Sven Krappmann

Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Bavaria, Germany

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Publications (68)285.79 Total impact

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    ABSTRACT: Fungal infections are of major relevance due to the increase of immunocompromised patients, the frequently delayed diagnosis, and limited therapeutics. To date, growth and nutritional requirements of the fungus during infection, which are relevant for invasion of the host, are poorly understood. This is particularly true for invasive pulmonary aspergillosis, as so far sources of (macro-)elements that are exploited during infection have been identified only to a limited extent. Here, we have investigated sulfur (S) utilization by the human-pathogenic mold Aspergillus fumigatus during invasive growth. Our data reveal that inorganic S-compounds or taurine are unlikely to serve as S-sources during invasive pulmonary aspergillosis since a sulfate transporter and a sulfite reductase mutant strain are fully virulent. In contrast, the S-containing amino acid cysteine is limiting for fungal growth, as proven by the reduced virulence of a cysteine auxotroph. Moreover, phenotypical characterization of this strain further revealed robustness of the subordinate glutathione redox system. Interestingly, we demonstrate that methionine synthase is essential for A. fumigatus virulence, defining the biosynthetic route of this proteinogenic amino acid as a potential antifungal target. In conclusion, we provide novel insights about the nutritional requirements of A. fumigatus during pathogenesis, a prerequisite to understanding and fighting infection.
    No preview · Article · Jan 2016 · Infection and immunity
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    ABSTRACT: Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease caused by the fungus Aspergillus fumigatus, and is a leading cause of invasive fungal infection-related mortality and morbidity in patients with hematological malignancies and bone marrow transplants. We developed and tested a novel probe for noninvasive detection of A. fumigatus lung infection based on antibody-guided positron emission tomography and magnetic resonance (immunoPET/MR) imaging. Administration of a [(64)Cu]DOTA-labeled A. fumigatus-specific monoclonal antibody (mAb), JF5, to neutrophil-depleted A. fumigatus-infected mice allowed specific localization of lung infection when combined with PET. Optical imaging with a fluorochrome-labeled version of the mAb showed colocalization with invasive hyphae. The mAb-based newly developed PET tracer [(64)Cu]DOTA-JF5 distinguished IPA from bacterial lung infections and, in contrast to [(18)F]FDG-PET, discriminated IPA from a general increase in metabolic activity associated with lung inflammation. To our knowledge, this is the first time that antibody-guided in vivo imaging has been used for noninvasive diagnosis of a fungal lung disease (IPA) of humans, an approach with enormous potential for diagnosis of infectious diseases and with potential for clinical translation.
    No preview · Article · Jan 2016 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Pathogenicity of the saprobe Aspergillus fumigatus strictly depends on nutrient acquisition during infection, as fungal growth determines colonisation and invasion of a susceptible host. Primary metabolism has to be considered as a valid target for antimycotic therapy, based on the fact that several fungal anabolic pathways are not conserved in higher eukaryotes. To test whether fungal proliferation during invasive aspergillosis relies on endogenous biosynthesis of aromatic amino acids, defined auxotrophic mutants of A. fumigatus were generated and assessed for their infectious capacities in neutropenic mice and found to be strongly attenuated in virulence. Moreover, essentiality of the complete biosynthetic pathway could be demonstrated, corroborated by conditional gene expression in infected animals and inhibitor studies. This brief report not only validates the aromatic amino acid biosynthesis pathway of A. fumigatus to be a promising antifungal target but furthermore demonstrates feasibility of conditional gene expression in a murine infection model of aspergillosis.
    No preview · Article · Nov 2015 · Virulence
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    ABSTRACT: In Aspergillus, controlled gene expression is often achieved using the reverse tetracycline-controlled transactivator (rtTA) dependent Tet-on system, whereby transcription is titratably activated by addition of the tetracycline derivative doxycycline. The complementary Tet-off system utilises the tetracycline-controlled transactivator (tTA) component to quantitatively reduce gene expression. In this study, we utilized a synthetic biological approach to engineer highly optimized Tet-off conditional expression systems in Aspergillus niger and Aspergillus fumigatus. Steps for delivery of these tools include utilizing codon optimized cassette components, testing several promoters for improved genetic stability and validating two modified luciferase reporters for highly accurate measurements of gene expression. The Tet-off cassettes developed in this study enable facile and quantitative functional analysis, as validated by Tet-off analysis of genes involved in chitin synthesis and cell wall polarity in A. niger, and para-aminobenzoic acid synthesis in A. fumigatus. We also used a racA(G18V) dominant allele to demonstrate that Tet-off in A. niger enables gene over-expression and downregulation in a single isolate. Additionally, we used the improved luciferase reporters to show that the Tet-off cassette in A. niger enables quantification of gene oscillations.In order to demonstrate that synthetic biological approaches developed here are broadly applicable to engineering transcriptional circuits in filamentous fungi, we used our strategy for improving cassette stability by promoter replacement in the A. niger Tet-on system, which resulted in a modified Tet-on cassette with higher stability in recipient genomes.
    No preview · Article · Nov 2015 · Fungal Genetics and Biology
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    Sven Krappmann

    Preview · Article · Nov 2015 · Virulence
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    ABSTRACT: Invasive aspergillosis is started after germination of Aspergillus fumigatus conidia that are inhaled by susceptible individuals. Fungal hyphae can grow in the lung through the epithelial tissue and disseminate hematogenously to invade into other organs. Low fungaemia indicates that fungal elements do not reside in the bloodstream for long. We analyzed whether blood represents a hostile environment to which the physiology of A. fumigatus has to adapt. An in vitro model of A. fumigatus infection was established by incubating mycelium in blood. Our model allowed to discern the changes of the gene expression profile of A. fumigatus at various stages of the infection. The majority of described virulence factors that are connected to pulmonary infections appeared not to be activated during the blood phase. Three active processes were identified that presumably help the fungus to survive the blood environment in an advanced phase of the infection: iron homeostasis, secondary metabolism, and the formation of detoxifying enzymes. We propose that A. fumigatus is hardly able to propagate in blood. After an early stage of sensing the environment, virtually all uptake mechanisms and energy-consuming metabolic pathways are shut-down. The fungus appears to adapt by trans-differentiation into a resting mycelial stage. This might reflect the harsh conditions in blood where A. fumigatus cannot take up sufficient nutrients to establish self-defense mechanisms combined with significant growth.
    Full-text · Article · Aug 2015 · BMC Genomics
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    ABSTRACT: Background: Platelets were recently identified as a part of innate immunity. They are activated by contact with Aspergillus fumigatus; putative consequences include antifungal defense but also thrombosis, excessive inflammation, and thrombocytopenia. We aimed to identify those fungal surface structures that mediate interaction with platelets. Methods: Human platelets were incubated with Aspergillus conidia and hyphae, isolated wall components, or fungal surface mutants. Interaction was visualized microscopically; activation was quantified by flow cytometry of specific markers. Results: The capacity of A. fumigatus conidia to activate platelets is at least partly due to melanin, because this effect can be mimicked with "melanin ghosts"; a mutant lacking melanin showed reduced platelet stimulating potency. In contrast, conidial hydrophobin masks relevant structures, because an A. fumigatus mutant lacking the hydrophobin protein induced stronger platelet activation than wild-type conidia. A. fumigatus hyphae also contain surface structures that interact with platelets. Wall proteins, galactomannan, chitin, and β-glucan are not the relevant hyphal components; instead, the recently identified fungal polysaccharide galactosaminogalactan potently triggered platelet activation. Conclusions: Conidial melanin and hydrophobin as well as hyphal galactosaminogalactan represent important pathogenicity factors that modulate platelet activity and thus might influence immune responses, inflammation, and thrombosis in infected patients.
    No preview · Article · Mar 2015 · The Journal of Infectious Diseases
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    ABSTRACT: Despite continuous contact with fungi, immunocompetent individuals rarely develop pro-inflammatory antifungal immune responses. The underlying tolerogenic mechanisms are incompletely understood. Using both mouse models and human patients, we show that infection with the human pathogenic fungi Aspergillus fumigatus and Candida albicans induces a distinct subset of neutrophilic myeloid-derived suppressor cells (MDSCs), which functionally suppress T and NK cell responses. Mechanistically, pathogenic fungi induce neutrophilic MDSCs through the pattern recognition receptor Dectin-1 and its downstream adaptor protein CARD9. Fungal MDSC induction is further dependent on pathways downstream of Dectin-1 signaling, notably reactive oxygen species (ROS) generation as well as caspase-8 activity and interleukin-1 (IL-1) production. Additionally, exogenous IL-1β induces MDSCs to comparable levels observed during C. albicans infection. Adoptive transfer and survival experiments show that MDSCs are protective during invasive C. albicans infection, but not A. fumigatus infection. These studies define an innate immune mechanism by which pathogenic fungi regulate host defense. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    No preview · Article · Mar 2015 · Cell host & microbe
  • Michaela Dümig · Sven Krappmann
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    ABSTRACT: Conditional expression of a gene of interest is fundamental for elucidating any cellular function of its gene product. To achieve this in the human-pathogenic mould Aspergillus fumigatus, established doxycycline-dependent systems were demonstrated to be highly suitable, therefore supporting the aim to identify virulence determinants of this pathogenic saprobe.
    No preview · Chapter · Jan 2015
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    ABSTRACT: The mould Aspergillus fumigatus is primarily an opportunistic pathogen of immunocompromised patients. Once fungal spores have been inhaled they encounter cells of the innate immune system, which include dendritic cells (DCs). DCs are the key antigen-presenting cells of the immune system and distinct subtypes, which differ in terms of origin, morphology and function. This study has systematically compared the interactions between A. fumigatus and myeloid DCs (mDCs), plasmacytoid DCs (pDCs) and monocyte-derived DCs (moDCs). Analyses were performed by time-lapse video microscopy, scanning electron microscopy, plating assays, flow cytometry, 25-plex ELISA and transwell assays. The three subsets of DCs displayed distinct responses to the fungus with mDCs and moDCs showing the greatest similarities. mDCs and moDCs both produced rough convolutions and occasionally phagocytic cups upon exposure to A. fumigatus whereas pDCs maintained a smooth appearance. Both mDCs and moDCs phagocytosed conidia and germ tubes, while pDCs did not phagocytose any fungi. Analysis of cytokine release and maturation markers revealed specific differences in pro- and anti-inflammatory patterns between the different DC subsets. These distinct characteristics between the DC subsets highlight their differences and suggest specific roles of moDCs, mDCs and pDCs during their interaction with A. fumigatus in vivo.
    No preview · Article · Aug 2014 · International Journal of Medical Microbiology
  • Sven Krappmann
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    ABSTRACT: Site-specific recombination mediates the rearrangement of nucleic acids by the virtue of an recombinase acting on specific recognition sequences. Recombining activities belong either to the tyrosine- or serine-type group, based on the presence of specific residues in the catalytic centre, which can be further subdivided into families due to additional criteria. The most prominent systems are the λ phage integrase acting on att sites; the Cre recombinase from bacteriophage P1 with its loxP attachment sites; the FLP/FTR system of fungal origin, where it is required for 2-μm plasmid replication/amplification in yeast; and the prokaryotic β-recombinase that recombines six sites specifically in cis. Each of these has been exploited in fungal hosts of biotechnological, medical or general relevance, mainly for cloning projects, approaches of gene targeting, genome modification or screening purposes. With their precise and defined mode of action are site-specific recombination systems eminently suited for genetic tasks in fungi, like they are executed in functional studies at high throughput or modern approaches of synthetic biology.
    No preview · Article · Jan 2014 · Applied Microbiology and Biotechnology
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    ABSTRACT: Platelets have been shown to cover a broad range of functions. Besides their role in hemostasis, they have immunological functions and thus participate in the interaction between pathogens and host defense. Platelets have a broad repertoire of receptor molecules that enable them to sense invading pathogens and infection-induced inflammation. Consequently, platelets exert antimicrobial effector mechanisms, but also initiate an intense crosstalk with other arms of the innate and adaptive immunity, including neutrophils, monocytes/macrophages, dendritic cells, B cells and T cells. There is a fragile balance between beneficial antimicrobial effects and detrimental reactions that contribute to the pathogenesis, and many pathogens have developed mechanisms to influence these two outcomes. This review aims to highlight aspects of the interaction strategies between platelets and pathogenic bacteria, viruses, fungi and parasites, in addition to the subsequent networking between platelets and other immune cells, and the relevance of these processes for the pathogenesis of infections.
    No preview · Article · Nov 2013 · Future Microbiology
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    Jorge Amich · Lukas Schafferer · Hubertus Haas · Sven Krappmann
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    ABSTRACT: The metRΔ strain displays reduced virulence in an alternative infection model. Larvae of the greater wax moth Galleria mellonella (n = 15 insects per group) were infected with conidial suspensions and survival was monitored. The metRΔ mutant shows a significant reduction in virulence, comparable to the established, avirulent pabaAΔ control strain. When injected in a solution containing 5 mM methionine, the mutant regained its ability to kill the larvae. The reconstituted strain recovered full virulence. Control mice either received no injection (‘untreated’), were pricked, but not injected (‘puncture’) or were mock injected using the solvent alone (‘NaCl/Tween’). (TIF)
    Preview · Dataset · Aug 2013
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    Jorge Amich · Lukas Schafferer · Hubertus Haas · Sven Krappmann
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    ABSTRACT: Plasmids used in the course of this study. (DOC)
    Preview · Dataset · Aug 2013
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    Jorge Amich · Lukas Schafferer · Hubertus Haas · Sven Krappmann
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    ABSTRACT: Construction of plasmids and recombinant A. fumigatus strains and chromatin immunoprecipitation (ChIP) protocol for identification of MetR targets. (DOC)
    Preview · Dataset · Aug 2013
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    Jorge Amich · Lukas Schafferer · Hubertus Haas · Sven Krappmann
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    ABSTRACT: Oligonucleotides used in this study. (DOC)
    Preview · Dataset · Aug 2013
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    Jorge Amich · Lukas Schafferer · Hubertus Haas · Sven Krappmann
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    ABSTRACT: Phenotypical characterisation of the metRΔ deletant with respect to various sources of nitrogen, carbon, or phosphorus. Conidia of the metRΔ deletion strain, its wild-type progenitor, and the reconstituted derivative were point inoculated on culture plates containing either methionine or sulphate as S-source and that were supplemented with the indicated sources of nitrogen, carbon, or phosphorus. Only for the carbon source galactose a pronounced influence on methionine utilisation became evident, whereas all other N-, C-, or P-sources have no influence on growth capacities of the deletant. (TIF)
    Preview · Dataset · Aug 2013
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    Jorge Amich · Lukas Schafferer · Hubertus Haas · Sven Krappmann
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    ABSTRACT: RNAseq data. (XLS)
    Preview · Dataset · Aug 2013
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    Jorge Amich · Lukas Schafferer · Hubertus Haas · Sven Krappmann
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    ABSTRACT: Deletion and reconstitution of the metR gene. (A) The complete metR ORF was replaced via homology recombination by a blaster cassette containing the hygromycin B resistance gene as selectable marker. Correct integration was checked by Southern blot hybridisation. Afterwards, the cassette was removed as a result of the action of the β-recombinase included in the cassette itself, expression of which is driven by a xylose-inducible promoter. Correct excision of the cassette was also checked by Southern analysis. (B) The metR gene was reintroduced at its original locus using its own 5′and 3′flanking sequences as homology regions. A silent punctual mutation was inserted to create an extra BstEII restriction site to allow differentiation between the reconstituted strain and its progenitor. Selection was performed by recovery of the sulphate utilization capacity, and correct integration was checked by Southern hybridisation. In both strategies the used probe is marked and only relevant restriction sites are shown. (TIF)
    Preview · Dataset · Aug 2013
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    Jorge Amich · Lukas Schafferer · Hubertus Haas · Sven Krappmann
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    ABSTRACT: Sulphur is an essential element that all pathogens have to absorb from their surroundings in order to grow inside their infected host. Despite its importance, the relevance of sulphur assimilation in fungal virulence is largely unexplored. Here we report a role of the bZIP transcription factor MetR in sulphur assimilation and virulence of the human pathogen Aspergillus fumigatus. The MetR regulator is essential for growth on a variety of sulphur sources; remarkably, it is fundamental for assimilation of inorganic S-sources but dispensable for utilization of methionine. Accordingly, it strongly supports expression of genes directly related to inorganic sulphur assimilation but not of genes connected to methionine metabolism. On a broader scale, MetR orchestrates the comprehensive transcriptional adaptation to sulphur-starving conditions as demonstrated by digital gene expression analysis. Surprisingly, A. fumigatus is able to utilize volatile sulphur compounds produced by its methionine catabolism, a process that has not been described before and that is MetR-dependent. The A. fumigatus MetR transcriptional activator is important for virulence in both leukopenic mice and an alternative mini-host model of aspergillosis, as it was essential for the development of pulmonary aspergillosis and supported the systemic dissemination of the fungus. MetR action under sulphur-starving conditions is further required for proper iron regulation, which links regulation of sulphur metabolism to iron homeostasis and demonstrates an unprecedented regulatory crosstalk. Taken together, this study provides evidence that regulation of sulphur assimilation is not only crucial for A. fumigatus virulence but also affects the balance of iron in this prime opportunistic pathogen.
    Full-text · Article · Aug 2013 · PLoS Pathogens

Publication Stats

2k Citations
285.79 Total Impact Points

Institutions

  • 2012-2015
    • Friedrich-Alexander Universität Erlangen-Nürnberg
      Erlangen, Bavaria, Germany
    • Universitätsklinikum Erlangen
      Erlangen, Bavaria, Germany
  • 2008-2011
    • University of Wuerzburg
      • Research Center for Infectious Diseases
      Würzburg, Bavaria, Germany
    • Universitätsmedizin Göttingen
      Göttingen, Lower Saxony, Germany
  • 2010
    • Institute of Molecular Biology
      Mayence, Rheinland-Pfalz, Germany
  • 1997-2008
    • Georg-August-Universität Göttingen
      • • Institute of Microbiology and Genetics
      • • Department of Molecular Microbiology and Genetics
      Göttingen, Lower Saxony, Germany
  • 1999
    • Whitehead Institute for Biomedical Research
      Cambridge, Massachusetts, United States