Functional genomic profiling of Aspergillus fumigatus biofilm reveals enhanced production of the mycotoxin gliotoxin
ABSTRACT: The opportunistic pathogenic mold Aspergillus fumigatus is an increasing cause of morbidity and mortality in immunocompromised and, in part, immunocompetent patients. Like bacteria or yeast, 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 24h and 48h. A biofilm- and time-dependent regulation of many proteins and genes of the primary metabolism indicates a developmental stage of the young biofilm at 24h, which demands energy. At a matured biofilm phase, metabolic activity seems to be reduced. However, genes encoding 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 RT-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 play also 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. Overall design: Comparison of biofilm and submers cultures at 24h and 48h after induction.
INSTRUMENT(S): JCVI PFGRC Aspergillus fumigatus 22K v3 array designed primarily based on strain Af293
Project description:The opportunistic pathogenic mold Aspergillus fumigatus is an increasing cause of morbidity and mortality in immunocompromised and, in part, immunocompetent patients. Like bacteria or yeast, 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 24h and 48h. A biofilm- and time-dependent regulation of many proteins and genes of the primary metabolism indicates a developmental stage of the young biofilm at 24h, which demands energy. At a matured biofilm phase, metabolic activity seems to be reduced. However, genes encoding 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 RT-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 play also 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. Comparison of biofilm and submers cultures at 24h and 48h after induction.
Project description:In patients with chronic pulmonary disease colonization with the mold Aspergillus fumigatus is associated with declining pulmonary function and obstructive airway disease. One potential effector of this inflammatory response is the pulmonary mast cell. In vitro studies have demonstrated that A. fumigatus contact induces IgE-independent mast cell degranulation. Conversely, the Aspergillus secondary metabolite gliotoxin has been shown to suppress mast cell activation. These contradictory results emphasize the need for a better understanding of the interactions between A. fumigatus and mast cells. Thus, the objective of this work was to identify A. fumigatus genes that are differentially regulated upon exposure to mast cells. Transcriptional profiling experiments indicated that, in addition to genes encoding for iron acquisition systems, allergens and putative virulence factors, genes from the gliotoxin biosynthesis cluster were significantly down-regulated upon exposure to mast cells. Globally, the results from this study provide insight into the A. fumigatus response to mast cells and suggest that one mechanism by which the host may circumvent the effects of gliotoxin is via the suppression of fungal gliotoxin synthesis by mast cells. Overall design: Aspergillus fumigatus hyphae were exposed either to RBL-2H3 mast cells or culture medium for 1.5hr or 3hrs. Hybridizations were performed with biological replicates and flip-dye pairs.
Project description:Aspergillus fumigatus is the most common opportunistic mold pathogen of humans, causing invasive diseases in immunocompromised patients. In these patients, the fungus can invade the lungs and other organs, causing severe damage. Penetration of the pulmonary epithelium is a key step in the infectious process. A. fumigatus produces extracellular proteases to degrade the structural barriers of the host. The A. fumigatus transcription factor PrtT controls the expression of multiple secreted proteases. PrtT shows similarity to the fungal Gal4-type Zn(2)-Cys(6) DNA-binding domain of several transcription factors. In this work, we further investigate the function of this transcription factor by performing a transcriptional and a proteomic analysis of the ΔprtT mutant. The microarray analysis revealed several unexpected findings. In addition to a decrease in protease expression, expression of genes involved in iron uptake and ergosterol synthesis was dramatically decreased in the ΔprtT mutant. A second finding of interest is that deletion of PrtT resulted in the upregulation of four secondary metabolite clusters, including genes for the biosynthesis of pseurotin A. The proteomic analysis identified 15 proteins secreted by the A. fumigatus WT strain when grown on protein-rich skim-milk medium. Of these, the ΔprtT mutant expressed reduced levels of ALP1 protease, TppA tripeptidyl peptidase and a serine peptidase and increased levels of FAD-oxygenase, AspF chitosinase, EglC endoglucanase and Bgt1 glucanosyltransferase compared to the WT strain. This report highlights the complexity of gene regulation by PrtT. Overall design: Three biological repeats of dual channel comparative transcriptome analysis between ΔprtT Protease-Deficient Mutant and wild type.
Project description:DCs play a central role for the immune response against the mold Aspergillus fumigatus. Hypoxic microenvironments occur during infection with A. fumigatus. Hypoxia and signaling via hypoxia inducible factor 1α may modulate the response of DCs; however, the role in fungal infections is unclear. We used microarrays to determine the influence of hypoxia and HIF-1α signaling on the immune response of human DCs towards A. fumigatus. HIF-1α silenced or non-silenced, human monocyte-derived DCs were cultivated for 6 h in normoxia or hypoxia (1 % O2) without stimulation or stimulated with inactivated germ tubes of A. fumigatus. Three independent experiments with DCs derived from different blood donors were performed. RNA was extracted and hybridized on Affymetrix microarrays.
Project description:CD14+ Monocytes from healthy volunteers were purified by MACS (negative selection) and FACSorting and either left untreated or stimulated for 24h and 48h with LPS. THP-1 cells were stimulated for 4h, 24h and 48h with LPS. Glycoproteins were captured with hydrazide chemistry and tryptic and PNGase F-released peptide fractions analyzed by MS/MS. Quantitative assessment revealed differential glycoprotein expression in activated/LPS-tolerized monocytes and naïve monocytes and THP-1 cells.
Project description:Blue mold, caused by Penicillium expansum, is responsible for postharvest losses of apple fruit, and threatens human health through production of the potent mycotoxin patulin. No major gene(s) providing resistance have as yet been identified, but recent studies indicate a quantitative control of the disease. An AryANE chip covering 60K apple transcripts was used to identify possible candidate gene(s) that are differentially regulated between resistant and susceptible cultivars upon P. expansum infection. Induction of cell wall related gene (PGIP1), and three genes involved in the ‘down-stream’ flavonoid biosynthesis pathway (CHS, FLS and LDOX), shows the fundamental role of cell wall as an important barrier, and contents of polyphenolic compounds of fruits as a quantitative components in enhancing disease resistance to blue mold. Moreover, exogenous application of Jasmonic acid hormone enhanced the defense mechanism in fruits. This is the first report linking Jasmonic acid and activation of cell wall and flavonoid pathway genes in apple fruit resistance to blue mold. Results provide an initial categorization of genes that are potentially involved in the resistance mechanism, and should be useful for developing tools for gene marker-assisted breeding of apple cultivars with an improved resistance to blue mold. Overall design: 4 cultivars (2 resistant and 2 susceptible), each in 3 technical replicates, were inoculated with fungal spores, and samples from control and inoculated fruits were taken in 3 time points.
Project description:Aspergillus fumigatus has to cope with a combination of several stress types in the human body, and the interplay between the different stress responses can significantly influence the survival of this human pathogen during the invasion of the host organism. In this study, we examined how the H2O2 induced oxidative stress response depends on iron availability. Surprisingly, the applied H2O2 treatment, which induced only a negligible stress response in iron fed cultures, deleteriously affected the fungus under iron starvation and the majority of observed transcriptome-level stress responses were characteristic only for the combined H2O2-iron starvation stress treatments. Our data suggest that the survival of the fungus highly depended on fragile balances, e.g. between siderophore and ergosterol productions or between economization on iron and production of essential iron containing proteins. The applied stress conditions also affected several processes related to virulence or drug susceptibility including secondary metabolism, zinc acquisition or antifungal drug transport. Our data clearly demonstrate that studying stress responses under single stress treatments is not sufficient at all to understand how fungal pathogens survive in a complex habitat and support the view that the evolutionary success of A. fumigatus as an opportunistic human pathogen is not the mere consequence of the productions of certain virulence factors. Importantly, this fungal pathogen is able to mount and coordinate high-complexity and outstandingly efficient responses to multiple and superpositioning stresses in various harsh habitats like the human body. Overall design: Control (untreated), hydrogen-peroxide treated, iron-starved as well as hydrogen-peroxide treated and iron-starved A. fumigatus cultures were studied in three independent experiments (3x4 RNAseq data sets).
Project description:Aspergillus fumigatus is a saprophytic mold capable of reaching the human deep respiratory system causing different diseases. In this study we tried to detect the virulence mechanisms developed by the fungus, using four days evolution murine intranasal infection model. For that, we develop three independent infections, in order to obtain three independent fungus samples from the lung of the infected mice per day study. Finally, we had 12 independent samples from 12 different infected mice.
Project description:The unfolded protein response (UPR) is a network of intracellular signaling pathways that supports the ability of the secretory pathway to maintain equilibrium between the load of proteins entering the endoplasmic reticulum (ER) and the protein folding capacity of the ER lumen. Current evidence suggests that human pathogenic fungi rely heavily on this pathway for virulence, but there is limited understanding of the mechanisms involved. The best known functional output of the UPR is transcriptional upregulation of mRNAs involved in ER homeostasis. However, this does not take into account mechanisms of translational regulation that involve differential recruitment of mRNAs to ribosomes. In this study, a global analysis of transcript-specific translational regulation was performed in the pathogenic mold Aspergillus fumigatus to determine the nature and scope of the translational response to ER stress.