Systems Biology of Fungal Infection

Elucidation of pathogenicity mechanisms of the most important human-pathogenic fungi, Aspergillus fumigatus and Candida albicans, has gained great interest in the light of the steadily increasing number of cases of invasive fungal infections. A key feature of these infections is the interaction of the different fungal morphotypes with epithelial and immune effector cells in the human host. Because of the high level of complexity, it is necessary to describe and understand invasive fungal infection by taking a systems biological approach, i.e., by a comprehensive quantitative analysis of the non-linear and selective interactions of a large number of functionally diverse, and frequently multifunctional, sets of elements, e.g., genes, proteins, metabolites, which produce coherent and emergent behaviors in time and space. The recent advances in systems biology will now make it possible to uncover the structure and dynamics of molecular and cellular cause-effect relationships within these pathogenic interactions. We review current efforts to integrate omics and image-based data of host-pathogen interactions into network and spatio-temporal models. The modeling will help to elucidate pathogenicity mechanisms and to identify diagnostic biomarkers and potential drug targets for therapy and could thus pave the way for novel intervention strategies based on novel antifungal drugs and cell therapy

Integrative analysis of the heat shock response in Aspergillus fumigatus

<p>Abstract</p> <p>Background</p> <p><it>Aspergillus fumigatus </it>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 <it>A. fumigatus </it>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 <it>A. fumigatus </it>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 <it>A. fumigatus</it>. 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.</p> <p>Results</p> <p>91 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 <it>A. fumigatus</it>. 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.</p> <p>Conclusions</p> <p>Our 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 <it>A. fumigatus </it>differs from already well-studied yeasts and other filamentous fungi.</p

Conidial Dihydroxynaphthalene Melanin of the Human Pathogenic Fungus Aspergillus fumigatus Interferes with the Host Endocytosis Pathway

Aspergillus fumigatus is the most important air-borne fungal pathogen of humans. The interaction of the pathogen with the host's immune system represents a key process to understand pathogenicity. For elimination of invading microorganisms, they need to be efficiently phagocytosed and located in acidified phagolysosomes. However, as shown previously, A. fumigatus is able to manipulate the formation of functional phagolysosomes. Here, we demonstrate that in contrast to pigmentless pksP mutant conidia of A. fumigatus, the gray-green wild-type conidia inhibit the acidification of phagolysosomes of alveolar macrophages, monocyte-derived macrophages, and human neutrophil granulocytes. Therefore, this inhibition is independent of the cell type and applies to the major immune effector cells required for defense against A. fumigatus. Studies with melanin ghosts indicate that the inhibitory effect of wild-type conidia is due to their dihydroxynaphthalene (DHN)-melanin covering the conidia, whereas the hydrophobin RodA rodlet layer plays no role in this process. This is also supported by the observation that pksP conidia still exhibit the RodA hydrophobin layer, as shown by scanning electron microscopy. Mutants defective in different steps of the DHN-melanin biosynthesis showed stronger inhibition than pksP mutant conidia but lower inhibition than wild-type conidia. Moreover, A. fumigatus and A. flavus led to a stronger inhibition of phagolysosomal acidification than A. nidulans and A. terreus. These data indicate that a certain type of DHN-melanin that is different in the various Aspergillus species, is required for maximal inhibition of phagolysosomal acidification. Finally, we identified the vacuolar ATPase (vATPase) as potential target for A. fumigatus based on the finding that addition of bafilomycin which inhibits vATPase, led to complete inhibition of the acidification whereas the fusion of phagosomes containing wild-type conidia and lysosomes was not affected

Phagocytosis of Aspergillus fumigatus conidia by murine macrophages involves recognition by the dectin-1 beta-glucan receptor and Toll-like receptor 2

Aspergillus fumigatus is a fungal pathogen causing severe infections in immunocompromised patients. For clearance of inhaled conidia, an efficient response of the innate immune system is required. Macrophages represent the first line of defence and ingest and kill conidia. C-type lectins represent a family of receptors, which recognize pathogen-specific carbohydrates. One of them is beta1-3 glucan, a major component of the fungal cell wall. Here we provide evidence that beta1-3 glucan plays an important role for the elimination of A. fumigatus conidia. Laminarin, a soluble beta1-3 glucan and antibodies to dectin-1, a well known beta1-3 glucan receptor, significantly inhibited conidial phagocytosis. On resting conidia low amounts of surface accessible beta1-3 glucan were detected, whereas high amounts were found on small spores that appear early during germination and infection as well as on resting conidia of a pksP mutant strain. Swollen conidia also display larger quantities of beta1-3 glucan, although in an irregular spotted pattern. Resting pksP mutant conidia and swollen wild-type conidia are phagocytosed with high efficiency thereby confirming the relevance of beta1-3 glucans for conidial phagocytosis. Additionally we found that TLR2 and the adaptor protein MyD88 are required for efficient conidial phagocytosis, suggesting a link between the TLR2-mediated recognition of A. fumigatus and the phagocytic response

Automated Image Analysis of the Host-Pathogen Interaction between Phagocytes and Aspergillus fumigatus

Aspergillus fumigatus is a ubiquitous airborne fungus and opportunistic human pathogen. In immunocompromised hosts, the fungus can cause life-threatening diseases like invasive pulmonary aspergillosis. Since the incidence of fungal systemic infections drastically increased over the last years, it is a major goal to investigate the pathobiology of A. fumigatus and in particular the interactions of A. fumigatus conidia with immune cells. Many of these studies include the activity of immune effector cells, in particular of macrophages, when they are confronted with conidia of A. fumigus wild-type and mutant strains. Here, we report the development of an automated analysis of confocal laser scanning microscopy images from macrophages coincubated with different A. fumigatus strains. At present, microscopy images are often analysed manually, including cell counting and determination of interrelations between cells, which is very time consuming and error-prone. Automation of this process overcomes these disadvantages and standardises the analysis, which is a prerequisite for further systems biological studies including mathematical modeling of the infection process. For this purpose, the cells in our experimental setup were differentially stained and monitored by confocal laser scanning microscopy. To perform the image analysis in an automatic fashion, we developed a ruleset that is generally applicable to phagocytosis assays and in the present case was processed by the software Definiens Developer XD. As a result of a complete image analysis we obtained features such as size, shape, number of cells and cell-cell contacts. The analysis reported here, reveals that different mutants of A. fumigatus have a major influence on the ability of macrophages to adhere and to phagocytose the respective conidia. In particular, we observe that the phagocytosis ratio and the aggregation behaviour of pksP mutant compared to wild-type conidia are both significantly increased

Redox Proteomic Analysis Reveals Oxidative Modifications of Proteins by Increased Levels of Intracellular Reactive Oxygen Species During Hypoxia Adaptation of Aspergillus fumigatus

We thank Silke Steinbach, Till Kindel, and Michael Cyrulies for their excellent technical assistance. Work of T.K., O.K. and A.A.B was supported by the Deutsche Forschungsge-meinschaft within the Collaborative Research Center TR124 FungiNet (project A1 and Z2).The work of E.S. was supported by the International Leibniz Research School for Microbial and Biomolecular Interactions (ILRS)and by the Medical Research Council Centre for Medical Mycology at the University of Aberdeen (MR/N006364/1).We thank Matthew Blango and Falk Hillmann for the critical reading of the manuscript.Peer reviewedPostprin

Salt and Metal Tolerance Involves Formation of Guttation Droplets in Species of the Aspergillus versicolor Complex

Three strains of the Aspergillus versicolor complex were isolated from a salty marsh at a former uranium mining site in Thuringia, Germany. The strains from a metal-rich environment were not only highly salt tolerant (up to 20% NaCl), but at the same time could sustain elevated Cs and Sr (both up to 100 mM) concentrations as well as other (heavy) metals present in the environment. During growth experiments when screening for differential cell morphology, the occurrence of guttation droplets was observed, specifically when elevated Sr concentrations of 25 mM were present in the media. To analyze the potential of metal tolerance being promoted by these excretions, proteomics and metabolomics of guttation droplets were performed. Indeed, proteins involved in up-regulated metabolic activities as well as in stress responses were identified. The metabolome verified the presence of amino sugars, glucose homeostasis-regulating substances, abscisic acid and bioactive alkaloids, flavones and quinones

Monte Carlo studies of two-dimensional polymer–solvent systems

<div><p>Allergic bronchopulmonary aspergillosis (ABPA) in asthma is a severe, life-affecting disease that potentially affects over 4.8 million people globally. In the UK, ABPA is predominantly caused by the fungus <i>Aspergillus fumigatus</i>. Phagocytosis is important in clearance of this fungus, and Early Endosome Antigen 1 (<i>EEA1</i>) has been demonstrated to be involved in phagocytosis of fungi. We sought to investigate the role of <i>EEA1</i> mutations and phagocytosis in ABPA. We used exome sequencing to identify variants in <i>EEA1</i> associated with ABPA. We then cultured monocyte-derived macrophages (MDMs) from 17 ABPA subjects with <i>A</i>. <i>fumigatus</i> conidia, and analyzed phagocytosis and phagolysosome acidification in relation to the presence of these variants. We found that variants in <i>EEA1</i> were associated with ABPA and with the rate of phagocytosis of <i>A</i>. <i>fumigatus</i> conidia and the acidification of phagolysosomes. MDMs from ABPA subjects carrying the disease associated genotype showed increased acidification and phagocytosis compared to those from ABPA subjects carrying the non-associated genotypes or healthy controls.The identification of ABPA-associated variants in EEA that have functional effects on MDM phagocytosis and phagolysosome acidification of <i>A</i>. <i>fumigatus</i> conidia revolutionizes our understanding of susceptibility to this disease, which may in future benefit patients by earlier identification or improved treatments. We suggest that the increased phagocytosis and acidification observed demonstrates an over-active MDM profile in these patients, resulting in an exaggerated cellular response to the presence of <i>A</i>. <i>fumigatus</i> in the airways.</p></div