Distinct Roles for Intra- and Extracellular Siderophores during Aspergillus fumigatus Infection

Siderophore biosynthesis by the highly lethal mould Aspergillus fumigatus is essential for virulence, but non-existent in humans, presenting a rare opportunity to strategize therapeutically against this pathogen. We have previously demonstrated that A. fumigatus excretes fusarinine C and triacetylfusarinine C to capture extracellular iron, and uses ferricrocin for hyphal iron storage. Here, we delineate pathways of intra- and extracellular siderophore biosynthesis and show that A. fumigatus synthesizes a developmentally regulated fourth siderophore, termed hydroxyferricrocin, employed for conidial iron storage. By inactivation of the nonribosomal peptide synthetase SidC, we demonstrate that the intracellular siderophores are required for germ tube formation, asexual sporulation, resistance to oxidative stress, catalase A activity, and virulence. Restoration of the conidial hydroxyferricrocin content partially rescues the virulence of the apathogenic siderophore null mutant ΔsidA, demonstrating an important role for the conidial siderophore during initiation of infection. Abrogation of extracellular siderophore biosynthesis following inactivation of the acyl transferase SidF or the nonribosomal peptide synthetase SidD leads to complete dependence upon reductive iron assimilation for growth under iron-limiting conditions, partial sensitivity to oxidative stress, and significantly reduced virulence, despite normal germ tube formation. Our findings reveal distinct cellular and disease-related roles for intra- and extracellular siderophores during mammalian Aspergillus infection

Ergothioneine Biosynthesis and Functionality in the Opportunistic Fungal Pathogen, Aspergillus fumigatus.

Ergothioneine (EGT; 2-mercaptohistidine trimethylbetaine) is a trimethylated and sulphurised histidine derivative which exhibits antioxidant properties. Here we report that deletion of Aspergillus fumigatus egtA (AFUA_2G15650), which encodes a trimodular enzyme, abrogated EGT biosynthesis in this opportunistic pathogen. EGT biosynthetic deficiency in A. fumigatus significantly reduced resistance to elevated H2O2 and menadione, respectively, impaired gliotoxin production and resulted in attenuated conidiation. Quantitative proteomic analysis revealed substantial proteomic remodelling in ΔegtA compared to wild-type under both basal and ROS conditions, whereby the abundance of 290 proteins was altered. Specifically, the reciprocal differential abundance of cystathionine γ-synthase and β-lyase, respectively, influenced cystathionine availability to effect EGT biosynthesis. A combined deficiency in EGT biosynthesis and the oxidative stress response regulator Yap1, which led to extreme oxidative stress susceptibility, decreased resistance to heavy metals and production of the extracellular siderophore triacetylfusarinine C and increased accumulation of the intracellular siderophore ferricrocin. EGT dissipated H2O2 in vitro, and elevated intracellular GSH levels accompanied abrogation of EGT biosynthesis. EGT deficiency only decreased resistance to high H2O2 levels which suggests functionality as an auxiliary antioxidant, required for growth at elevated oxidative stress conditions. Combined, these data reveal new interactions between cellular redox homeostasis, secondary metabolism and metal ion homeostasis

Metallothionein – overexpression as a highly significant prognostic factor in melanoma: a prospective study on 1270 patients

Metallothioneins (MT) are ubiquitous, intracellular small proteins with high affinity for heavy metal ions. In the last decades, it was shown that MT overexpression in a variety of cancers is associated with resistance to anticancer drugs and is combined with a poor prognosis. In this prospective study, we examined the role of MT overexpression in melanoma patients as a prognostic factor for progression and survival. Between 1993 and 2004, 3386 patients with primary cutaneous melanoma were investigated by using a monoclonal antibody against MT on routinely fixed, paraffin-embedded tissues. In all, 1270 patients could be followed up for further statistical analysis (Fisher's exact test, Mantel–Haenszel χ2 test, Kaplan–Meier curves). The MT data of disease-free interval and overall survival were compared univariately and multivariately in Cox regression analysis. Immunohistochemical overexpression of MT in tumour cells of patients with primary melanoma (310 of 1270; 24.4%) was associated with a higher risk for progression (117 of 167; 70.1%) and reduced survival (80 of 110; 72.7%) of the disease (P<0.0001). Similarly, Kaplan–Meier curves gave highly significant disadvantages for the MT-positive group. Univariate analysis (relative risk 7.4; 95% confidence interval (CI) 5.2–10.2; P<0.0001 for progression; relative risk 7.1; 95% CI 4.7–10.9; P<0.0001 for survival), as well as multivariate analysis with other prognostic markers resulted in MT overexpression as a highly significant and independent factor for prognosis in primary melanoma

Regulation of Petrobactin and Bacillibactin Biosynthesis in Bacillus anthracis under Iron and Oxygen Variation

siderophore biosynthetic operons that are responsible for synthesis of petrobactin and bacillibactin, during variable growth conditions., a member of the bacillibactin biosynthetic operon, was only transcribed under conditions of iron-depletion, regardless of growth aeration.

Identification of metabolic pathways influenced by the G-protein coupled receptors GprB and GprD in Aspergillus nidulans

Heterotrimeric G-protein-mediated signaling pathways play a pivotal role in transmembrane signaling in eukaryotes. Our main aim was to identify signaling pathways regulated by A. nidulans GprB and GprD G-protein coupled receptors (GPCRs). When these two null mutant strains were compared to the wild-type strain, the DeltagprB mutant showed an increased protein kinase A (PKA) activity while growing in glucose 1% and during starvation. In contrast, the DeltagprD has a much lower PKA activity upon starvation. Transcriptomics and (1)H NMR-based metabolomics were performed on two single null mutants grown on glucose. We noted modulation in the expression of 11 secondary metabolism gene clusters when the DeltagprB and DeltagprD mutant strains were grown in 1% glucose. Several members of the sterigmatocystin-aflatoxin gene cluster presented down-regulation in both mutant strains. The genes of the NR-PKS monodictyphenone biosynthesis cluster had overall increased mRNA accumulation in DeltagprB, while in the DeltagprD mutant strain the genes had decreased mRNA accumulation. Principal component analysis of the metabolomic data demonstrated that there was a significant metabolite shift in the DeltagprD strain. The (1)H NMR analysis revealed significant expression of essential amino acids with elevated levels in the DeltagprD strain, compared to the wild-type and DeltagprB strains. With the results, we demonstrated the differential expression of a variety of genes related mainly to secondary metabolism, sexual development, stress signaling, and amino acid metabolism. We propose that the absence of GPCRs triggered stress responses at the genetic level. The data suggested an intimate relationship among different G-protein coupled receptors, fine-tune regulation of secondary and amino acid metabolisms, and fungal development

DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work

Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding) requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants) are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%) in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized for future collaborative programs. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilisation of metabarcoding in aquatic biomonitoring.This paper is a deliverable of the European Cooperation in Science and Technology (COST) Action DNAqua-Net (CA15219) Working Group 1, led by Torbjørn Ekrem and Fedor Čiampor. Thanks to the University of Minho and University of Pécs for hosting workshops and working group meetings. We also thank staff at National Environment Agencies and others that provided national checklists of taxa used in biomonitoring, and otherwise assisted with checklist proof-reading: Jarmila Makovinská and Emília Mišíková Elexová (Slovakia); Steinar Sandøy and Dag Rosland (Norway); Mišel Jelič (Croatia); Marlen Vasquez (Cyprus); Adam Petrusek (Czech Republic); Kristel Panksep (Estonia); Panagiotis Kaspiditis (Greece); Matteo Montagna (Italy); Marija Katarzyte (Lithuania); Ana Rotter (Slovenia); Rosa Trabajo (Spain); Florian Altermatt (Switzerland); Kristian Meissner (Finland), Rigers Bakiu (Albania), Valentina Stamenkovic and Jelena Hinic (Macedonia); Patricia Mergen (Belgium); Gael Denys & the French Biodiversity Agency (France); Mary Kelly-Quinn (Ireland); Piotr Panek and Andrzej Zawal (Poland); Cesare Mario Puzzi (Italy); Carole Fitzpatrick (United Kingdom); Simon Vitecek (Austria); Ana Filipa Filipe (Portugal); Peter Anton Stæhr & Anne Winding (Denmark); Michael Monaghan (Germany); Alain Dohet, Lionel L'Hoste, Nora Welschbillig & Luc Ector (Luxembourg), Lujza Keresztes, (Romania). The authors also want to thank Dirk Steinke for providing the original European ERMS list for marine taxa and Florian Malard for comments on the manuscript. The preparation of the AMBI checklist was carried out in the scope of a Short-term Scientific Mission (ECOST-STSM-CA15219-150217- 082111) granted to SD visiting AZTI, Spain. ZC was supported by grants EFOP-3.6.1.-16-2016-00004 and 20765-3/2018/FEKUTSTRAT. TE was supported by the NorBOL-grant (226134/F50) from the Research Coun cil of Norway. BR, FL and MFG contributed through support from the GBOL project, which is generously funded by the German Federal Min istry of Education and Research (FKZ 01LI1101 and 01LI1501). MG contributed through support of the Polish National Science Centre, grants N N303 5794 39 and 2014/15/B/NZ8/00266. SF was funded by the project PORBIOTA - Portuguese E-Infrastructure for Information and Research on Biodiversity (POCI-01-0145-FEDER-022127), supported by Operational Thematic Program for Competitiveness and Internationalization (POCI), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER)

Analysis of the Aspergillus fumigatus Proteome Reveals Metabolic Changes and the Activation of the Pseurotin A Biosynthesis Gene Cluster in Response to Hypoxia

The mold Aspergillus fumigatus is the most important airborne fungal pathogen. Adaptation to hypoxia represents an important virulence attribute for A. fumigatus. Therefore, we aimed at obtaining a comprehensive overview about this process on the proteome level. To ensure highly reproducible growth conditions, an oxygen-controlled, glucose-limited chemostat cultivation was established. Two-dimensional gel electrophoresis analysis of mycelial and mitochondrial proteins as well as two-dimensional Blue Native/SDS-gel separation of mitochondrial membrane proteins led to the identification of 117 proteins with an altered abundance under hypoxic in comparison to normoxic conditions. Hypoxia induced an increased activity of glycolysis, the TCA-cycle, respiration, and amino acid metabolism. Consistently, the cellular contents in heme, iron, copper, and zinc increased. Furthermore, hypoxia induced biosynthesis of the secondary metabolite pseurotin A as demonstrated at proteomic, transcriptional, and metabolite levels. The observed and so far not reported stimulation of the biosynthesis of a secondary metabolite by oxygen depletion may also affect the survival of A. fumigatus in hypoxic niches of the human host. Among the proteins so far not implicated in hypoxia adaptation, an NO-detoxifying flavohemoprotein was one of the most highly up-regulated proteins which indicates a link between hypoxia and the generation of nitrosative stress in A. fumigatus

The genetic study of three population microisolates in South Tyrol (MICROS): study design and epidemiological perspectives

<p>Abstract</p> <p>Background</p> <p>There is increasing evidence of the important role that small, isolated populations could play in finding genes involved in the etiology of diseases. For historical and political reasons, South Tyrol, the northern most Italian region, includes several villages of small dimensions which remained isolated over the centuries.</p> <p>Methods</p> <p>The MICROS study is a population-based survey on three small, isolated villages, characterized by: old settlement; small number of founders; high endogamy rates; slow/null population expansion. During the stage-1 (2002/03) genealogical data, screening questionnaires, clinical measurements, blood and urine samples, and DNA were collected for 1175 adult volunteers. Stage-2, concerning trait diagnoses, linkage analysis and association studies, is ongoing. The selection of the traits is being driven by expert clinicians. Preliminary, descriptive statistics were obtained. Power simulations for finding linkage on a quantitative trait locus (QTL) were undertaken.</p> <p>Results</p> <p>Starting from participants, genealogies were reconstructed for 50,037 subjects, going back to the early 1600s. Within the last five generations, subjects were clustered in one pedigree of 7049 subjects plus 178 smaller pedigrees (3 to 85 subjects each). A significant probability of familial clustering was assessed for many traits, especially among the cardiovascular, neurological and respiratory traits. Simulations showed that the MICROS pedigree has a substantial power to detect a LOD score ≥ 3 when the QTL specific heritability is ≥ 20%.</p> <p>Conclusion</p> <p>The MICROS study is an extensive, ongoing, two-stage survey aimed at characterizing the genetic epidemiology of Mendelian and complex diseases. Our approach, involving different scientific disciplines, is an advantageous strategy to define and to study population isolates. The isolation of the Alpine populations, together with the extensive data collected so far, make the MICROS study a powerful resource for the study of diseases in many fields of medicine. Recent successes and simulation studies give us confidence that our pedigrees can be valuable both in finding new candidates loci and to confirm existing candidate genes.</p