, as well as ethanol and beta-bisabolol in the dikaryon. Putatively involved biosynthetic proteins like alcohol dehydrogenases, Ppo-like oxygenases, or sesquiterpene synthases showed correlating transcriptional regulation depending on either mono- or dikaryotic stages.

Authors: D. Freihorst, M. Brunsch, S. Wirth, K. Krause, , , M. Kunert, W. Boland, E. Kothe

Date Published: 7th Sep 2016

Publication Type: Not specified

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