Sven Krappmann

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

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Publications (52)239.71 Total impact

  • 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.
    Applied Microbiology and Biotechnology 01/2014; · 3.81 Impact Factor
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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.
    International Journal of Medical Microbiology. 01/2014;
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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.
    Future Microbiology 11/2013; 8:1431-51. · 4.02 Impact Factor
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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.
    PLoS Pathogens 08/2013; 9(8):e1003573. · 8.14 Impact Factor
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    Sven Krappmann, Gordon Ramage
    Frontiers in Microbiology 01/2013; 4:159. · 3.90 Impact Factor
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    ABSTRACT: Conditional gene expression is key for functional studies in any given microorganism. To allow tight regulation in the pathogenic mold Aspergillus fumigatus, improved versions of the doxycycline-dependent Tet-On system were generated by replacing functional elements of the precursor module, thereby circumventing the former problem of leakiness due to intra-molecular recombination.
    Applied and Environmental Microbiology 12/2012; · 3.95 Impact Factor
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    ABSTRACT: Aspergillus fumigatus has two chitin synthases (AfCSMA and AfCSMB) with a myosin motor-like domain (MMD) arranged in a head-to-head configuration. To understand the function of these chitin synthases, single and double csm mutants were constructed and analysed. Although there was a slight reduction in mycelial growth of the mutants, the total chitin synthase activity and the cell wall chitin content were similar in the mycelium of all the mutants and the parental strain. In the conidia, chitin content in the ΔcsmA cell wall was less than half the amount found in the parental strain. In contrast, ΔcsmB and unexpectedly the ΔcsmA/ΔcsmB mutants did not show any modification of the chitin content in their conidial cell-walls. In contrast to the hydrophobic conidia of the parental strain, conidia of all the csm mutants were hydrophilic due to the presence of an amorphous material covering the hydrophobic surface-rodlet layer. The deletion of CSM genes resulted also in an increased susceptibility of resting and germinating conidia to echinocandins. These results show that the deletion of the CSMA and CSMB genes induced a significant disorganisation of the cell wall structure even though they contribute only weakly to the overall cell wall chitin synthesis.
    Antimicrobial Agents and Chemotherapy 09/2012; · 4.57 Impact Factor
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    Jorge Amich, Sven Krappmann
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    ABSTRACT: Incidence rates of infections caused by environmental opportunistic fungi have risen over recent decades. Aspergillus species have emerged as serious threat for the immunecompromised, and detailed knowledge about virulence-determining traits is crucial for drug target identification. As a prime saprobe, A. fumigatus has evolved to efficiently adapt to various stresses and to sustain nutritional supply by osmotrophy, which is characterized by extracellular substrate digestion followed by efficient uptake of breakdown products that are then fed into the fungal primary metabolism. These intrinsic metabolic features are believed to be related with its virulence ability. The plethora of genes that encode underlying effectors has hampered their in-depth analysis with respect to pathogenesis. Recent developments in Aspergillus molecular biology allow conditional gene expression or comprehensive targeting of gene families to cope with redundancy. Furthermore, identification of essential genes that are intrinsically connected to virulence opens accurate perspectives for novel targets in antifungal therapy.
    Frontiers in Microbiology 01/2012; 3:414. · 3.90 Impact Factor
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    ABSTRACT: Non-invasive imaging techniques in microbial disease models have delivered valuable insights in the intimate pathogen-host interplay during infection. Here we describe evaluation and validation of a transgenic bioluminescence reporter strain of the human-pathogenic mold Aspergillus fumigatus, one of the main fungal pathogens affecting immunocompromised individuals. Expression and surface display of the Gaussia princeps luciferase allowed sensitive and rapid detection of luminescence emitted from this strain after substrate addition, with photon fluxes strongly correlating to the amounts of fungal conidia or germlings. The reporter strain allowed spatio-temporal monitoring of infection in a cutaneous model of aspergillosis, where neutropenic mice maintained the fungal burden while immunocompetent ones were able to clear it entirely. Most importantly, antifungal therapy could be followed in this type of disease model making use of the bioluminescent A. fumigatus strain. In conclusion, combining sensitivity of the Gaussia luciferase with a surface display expression system in the fungal host allows longitudinal infection studies on cutaneous forms of aspergillosis, providing perspective on drug screening approaches at high-throughput.
    Virulence 01/2012; 3(1):51-61. · 2.79 Impact Factor
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    ABSTRACT: Moulds are characterized by their saprophytic lifestyle that is based on osmotrophy. Among them, Aspergillus fumigatus has emerged as the leading cause of fungal infections in the presence of an underlying immunodeficiency. To assess the role of its nutritional versatility for virulence, transcriptional profiling studies in the presence of varying sources of nitrogen were carried out and revealed an extensive reprogramming of the fungal transcriptome when shifting to a proteinaceous growth substrate. Transcripts encoding metabolic activities were predominantly upregulated, as were proteinases and transport activities. To probe whether fundamental aspects of its osmotrophic lifestyle, that is, extracellular proteolysis and uptake of oligopeptides, are required for A. fumigatus pathogenicity, serial gene replacements were carried out, which eventually yielded an octuple deletion mutant ablated for the opt gene family. This strain displayed no growth defect on various substrates, but supplementary reduction of extracellular proteolytic activity by additional deletion of the prtT gene revealed a synthetic phenotype on porcine lung tissue agar. Virulence studies in a murine model of pulmonary aspergillosis did not disclose any attenuation in virulence of these deletants. Our data emphasize a high degree of redundancy encoded by the A. fumigatus genome that secures nutrient supply for growth and, therefore, virulence.
    Molecular Microbiology 11/2011; 82(4):917-35. · 5.03 Impact Factor
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    ABSTRACT: Viral and fungal infections remain a leading cause of mortality in patients after hematopoietic stem cell transplantation (HSCT). Adoptive transfer of multipathogen-specific T cells is promising in restoring immunity and thereby preventing and treating infections, but approaches are currently limited because of time-consuming and laborious procedures. Therefore, we investigated a new strategy to simultaneously select T cells specific for viral and fungal pathogens based on activation-dependent expression of CD154. Single- and multipathogen-specific T-cell lines with high specificity for adenovirus (AdV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), Candida albicans, and/or Aspergillus fumigatus could be readily generated within 14 days irrespective of the precursor frequency. The T-cell lines responded reproducibly to endogenously processed antigen and specifically proliferated upon antigenic stimulation. Although isolation based on CD154 favors enrichment of CD4(+) T cells, AdV-, EBV- and CMV-specific CD8(+) T cells could be expanded and demonstrated lysis of target cells. Conversely, T cell-mediated alloreactivity was almost abrogated compared with the starting fraction. This selection and/or expansion strategy may form the basis for future adoptive immunotherapy trials in patients at risk for multiple infections and may be translated to other antigens.
    Blood 06/2011; 118(4):1121-31. · 9.78 Impact Factor
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    ABSTRACT: The saprophytic fungus Aspergillus fumigatus is a mold which is ubiquitously present in the environment. It produces large numbers of spores, called conidia that we constantly inhale with the breathing air. Healthy individuals normally do not suffer from true fungal infections with this pathogen. A normally robust resistance against Aspergillus is based on the presence of a very effective immunological defense system in the vertebrate body. Inhaled conidia are first encountered by lung-resident alveolar macrophages and then by neutrophil granulocytes. Both cell types are able to effectively ingest and destroy the fungus. Although some responses of the adaptive immune system develop, the key protection is mediated by innate immunity. The importance of phagocytes for defense against aspergillosis is also supported by large numbers of animal studies. Despite the production of aggressive chemicals that can extracellularly destroy fungal pathogens, the main effector mechanism of the innate immune system is phagocytosis. Very recently, the production of extracellular neutrophil extracellular traps (NETs) consisting of nuclear DNA has been added to the armamentarium that innate immune cells use against infection with Aspergillus. Phagocyte responses to Aspergillus are very broad, and a number of new observations have added to this complexity in recent years. To summarize established and newer findings, we will give an overview on current knowledge of the phagocyte system for the protection against Aspergillus.
    International journal of medical microbiology: IJMM 06/2011; 301(5):436-44. · 4.54 Impact Factor
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    ABSTRACT: Aspergillus fumigatus as prime pathogen to cause aspergillosis has evolved as a saprophyte, but is also able to infect and colonise immunocompromised hosts. Based on the 'dual use' hypothesis of fungal pathogenicity, general characteristics have to be considered as unspecific virulence determinants, among them stress adaptation capacities. The susceptible, warm-blooded mammalian host represents a specific ecological niche that poses several kinds of stress conditions to the fungus during the course of infection. Detailed knowledge about the cellular pathways and adaptive traits that have evolved in A. fumigatus to counteract situations of stress and varying environmental conditions is crucial for the identification of novel and specific antifungal targets. Comprehensive profiling data accompanied by mutant analyses have shed light on such stressors, and nutritional deprivation, oxidative stress, hypoxia, elevated temperature, alkaline pH, extensive secretion, and, in particular during treatment with antifungals, cell membrane perturbations appear to represent the major hazards A. fumigatus has to cope with during infection. Further efforts employing innovative approaches and advanced technologies will have to be made to expand our knowledge about the scope of the A. fumigatus adaptome that is relevant for disease.
    International journal of medical microbiology: IJMM 06/2011; 301(5):408-16. · 4.54 Impact Factor
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    ABSTRACT: T cell-mediated heterologous immunity to different pathogens is promising for the development of immunotherapeutic strategies. Aspergillus fumigatus and Candida albicans, the 2 most common fungal pathogens causing severe infections in immunocompromised patients, are controlled by CD4+ type 1 helper T (T(H)1) cells in humans and mice, making induction of fungus-specific CD4+ T(H)1 immunity an appealing strategy for antifungal therapy. We identified an immunogenic epitope of the A fumigatus cell wall glucanase Crf1 that can be presented by 3 common major histocompatibility complex class II alleles and that induces memory CD4+ T(H)1 cells with a diverse T-cell receptor repertoire that is cross-reactive to C albicans. In BALB/c mice, the Crf1 protein also elicits cross-protection against lethal infection with C albicans that is mediated by the same epitope as in humans. These data illustrate the existence of T cell-based cross-protection for the 2 distantly related clinically relevant fungal pathogens that may foster the development of immunotherapeutic strategies.
    Blood 03/2011; 117(22):5881-91. · 9.78 Impact Factor
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    ABSTRACT: Dendritic cells (DC) are the most important antigen presenting cells and play a pivotal role in host immunity to infectious agents by acting as a bridge between the innate and adaptive immune systems. Monocyte-derived immature DCs (iDC) were infected with viable resting conidia of Aspergillus fumigatus (Af293) for 12 hours at an MOI of 5; cells were sampled every three hours. RNA was extracted from both organisms at each time point and hybridised to microarrays. iDC cell death increased at 6 h in the presence of A. fumigatus which coincided with fungal germ tube emergence; >80% of conidia were associated with iDC. Over the time course A. fumigatus differentially regulated 210 genes, FunCat analysis indicated significant up-regulation of genes involved in fermentation, drug transport, pathogenesis and response to oxidative stress. Genes related to cytotoxicity were differentially regulated but the gliotoxin biosynthesis genes were down regulated over the time course, while Aspf1 was up-regulated at 9 h and 12 h. There was an up-regulation of genes in the subtelomeric regions of the genome as the interaction progressed. The genes up-regulated by iDC in the presence of A. fumigatus indicated that they were producing a pro-inflammatory response which was consistent with previous transcriptome studies of iDC interacting with A. fumigatus germ tubes. This study shows that A. fumigatus adapts to phagocytosis by iDCs by utilising genes that allow it to survive the interaction rather than just up-regulation of specific virulence genes.
    PLoS ONE 01/2011; 6(1):e16016. · 3.53 Impact Factor
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    ABSTRACT: Zusammenfassung Die Kombination massiv-paralleler long-read-Sequenzierungstechnologie mit short-read-Technologie eröffnet neue Wege in der Identifikation und quantitativen Analyse von mRNA-Transkripten und hilft so beim Verständnis von Genexpression.
    BioSpektrum 01/2011; 17(4).
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    ABSTRACT: Recyclable markers based on site-specific recombination allow repetitive gene targeting in filamentous fungi. Here we describe for the first time functionality of the bacterial recombination system employing beta serine recombinase acting on six recognition sequences (beta-rec/six) in a fungal host, the human pathogen Aspergillus fumigatus, and its use in establishing a self-excising resistance marker cassette for serial gene replacement.
    Applied and Environmental Microbiology 09/2010; 76(18):6313-7. · 3.95 Impact Factor
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    Edyta Szewczyk, Sven Krappmann
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    ABSTRACT: Sexual reproduction of the human pathogen Aspergillus fumigatus (teleomorph: Neosartorya fumigata) was assumed to be absent or cryptic until recently, when fertile crosses among geographically restricted environmental isolates were described. Here, we provide evidence for mating, fruiting body development, and ascosporogenesis accompanied by genetic recombination between unrelated, clinical isolates of A. fumigatus, and this evidence demonstrates the generality and reproducibility of this long-time-undisclosed phase in the life cycle of this heterothallic fungus. Successful mating requires the presence of both mating-type idiomorphs MAT1-1 and MAT1-2, as does expression of genes encoding factors presumably involved in this process. Moreover, analysis of an A. fumigatus mutant deleted for the nsdD gene suggests a role of this conserved regulator of cleistothecium development in hyphal fusion and hence heterokaryon formation.
    Eukaryotic Cell 03/2010; 9(5):774-83. · 3.59 Impact Factor
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    ABSTRACT: Virulence of the fungal pathogen Aspergillus fumigatus is in part based on the saprophytic lifestyle that this mold has evolved. A crucial function for saprophytism resides in secreted proteases that allow assimilation of proteinaceous substrates. The impact of extracellular proteolytic activities on the pathogenesis of aspergillosis, however, remains controversial. In order to address this issue, characterization of a conserved regulatory factor, PrtT, that acts on expression of secreted proteases was pursued. Expression of PrtT appears to be regulated posttranscriptionally, and the existence of an mRNA leader sequence implies translational control via eIF2alpha kinase signaling. Phenotypic classification of a prtTDelta deletion mutant revealed that expression of several major extracellular proteases is PrtT dependent, resulting in the inability to utilize protein as a nutritional source. Certain genes encoding secreted proteases are not regulated by PrtT. Most strikingly, the deletant strain is not attenuated in virulence when tested in a leukopenic mouse model, which makes a strong case for reconsidering any impact of secreted proteases in pulmonary aspergillosis.
    Infection and immunity 07/2009; 77(9):4041-50. · 4.21 Impact Factor

Publication Stats

1k Citations
239.71 Total Impact Points

Institutions

  • 2012–2014
    • Friedrich-Alexander Universität Erlangen-Nürnberg
      Erlangen, Bavaria, Germany
  • 2009–2013
    • University of Wuerzburg
      • • Research Center for Infectious Diseases
      • • Institute for Molecular Infection Biology
      Würzburg, Bavaria, Germany
  • 1997–2008
    • Georg-August-Universität Göttingen
      • • Institute of Microbiology and Genetics
      • • Department of Molecular Microbiology and Genetics
      Göttingen, Lower Saxony, Germany
  • 2005–2006
    • Gesellschaft für wissenschaftliche Datenverarbeitung mbH Göttingen
      Göttingen, Lower Saxony, Germany
    • Rhein Biotech GmbH
      Düsseldorf, North Rhine-Westphalia, Germany
  • 1999
    • Whitehead Institute for Biomedical Research
      Cambridge, Massachusetts, United States