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Proteomics of Aspergillus fumigatus Conidia-containing Phagolysosomes Identifies Processes Governing Immune Evasion.
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Abstract (Expand)

Invasive infections by the human pathogenic fungus Aspergillus fumigatus start with the outgrowth of asexual, airborne spores (conidia) into the lung tissue of immunocompromised patients. The resident … alveolar macrophages phagocytose conidia, which end up in phagolysosomes. However, A. fumigatus conidia resist phagocytic degradation to a certain degree. This is mainly attributable to the pigment 1,8-dihydroxynaphthalene (DHN) melanin located in the cell wall of conidia, which manipulates the phagolysosomal maturation and prevents their intracellular killing. To get insight in the underlying molecular mechanisms, we comparatively analyzed proteins of mouse macrophage phagolysosomes containing melanized wild-type (wt) or nonmelanized pksP mutant conidia. For this purpose, a protocol to isolate conidia-containing phagolysosomes was established and a reference protein map of phagolysosomes was generated. We identified 637 host and 22 A. fumigatus proteins that were differentially abundant in the phagolysosome. 472 of the host proteins were overrepresented in the pksP mutant and 165 in the wt conidia-containing phagolysosome. Eight of the fungal proteins were produced only in pksP mutant and 14 proteins in wt conidia-containing phagolysosomes. Bioinformatical analysis compiled a regulatory module, which indicates host processes affected by the fungus. These processes include vATPase-driven phagolysosomal acidification, Rab5 and Vamp8-dependent endocytic trafficking, signaling pathways, as well as recruitment of the Lamp1 phagolysosomal maturation marker and the lysosomal cysteine protease cathepsin Z. Western blotting and immunofluorescence analyses confirmed the proteome data and moreover showed differential abundance of the major metabolic regulator mTOR. Taken together, with the help of a protocol optimized to isolate A. fumigatus conidia-containing phagolysosomes and a potent bioinformatics algorithm, we were able to confirm A. fumigatus conidia-dependent modification of phagolysosomal processes that have been described before and beyond that, identify pathways that have not been implicated in A. fumigatus evasion strategy, yet.Mass spectrometry proteomics data are available via ProteomeXchange with identifiers PXD005724 and PXD006134.

Authors: H. Schmidt, S. Vlaic, T. Kruger, F. Schmidt, J. Balkenhol, T. Dandekar, R. Guthke, O. Kniemeyer, T. Heinekamp, A. A. Brakhage

Date Published: 7th Mar 2018

Journal: Mol Cell Proteomics

Integrated inference and evaluation of host-fungi interaction networks.
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Abstract (Expand)

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.

Authors: C. W. Remmele, C. H. Luther, J. Balkenhol, T. Dandekar, T. Muller, M. T. Dittrich

Date Published: 25th Aug 2015

Journal: Front Microbiol

Evaluation of Aspergillus and Mucorales specific T-cells and peripheral blood mononuclear cell cytokine signatures as biomarkers of environmental mold exposure.
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Abstract (Expand)

Mold specific T-cells have been described as a supportive biomarker to monitor invasive mycoses and mold exposure. This study comparatively evaluated frequencies and cytokine profiles of Aspergillus … fumigatus and Mucorales reactive T-cells depending on environmental mold exposure. Peripheral blood mononuclear cells (PBMCs) obtained from 35 healthy donors were stimulated with mycelial lysates of A. fumigatus and three human pathogenic Mucorales species. CD154(+) specific T-cells were quantified by flow cytometry. In a second cohort of 20 additional donors, flow cytometry was complemented by 13-plex cytokine assays. Mold exposure of the subjects was determined using a previously established questionnaire. Highly exposed subjects exhibited significantly greater CD154(+)A. fumigatus and Mucorales specific naive and memory T-helper cell frequencies. Significant correlation (r = 0.48 - 0.79) was found between A. fumigatus and Mucorales specific T-cell numbers. Logistic regression analyses revealed that combined analysis of mold specific T-cell frequencies and selected cytokine markers (A. fumigatus: IL-5 and TNF-alpha, R. arrhizus: IL-17A and IL-13) significantly improves classification performance, resulting in 75-90 % predictive power using 10-fold cross-validation. In conclusion, mold specific T-cell frequencies and their cytokine signatures offer promising potential in the assessment of environmental mold exposure. The cytokines identified in this pilot study should be validated in the clinical setting, e. g. in patients with hypersensitivity pneumonitis.

Authors: L. Page, P. Weis, T. Muller, M. Dittrich, M. Lazariotou, M. Dragan, A. M. Waaga-Gasser, J. Helm, T. Dandekar, H. Einsele, J. Loffler, A. J. Ullmann, S. Wurster

Date Published: 12th Sep 2018

Journal: Int J Med Microbiol

The role of Arabidopsis ABA receptors from the PYR/PYL/RCAR family in stomatal acclimation and closure signal integration.
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Abstract (Expand)

Stomata are microscopic pores found on the surfaces of leaves that act to control CO2 uptake and water loss. By integrating information derived from endogenous signals with cues from the surrounding … environment, the guard cells, which surround the pore, 'set' the stomatal aperture to suit the prevailing conditions. Much research has concentrated on understanding the rapid intracellular changes that result in immediate changes to the stomatal aperture. In this study, we look instead at how stomata acclimate to longer timescale variations in their environment. We show that the closure-inducing signals abscisic acid (ABA), increased CO2, decreased relative air humidity and darkness each access a unique gene network made up of clusters (or modules) of common cellular processes. However, within these networks some gene clusters are shared amongst all four stimuli. All stimuli modulate the expression of members of the PYR/PYL/RCAR family of ABA receptors. However, they are modulated differentially in a stimulus-specific manner. Of the six members of the PYR/PYL/RCAR family expressed in guard cells, PYL2 is sufficient for guard cell ABA-induced responses, whereas in the responses to CO2, PYL4 and PYL5 are essential. Overall, our work shows the importance of ABA as a central regulator and integrator of long-term changes in stomatal behaviour, including sensitivity, elicited by external signals. Understanding this architecture may aid in breeding crops with improved water and nutrient efficiency.

Authors: M. Dittrich, H. M. Mueller, H. Bauer, M. Peirats-Llobet, P. L. Rodriguez, C. M. Geilfus, S. C. Carpentier, K. A. S. Al Rasheid, H. Kollist, E. Merilo, J. Herrmann, T. Muller, P. Ache, A. M. Hetherington, R. Hedrich

Date Published: 28th Aug 2019

Journal: Nat Plants

Comparison of the central human and mouse platelet signaling cascade by systems biological analysis.
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Abstract (Expand)

BACKGROUND: Understanding the molecular mechanisms of platelet activation and aggregation is of high interest for basic and clinical hemostasis and thrombosis research. The central platelet protein … interaction network is involved in major responses to exogenous factors. This is defined by systemsbiological pathway analysis as the central regulating signaling cascade of platelets (CC). RESULTS: The CC is systematically compared here between mouse and human and major differences were found. Genetic differences were analysed comparing orthologous human and mouse genes. We next analyzed different expression levels of mRNAs. Considering 4 mouse and 7 human high-quality proteome data sets, we identified then those major mRNA expression differences (81%) which were supported by proteome data. CC is conserved regarding genetic completeness, but we observed major differences in mRNA and protein levels between both species. Looking at central interactors, human PLCB2, MMP9, BDNF, ITPR3 and SLC25A6 (always Entrez notation) show absence in all murine datasets. CC interactors GNG12, PRKCE and ADCY9 occur only in mice. Looking at the common proteins, TLN1, CALM3, PRKCB, APP, SOD2 and TIMP1 are higher abundant in human, whereas RASGRP2, ITGB2, MYL9, EIF4EBP1, ADAM17, ARRB2, CD9 and ZYX are higher abundant in mouse. Pivotal kinase SRC shows different regulation on mRNA and protein level as well as ADP receptor P2RY12. CONCLUSIONS: Our results highlight species-specific differences in platelet signaling and points of specific fine-tuning in human platelets as well as murine-specific signaling differences.

Authors: J. Balkenhol, K. V. Kaltdorf, E. Mammadova-Bach, A. Braun, B. Nieswandt, M. Dittrich, T. Dandekar

Date Published: 22nd Dec 2020

Journal: BMC Genomics

Identification of Antifungal Targets Based on Computer Modeling.
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Abstract (Expand)

Aspergillus fumigatus is a saprophytic, cosmopolitan fungus that attacks patients with a weak immune system. A rational solution against fungal infection aims to manipulate fungal metabolism or to block … enzymes essential for Aspergillus survival. Here we discuss and compare different bioinformatics approaches to analyze possible targeting strategies on fungal-unique pathways. For instance, phylogenetic analysis reveals fungal targets, while domain analysis allows us to spot minor differences in protein composition between the host and fungi. Moreover, protein networks between host and fungi can be systematically compared by looking at orthologs and exploiting information from host(-)pathogen interaction databases. Further data—such as knowledge of a three-dimensional structure, gene expression data, or information from calculated metabolic fluxes—refine the search and rapidly put a focus on the best targets for antimycotics. We analyzed several of the best targets for application to structure-based drug design. Finally, we discuss general advantages and limitations in identification of unique fungal pathways and protein targets when applying bioinformatics tools.

Authors: E. Bencurova, S. K. Gupta, E. Sarukhanyan, T. Dandekar

Date Published: 4th Jul 2018

Journal: J Fungi (Basel)

Analyzing pharmacological intervention points: A method to calculate external stimuli to switch between steady states in regulatory networks.
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Abstract (Expand)

Once biological systems are modeled by regulatory networks, the next step is to include external stimuli, which model the experimental possibilities to affect the activity level of certain network's … nodes, in a mathematical framework. Then, this framework can be interpreted as a mathematical optimal control framework such that optimization algorithms can be used to determine external stimuli which cause a desired switch from an initial state of the network to another final state. These external stimuli are the intervention points for the corresponding biological experiment to obtain the desired outcome of the considered experiment. In this work, the model of regulatory networks is extended to controlled regulatory networks. For this purpose, external stimuli are considered which can affect the activity of the network's nodes by activation or inhibition. A method is presented how to calculate a selection of external stimuli which causes a switch between two different steady states of a regulatory network. A software solution based on Jimena and Mathworks Matlab is provided. Furthermore, numerical examples are presented to demonstrate application and scope of the software on networks of 4 nodes, 11 nodes and 36 nodes. Moreover, we analyze the aggregation of platelets and the behavior of a basic T-helper cell protein-protein interaction network and its maturation towards Th0, Th1, Th2, Th17 and Treg cells in accordance with experimental data.

Authors: T. Breitenbach, C. Liang, N. Beyersdorf, T. Dandekar

Date Published: 17th Jul 2019

Journal: PLoS Comput Biol

The Definition of Open Reading Frame Revisited.
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Abstract (Expand)

The term open reading frame (ORF) is of central importance to gene finding. Surprisingly, at least three definitions are in use. We discuss several molecular biological and bioinformatics aspects, and … we recommend using the definition in which an ORF is bounded by stop codons.

Authors: Patricia Sieber, M. Platzer, Stefan Schuster

Date Published: 26th Jan 2018

Journal: Trends Genet

Salt generates antiinflammatory Th17 cells but amplifies pathogenicity in proinflammatory cytokine microenvironments.
FungiNet C - Candida projects, C5

Abstract (Expand)

Th cells integrate signals from their microenvironment to acquire distinct specialization programs for efficient clearance of diverse pathogens or for immunotolerance. Ionic signals have recently been … demonstrated to affect T cell polarization and function. Sodium chloride (NaCl) was proposed to accumulate in peripheral tissues upon dietary intake and to promote autoimmunity via the Th17 cell axis. Here, we demonstrate that high-NaCl conditions induced a stable, pathogen-specific, antiinflammatory Th17 cell fate in human T cells in vitro. The p38/MAPK pathway, involving NFAT5 and SGK1, regulated FoxP3 and IL-17A expression in high-NaCl conditions. The NaCl-induced acquisition of an antiinflammatory Th17 cell fate was confirmed in vivo in an experimental autoimmune encephalomyelitis (EAE) mouse model, which demonstrated strongly reduced disease symptoms upon transfer of T cells polarized in high-NaCl conditions. However, NaCl was coopted to promote murine and human Th17 cell pathogenicity, if T cell stimulation occurred in a proinflammatory and TGF-beta-low cytokine microenvironment. Taken together, our findings reveal a context-dependent, dichotomous role for NaCl in shaping Th17 cell pathogenicity. NaCl might therefore prove beneficial for the treatment of chronic inflammatory diseases in combination with cytokine-blocking drugs.

Authors: J. Matthias, S. Heink, F. Picard, J. Zeitrag, A. Kolz, Y. Y. Chao, D. Soll, G. P. de Almeida, E. Glasmacher, Ilse Jacobsen, T. Riedel, A. Peters, S. Floess, J. Huehn, D. Baumjohann, M. Huber, T. Korn, C. E. Zielinski

Date Published: 1st Sep 2020

Journal: J Clin Invest

The impact of the Fungus-Host-Microbiota interplay upon Candida albicans infections: current knowledge and new perspectives.
FungiNet C - Candida projects, C5

Abstract (Expand)

Candida albicans is a major fungal pathogen of humans. It exists as a commensal in the oral cavity, gut or genital tract of most individuals, constrained by the local microbiota, epithelial barriers … and immune defences. Their perturbation can lead to fungal outgrowth and the development of mucosal infections such as oropharyngeal or vulvovaginal candidiasis, and patients with compromised immunity are susceptible to life-threatening systemic infections. The importance of the interplay between fungus, host and microbiota in driving the transition from C. albicans commensalism to pathogenicity is widely appreciated. However, the complexity of these interactions, and the significant impact of fungal, host and microbiota variability upon disease severity and outcome, are less well understood. Therefore, we summarise the features of the fungus that promote infection, and how genetic variation between clinical isolates influences pathogenicity. We discuss antifungal immunity, how this differs between mucosae, and how individual variation influences a person's susceptibility to infection. Also, we describe factors that influence the composition of gut, oral and vaginal microbiotas, and how these affect fungal colonisation and antifungal immunity. We argue that a detailed understanding of these variables, which underlie fungal-host-microbiota interactions, will present opportunities for directed antifungal therapies that benefit vulnerable patients.

Authors: C. d'Enfert, A. K. Kaune, L. R. Alaban, S. Chakraborty, N. Cole, M. Delavy, D. Kosmala, B. Marsaux, R. Frois-Martins, M. Morelli, D. Rosati, M. Valentine, Z. Xie, Y. Emritloll, P. A. Warn, F. Bequet, M. E. Bougnoux, S. Bornes, Mark Gresnigt, Bernhard Hube, Ilse Jacobsen, M. Legrand, S. Leibundgut-Landmann, C. Manichanh, C. A. Munro, M. G. Netea, K. Queiroz, K. Roget, V. Thomas, C. Thoral, P. Van den Abbeele, A. W. Walker, A. J. P. Brown

Date Published: 24th Nov 2020

Journal: FEMS Microbiol Rev

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