Daniela Albrecht

Integrative analysis of the heat shock response in Aspergillus fumigatus

BackgroundAspergillus fumigatus is a thermotolerant human-pathogenic mold and the most common cause of invasive aspergillosis (IA) in immunocompromised patients. Its predominance is based on several factors most of which are still unknown. The thermotolerance of A. fumigatus is one of the traits which have been assigned to pathogenicity. It allows the fungus to grow at temperatures up to and above that of a fevered human host. To elucidate the mechanisms of heat resistance, we analyzed the change of the A. fumigatus proteome during a temperature shift from 30°C to 48°C by 2D-fluorescence difference gel electrophoresis (DIGE). To improve 2D gel image analysis results, protein spot quantitation was optimized by missing value imputation and normalization. Differentially regulated proteins were compared to previously published transcriptome data of A. fumigatus. The study was augmented by bioinformatical analysis of transcription factor binding sites (TFBSs) in the promoter region of genes whose corresponding proteins were differentially regulated upon heat shock.Results91 differentially regulated protein spots, representing 64 different proteins, were identified by mass spectrometry (MS). They showed a continuous up-, down- or an oscillating regulation. Many of the identified proteins were involved in protein folding (chaperones), oxidative stress response, signal transduction, transcription, translation, carbohydrate and nitrogen metabolism. A correlation between alteration of transcript levels and corresponding proteins was detected for half of the differentially regulated proteins. Interestingly, some previously undescribed putative targets for the heat shock regulator Hsf1 were identified. This provides evidence for Hsf1-dependent regulation of mannitol biosynthesis, translation, cytoskeletal dynamics and cell division in A. fumigatus. Furthermore, computational analysis of promoters revealed putative binding sites for an AP-2alpha-like transcription factor upstream of some heat shock induced genes. Until now, this factor has only been found in vertebrates.ConclusionsOur newly established DIGE data analysis workflow yields improved data quality and is widely applicable for other DIGE datasets. Our findings suggest that the heat shock response in A. fumigatus differs from already well-studied yeasts and other filamentous fungi.

FungiFun: A web-based application for functional categorization of fungal genes and proteins

FungiFun assigns functional annotations to fungal genes or proteins and performs gene set enrichment analysis. Based on three different classification methods (FunCat, GO and KEGG), FungiFun categorizes genes and proteins for several fungal species on different levels of annotation detail. It is web-based and accessible to users without any programming skills. FungiFun is the first tool offering gene set enrichment analysis including the FunCat categorization. Two biological datasets for Aspergillus fumigatus and Candida albicans were analyzed using FungiFun, providing an overview of the usage and functions of the tool. FungiFun is freely accessible at https://www.omnifung.hki-jena.de/FungiFun/.

Two-dimensional proteome reference maps for the human pathogenic filamentous fungus Aspergillus fumigatus

The filamentous fungus Aspergillus fumigatus has become the most important airborne fungal pathogen causing life‐threatening infections in immunosuppressed patients. We established a 2‐D reference map for A. fumigatus. Using MALDI‐TOF‐MS/MS, we identified 381 spots representing 334 proteins. Proteins involved in cellular metabolism, protein synthesis, transport processes and cell cycle were most abundant. Furthermore, we established a protocol for the isolation of mitochondria of A. fumigatus and developed a mitochondrial proteome reference map. 147 proteins represented by 234 spots were identified.

Functional genomic profiling of Aspergillus fumigatus biofilm reveals enhanced production of the mycotoxin gliotoxin

The opportunistic pathogenic mold Aspergillus fumigatus is an increasing cause of morbidity and mortality in immunocompromized and in part immunocompetent patients. A. fumigatus can grow in multicellular communities by the formation of a hyphal network encased in an extracellular matrix. Here, we describe the proteome and transcriptome of planktonic‐ and biofilm‐grown A. fumigatus mycelium after 24 and 48 h. A biofilm‐ and time‐dependent regulation of many proteins and genes of the primary metabolism indicates a developmental stage of the young biofilm at 24 h, which demands energy. At a matured biofilm phase, metabolic activity seems to be reduced. However, genes, which code for hydrophobins, and proteins involved in the biosynthesis of secondary metabolites were significantly upregulated. In particular, proteins of the gliotoxin secondary metabolite gene cluster were induced in biofilm cultures. This was confirmed by real‐time PCR and by detection of this immunologically active mycotoxin in culture supernatants using HPLC analysis. The enhanced production of gliotoxin by in vitro formed biofilms reported here may also play a significant role under in vivo conditions. It may confer A. fumigatus protection from the host immune system and also enable its survival and persistence in chronic lung infections such as aspergilloma.

Missing values in gel-based proteomics.

Gel‐based proteomics is a widely applied technique to measure abundances of proteins in various biological systems. Comparison of two or more biological groups involves matching of 2‐D gels. Depending on the software, this can result in spots showing missing values on several gels. Most studies ignore this fact or substitute all missing data by zero. Since a couple of years, scientists have realized that this is not the optimal way of analyzing their data and several studies were published presenting methods of imputing missing proteomics data. Most of these methods have already been applied to microarray data before; the phenomenon of missing data is well known in this field, too. With this review, we intend to further raise awareness of the problem of missing values in gel‐based proteomics. We summarize reasons for missing values and explore their distribution in data sets. We also provide a comparison and evaluation of hitherto proposed imputation methods for gel‐based proteomics data.

Differential RelA- and RelB-dependent gene transcription in LTβR-stimulated mouse embryonic fibroblasts

BackgroundLymphotoxin signaling via the lymphotoxin-β receptor (LTβR) has been implicated in biological processes ranging from development of secondary lymphoid organs, maintenance of spleen architecture, host defense against pathogens, autoimmunity, and lipid homeostasis. The major transcription factor that is activated by LTβR crosslinking is NF-κB. Two signaling pathways have been described, the classical inhibitor of NF-κB α (IκBα)-regulated and the alternative p100-regulated pathway that result in the activation of p50-RelA and p52-RelB NF-κB heterodimers, respectively.ResultsUsing microarray analysis, we investigated the transcriptional response downstream of the LTβR in mouse embryonic fibroblasts (MEFs) and its regulation by the RelA and RelB subunits of NF-κB. We describe novel LTβR-responsive genes that were regulated by RelA and/or RelB. The majority of LTβR-regulated genes required the presence of both RelA and RelB, revealing significant crosstalk between the two NF-κB activation pathways. Gene Ontology (GO) analysis confirmed that LTβR-NF-κB target genes are predominantly involved in the regulation of immune responses. However, other biological processes, such as apoptosis/cell death, cell cycle, angiogenesis, and taxis were also regulated by LTβR signaling. Moreover, LTβR activation inhibited expression of a key adipogenic transcription factor, peroxisome proliferator activated receptor-γ (pparg), suggesting that LTβR signaling may interfere with adipogenic differentiation.ConclusionMicroarray analysis of LTβR-stimulated fibroblasts provided comprehensive insight into the transcriptional response of LTβR signaling and its regulation by the NF-κB family members RelA and RelB.

Discovery of gene regulatory networks in aspergillus fumigatus

Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immuno suppressed patients. During the infection process, A. fumigatus has to cope with a dramatic change of environmental conditions, such as temperature shifts. Recently, gene expression data monitoring the stress response to a temperature shift from 30°C to 48°C was published. In the present work, these data were analyzed by reverse engineering to discover gene regulatory mechanisms of temperature resistance of A. fumigatus. Time series data, i.e. expression profiles of 1926 differentially expressed genes, were clustered by fuzzy c-means. The number of clusters was optimized using a set of optimization criteria. From each cluster a representative gene was selected by text mining in the gene descriptions and evaluating gene ontology terms. The expression profiles of these genes were simulated by a differential equation system, whose structure and parameters were optimized minimizing both the number of non-vanishing parameters and the mean square error of model fit to the microarray data.

Integration of Transcriptome and Proteome Data from Human-Pathogenic Fungi by Using a Data Warehouse

Summary A data warehouse for the integrated storage and visualisation of genome and experimental transcriptome and proteome data of human-pathogenic fungi was established. It provides tools for uploading images and corresponding data from microarray experiments, two-dimensional (2D) gel experiments and mass spectrometry (MS) analyses. All data are cross-linked. A user can find out, on which gels in the database an interesting protein was detected. Additionally, he can see on which microarrays the corresponding mRNA had been spotted and whether these spots show interesting intensity values. So the data warehouse enables an integrated analysis of both transcriptome and proteome data. Some of the uploaded data were transcriptome and proteome time series data of temperature shift experiments obtained from Aspergillus fumigatus. Several proteins were differentially regulated at different times after the temperature shift. For a couple of them also the respective transcripts were found to be differentially expressed. For even more of those proteins the transcripts did not show differential regulation and vice versa. So both kinds of data clearly complement each other and should be analysed together.

p100 Deficiency Is Insufficient for Full Activation of the Alternative NF-κB Pathway: TNF Cooperates with p52-RelB in Target Gene Transcription

Background Constitutive activation of the alternative NF-κB pathway leads to marginal zone B cell expansion and disorganized spleen microarchitecture. Furthermore, uncontrolled alternative NF-κB signaling may result in the development and progression of cancer. Here, we focused on the question how does the constitutive alternative NF-κB signaling exert its effects in these malignant processes. Methodology/Principal Findings To explore the consequences of unrestricted alternative NF-κB activation on genome-wide transcription, we compared gene expression profiles of wild-type and NF-κB2/p100-deficient (p100−/−) primary mouse embryonic fibroblasts (MEFs) and spleens. Microarray experiments revealed only 73 differentially regulated genes in p100−/− vs. wild-type MEFs. Chromatin immunoprecipitation (ChIP) assays showed in p100−/− MEFs direct binding of p52 and RelB to the promoter of the Enpp2 gene encoding ENPP2/Autotaxin, a protein with an important role in lymphocyte homing and cell migration. Gene ontology analysis revealed upregulation of genes with anti-apoptotic/proliferative activity (Enpp2/Atx, Serpina3g, Traf1, Rrad), chemotactic/locomotory activity (Enpp2/Atx, Ccl8), and lymphocyte homing activity (Enpp2/Atx, Cd34). Most importantly, biochemical and gene expression analyses of MEFs and spleen, respectively, indicated a marked crosstalk between classical and alternative NF-κB pathways. Conclusions/Significance Our results show that p100 deficiency alone was insufficient for full induction of genes regulated by the alternative NF-κB pathway. Moreover, alternative NF-κB signaling strongly synergized both in vitro and in vivo with classical NF-κB activation, thereby extending the number of genes under the control of the p100 inhibitor of the alternative NF-κB signaling pathway.

On the way toward systems biology of Aspergillus fumigatus infection

Pathogenicity of Aspergillus fumigatus is multifactorial. Thus, global studies are essential for the understanding of the infection process. Therefore, a data warehouse was established where genome sequence, transcriptome and proteome data are stored. These data are analyzed for the elucidation of virulence determinants. The data analysis workflow starts with pre-processing including imputing of missing values and normalization. Last step is the identification of differentially expressed genes/proteins as interesting candidates for further analysis, in particular for functional categorization and correlation studies. Sequence data and other prior knowledge extracted from databases are integrated to support the inference of gene regulatory networks associated with pathogenicity. This knowledge-assisted data analysis aims at establishing mathematical models with predictive strength to assist further experimental work. Recently, first steps were done to extend the integrative data analysis and computational modeling by evaluating spatio-temporal data (movies) that monitor interactions of A. fumigatus morphotypes (e.g. conidia) with host immune cells.