21 results on '"Marc Schulte"'
Search Results
2. Exposure to stressors and antimicrobials induces cell-autonomous ultrastructural heterogeneity of an intracellular bacterial pathogen
- Author
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Marc Schulte, Michael Hensel, and Katarzyna Miskiewicz
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intracellular pathogen ,bacterial cytoplasm ,CLEM ,bacterial heterogeneity ,stress response ,Microbiology ,QR1-502 - Abstract
Despite their clonality, intracellular bacterial pathogens commonly show remarkable physiological heterogeneity during infection of host cells. Physiological heterogeneity results in distinct ultrastructural morphotypes, but the correlation between bacterial physiological state and ultrastructural appearance remains to be established. In this study, we showed that individual cells of Salmonella enterica serovar Typhimurium are heterogeneous in their ultrastructure. Two morphotypes based on the criterion of cytoplasmic density were discriminated after growth under standard culture conditions, as well as during intracellular lifestyle in mammalian host cells. We identified environmental conditions which affect cytoplasmic densities. Using compounds generating oxygen radicals and defined mutant strains, we were able to link the occurrence of an electron-dense ultrastructural morphotype to exposure to oxidative stress and other stressors. Furthermore, by combining ultrastructural analyses of Salmonella during infection and fluorescence reporter analyses for cell viability, we provided evidence that two characterized ultrastructural morphotypes with electron-lucent or electron-dense cytoplasm represent viable cells. Moreover, the presence of electron-dense types is stress related and can be experimentally induced only when amino acids are available in the medium. Our study proposes ultrastructural morphotypes as marker for physiological states of individual intracellular pathogens providing a new marker for single cell analyses. more...
- Published
- 2022
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3. The protected physiological state of intracellular Salmonella enterica persisters reduces host cell-imposed stress
- Author
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Marc Schulte, Katharina Olschewski, and Michael Hensel
- Subjects
Biology (General) ,QH301-705.5 - Abstract
Schulte et al. show that non-replicating Salmonella enterica serovar Typhimurium persisters, which are tolerant to antibiotics, sense their environment and respond to stressors. This study suggests that stress sensing and response of persisters may be targeted as an antimicrobial strategy. more...
- Published
- 2021
- Full Text
- View/download PDF
4. Proteomics of intracellular Salmonella enterica reveals roles of Salmonella pathogenicity island 2 in metabolism and antioxidant defense.
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Janina Noster, Tzu-Chiao Chao, Nathalie Sander, Marc Schulte, Tatjana Reuter, Nicole Hansmeier, and Michael Hensel
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Intracellular Salmonella enterica serovar Typhimurium (STM) deploy the Salmonella Pathogenicity Island 2-encoded type III secretion system (SPI2-T3SS) for the massive remodeling of the endosomal system for host cells. This activity results in formation of an extensive interconnected tubular network of Salmonella-induced filaments (SIFs) connected to the Salmonella-containing vacuole (SCV). Such network is absent in cells infected with SPI2-T3SS-deficient mutant strains such as ΔssaV. A tubular network with reduced dimensions is formed if SPI2-T3SS effector protein SseF is absent. Previous single cell live microscopy-based analyses revealed that intracellular proliferation of STM is directly correlated to the ability to transform the host cell endosomal system into a complex tubular network. This network may also abrogate host defense mechanisms such as delivery of antimicrobial effectors to the SCV. To test the role of SIFs in STM patho-metabolism, we performed quantitative comparative proteomics of STM recovered from infected murine macrophages. We infected RAW264.7 cells with STM wild type (WT), ΔsseF or ΔssaV strains, recovered bacteria 12 h after infection and determined proteome compositions. Increased numbers of proteins characteristic for nutritional starvation were detected in STM ΔsseF and ΔssaV compared to WT. In addition, STM ΔssaV, but not ΔsseF showed signatures of increased exposure to stress by antimicrobial defenses, in particular reactive oxygen species, of the host cells. The proteomics analyses presented here support and extend the role of SIFs for the intracellular lifestyle of STM. We conclude that efficient manipulation of the host cell endosomal system by effector proteins of the SPI2-T3SS contributes to nutrition, as well as to resistance against antimicrobial host defense mechanisms. more...
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- 2019
- Full Text
- View/download PDF
5. Models of intestinal infection by Salmonella enterica: introduction of a new neonate mouse model [version 1; referees: 2 approved]
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Marc Schulte and Michael Hensel
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Bacterial Infections ,Cellular Microbiology & Pathogenesis ,Epidemiology ,Immunity to Infections ,Innate Immunity ,Medical Microbiology ,Microbial Evolution & Genomics ,Medicine ,Science - Abstract
Salmonella enterica serovar Typhimurium is a foodborne pathogen causing inflammatory disease in the intestine following diarrhea and is responsible for thousands of deaths worldwide. Many in vitro investigations using cell culture models are available, but these do not represent the real natural environment present in the intestine of infected hosts. Several in vivo animal models have been used to study the host-pathogen interaction and to unravel the immune responses and cellular processes occurring during infection. An animal model for Salmonella-induced intestinal inflammation relies on the pretreatment of mice with streptomycin. This model is of great importance but still shows limitations to investigate the host-pathogen interaction in the small intestine in vivo. Here, we review the use of mouse models for Salmonella infections and focus on a new small animal model using 1-day-old neonate mice. The neonate model enables researchers to observe infection of both the small and large intestine, thereby offering perspectives for new experimental approaches, as well as to analyze the Salmonella-enterocyte interaction in the small intestine in vivo. more...
- Published
- 2016
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6. Flow Cytometry-Based Single Cell Analyses of Bacterial Adaptation to Intracellular Environments
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Marc, Schulte and Michael, Hensel
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Bacteria ,Acclimatization ,Single-Cell Analysis ,Flow Cytometry ,Adaptation, Physiological - Abstract
Since decades, flow cytometry (FC) is a powerful technique to perform single cell analyses with high accuracy and throughput. Moreover, FC is the method of choice to study bacterial cell heterogeneity and complements single-cell imaging techniques. The complex experimental approaches for FC sample preparation and the subsequent FC adjustment and gating strategy demand careful considerations to be successful when analyzing complex microbial populations, especially when liberated populations of intracellular bacterial pathogens, or bacterial pathogens inside intact host cells are analyzed. Here, we provide a set of experimental protocols for FC sample preparation of (1) in vitro cultured bacterial cells, (2) liberated intracellular bacteria from host cells, or (3) preparation of intact infected phagocytic or epithelial cells commonly used as host cells in infection biology. Since sample preparation, cytometer adjustment, and gating strategy are essential for experimental success, we aim to provide our expertise to support application of FC by other researchers. more...
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- 2022
7. Single molecule analyses of Salmonella translocated effector proteins reveal targeting to and dynamics in host cell endomembranes
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Vera Göser, Marc Schulte, Felix Scharte, Rico Franzkoch, Viktoria Liss, Olympia E. Psathaki, and Michael Hensel
- Abstract
Bacterial pathogens deliver proteins in temporal and spatial coordinated manner to manipulate mammalian host cells. The facultative intracellular pathogen Salmonella enterica remodels the host endosomal system for survival and proliferation inside host cells. The pathogen resides in a membrane-bound compartment termed Salmonella-containing vacuole (SCV). By Salmonella- induced fusions of host endomembranes, the SCV is connected with extensive tubular structures termed Salmonella-induced filaments (SIF). The intracellular lifestyle of Salmonella critically depends on effector molecules translocated by the SPI2-encoded type III secretion system (SPI2-T3SS) into host cells. A subset of these effectors is associated with, or integral in SCV and SIF membranes. It remained to be determined how SPI2-T3SS effectors reach their subcellular destination, and how these effectors interact with endomembranes remodeled by Salmonella. We deployed self-labeling enzyme (SLE) tags as novel approach to label translocated effector proteins in living host cells, and analyzed their dynamics on single molecule level. We found that SPI2-T3SS effector proteins diffuse in membranes of SIF with mobility comparable to membrane-integral host proteins in endomembranes. Dynamics differed between various effector proteins investigated and was dependent on membrane architecture of SIF. In the early infection, we observed host endosomal vesicles associated with Salmonella effector proteins. Effector-positive vesicles continuously fused with SCV and SIF membranes, providing a route of effector delivery by SPI2-T3SS translocation, interaction with endosomal vesicles, and ultimately fusion with the continuum of SCV/SIF membranes. This novel mechanism controls membrane deformation and vesicular fusion to generate the specific intracellular niche for bacterial survival and proliferation. more...
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- 2022
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8. Flow Cytometry–Based Single Cell Analyses of Bacterial Adaptation to Intracellular Environments
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Marc Schulte and Michael Hensel
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- 2022
- Full Text
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9. The protected physiological status of intracellular Salmonella enterica persisters reduces host cell-imposed stress
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Michael Hensel, Marc Schulte, and Katharina Olschewski
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Immune system ,Plasmid ,biology ,Multidrug tolerance ,Salmonella enterica ,Phenotypic switching ,biology.organism_classification ,Pathogen ,Bacteria ,Intracellular ,Cell biology - Abstract
Today, we are faced with increasingly occurring bacterial infections that are hard to treat and often tend to relapse. These recurrent infections can occur possibly due to antibiotic-tolerant persister cells. Antibiotic persistent bacteria represent a small part of a bacterial population that enters a non-replicating (NR) state arising from phenotypic switching. Intracellularly, after uptake by phagocytic cells, Salmonella enterica serovar Typhimurium (STM) forms persister cells that are able to subvert immune defenses of the host. However, the clear physiological state and perceptual properties are still poorly understood and many questions remain unanswered. Here we describe further development of fluorescent protein-based reporter plasmids that were used to detect intracellular NR persister cells and monitor the expression of stress response genes via extensive flow cytometric analyses. Moreover, we performed extensive measurements of the metabolic properties of NR STM at the early course of infection. Our studies demonstrate that NR STM persister cells perceive their environment and are capable respond to stress factors. Since persisters showed a lower stress response compared to replicating (R) STM, which was not a consequence of a lower metabolic capacity, the persistent status of STM serves as protective niche. Furthermore, up to 95% of NR STM were metabolically active at the beginning of infection additionally showing no difference in the metabolic capacity compared to R STM. The accessory capability of NR STM persisters to sense and to react to stress with constant metabolic activity may supports the pathogen to create a more permissive environment for recurrent infections. more...
- Published
- 2020
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10. Fluorescent protein-based reporters reveal stress response of intracellular Salmonella enterica on single cell level
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Marc Schulte, Michael Hensel, and Katharina Olschewski
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Cytosol ,medicine.diagnostic_test ,biology ,Cytoplasm ,Salmonella enterica ,Intracellular parasite ,medicine ,Virulence ,Periplasmic space ,biology.organism_classification ,Intracellular ,Flow cytometry ,Cell biology - Abstract
Intracellular bacteria such as Salmonella enterica are confronted with a broad array of defense mechanisms of their mammalian host cells. The ability to sense host cell-imposed damages, and to mount efficient stress responses are crucial for survival and proliferation of intracellular pathogens. The various combinations of host defense mechanisms acting on intracellular bacteria and their individual response also explain the occurrence of distinct subpopulations of intracellular S. enterica such as dormant or persisting, slowly or rapidly replicating cells. Here we describe a set of fluorescence protein (FP)-based reporter strains that were used to monitor the expression of cytoplasmic or periplasmic stress response systems on a single cell level. This is mediated by a fast maturing FP as reporter for induction of stress response genes. We evaluated slower maturing FPs for a second function, i.e. the analyses of the status of intracellular proliferation of pathogens. The combination of two FPs allows, on a single cell level, the interrogation of stress response and intracellular proliferation. Application of these reporters to S. enterica allowed us to detect and quantify distinct intracellular subpopulations with different levels of stress response and proliferation.ImportanceSensing of, and responding to host-mediated damages are important defensive virulence traits of bacterial pathogens. Intracellular pathogens such as Salmonella enterica are exposed to various types of antimicrobial host cell defenses that impose, among other, periplasmic and cytosolic stresses. Intracellular S. enterica form distinct subpopulations that differ in proliferation rate, metabolic activity and persister formation. Here we deploy fluorescence protein-based reporter strains to monitor, on a single cell level, the response of intracellular S. enterica to periplasmic or cytoplasmic stress. A second fluorescent protein reports the biosynthetic capacity of individual intracellular S. enterica. The dual fluorescence reporters can be deployed to characterize by flow cytometry phenotypically diverse subpopulations and stress responses in intracellular bacteria. more...
- Published
- 2020
- Full Text
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11. Fluorescent protein-based reporters reveal stress response of intracellular Salmonella enterica at level of single bacterial cells
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Michael Hensel, Katharina Olschewski, and Marc Schulte
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Paraquat ,Salmonella typhimurium ,Immunology ,Biology ,Microbiology ,Flow cytometry ,03 medical and health sciences ,Mice ,Genes, Reporter ,Stress, Physiological ,Virology ,medicine ,Type III Secretion Systems ,Animals ,Humans ,Gene ,030304 developmental biology ,Cell Proliferation ,Polymyxin B ,0303 health sciences ,medicine.diagnostic_test ,030306 microbiology ,Intracellular parasite ,Macrophages ,Epithelial Cells ,Periplasmic space ,Gene Expression Regulation, Bacterial ,Hydrogen Peroxide ,biology.organism_classification ,Cell biology ,Dithiothreitol ,Luminescent Proteins ,RAW 264.7 Cells ,Salmonella enterica ,Cytoplasm ,Genes, Bacterial ,Single-Cell Analysis ,Intracellular ,Function (biology) ,HeLa Cells - Abstract
Intracellular bacteria such as Salmonella enterica are confronted with a broad array of defence mechanisms of their mammalian host cells. The ability to sense host cell-imposed damages, and to mount efficient stress responses are crucial for survival and proliferation of intracellular pathogens. The various combinations of host defence mechanisms acting on intracellular bacteria and their individual response also explain the occurrence of distinct subpopulations of intracellular S. enterica such as dormant or persisting, slowly or rapidly replicating cells. Here we describe a set of fluorescence protein (FP)-based reporter strains that were used to monitor the expression of cytoplasmic or periplasmic stress response systems of single bacterial cells. This is mediated by a fast-maturing FP as reporter for induction of stress response genes. We evaluated slower maturing FPs for a second function, that is, the analysis of the status of intracellular proliferation of pathogens. The combination of two FPs allows, at level of single bacterial cells, the interrogation of stress response and intracellular proliferation. Application of these reporters to S. enterica allowed us to detect and quantify distinct intracellular subpopulations with different levels of stress response and proliferation. more...
- Published
- 2020
12. Proteomics of intracellular Salmonella enterica reveals roles of Salmonella pathogenicity island 2 in metabolism and antioxidant defense
- Author
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Tzu-Chiao Chao, Tatjana Reuter, Janina Noster, Nicole Hansmeier, Nathalie Sander, Michael Hensel, and Marc Schulte
- Subjects
Proteomics ,Salmonella ,Proteome ,Proteomes ,Centrifugation ,medicine.disease_cause ,Pathology and Laboratory Medicine ,Biochemistry ,Antioxidants ,Type three secretion system ,White Blood Cells ,Mice ,Animal Cells ,Medicine and Health Sciences ,Protein Interaction Maps ,Biology (General) ,Cells, Cultured ,Protein Metabolism ,0303 health sciences ,biology ,Chemistry ,Effector ,030302 biochemistry & molecular biology ,Cell biology ,Mutant Strains ,Separation Processes ,Intracellular Pathogens ,Salmonella enterica ,Cellular Types ,Pathogens ,Intracellular ,Research Article ,Genomic Islands ,QH301-705.5 ,Immune Cells ,Immunology ,Research and Analysis Methods ,Microbiology ,03 medical and health sciences ,Bacterial Proteins ,Virology ,medicine ,Genetics ,Animals ,Typhoid Fever ,Molecular Biology ,030304 developmental biology ,Blood Cells ,Intracellular parasite ,Macrophages ,Host Cells ,Biology and Life Sciences ,Proteins ,Computational Biology ,Cell Biology ,RC581-607 ,Salmonella typhi ,biology.organism_classification ,Pathogenicity island ,Metabolism ,Mutation ,Parasitology ,Immunologic diseases. Allergy ,Viral Transmission and Infection - Abstract
Intracellular Salmonella enterica serovar Typhimurium (STM) deploy the Salmonella Pathogenicity Island 2-encoded type III secretion system (SPI2-T3SS) for the massive remodeling of the endosomal system for host cells. This activity results in formation of an extensive interconnected tubular network of Salmonella-induced filaments (SIFs) connected to the Salmonella-containing vacuole (SCV). Such network is absent in cells infected with SPI2-T3SS-deficient mutant strains such as ΔssaV. A tubular network with reduced dimensions is formed if SPI2-T3SS effector protein SseF is absent. Previous single cell live microscopy-based analyses revealed that intracellular proliferation of STM is directly correlated to the ability to transform the host cell endosomal system into a complex tubular network. This network may also abrogate host defense mechanisms such as delivery of antimicrobial effectors to the SCV. To test the role of SIFs in STM patho-metabolism, we performed quantitative comparative proteomics of STM recovered from infected murine macrophages. We infected RAW264.7 cells with STM wild type (WT), ΔsseF or ΔssaV strains, recovered bacteria 12 h after infection and determined proteome compositions. Increased numbers of proteins characteristic for nutritional starvation were detected in STM ΔsseF and ΔssaV compared to WT. In addition, STM ΔssaV, but not ΔsseF showed signatures of increased exposure to stress by antimicrobial defenses, in particular reactive oxygen species, of the host cells. The proteomics analyses presented here support and extend the role of SIFs for the intracellular lifestyle of STM. We conclude that efficient manipulation of the host cell endosomal system by effector proteins of the SPI2-T3SS contributes to nutrition, as well as to resistance against antimicrobial host defense mechanisms., Author summary The facultative intracellular bacterium Salmonella enterica has evolved sophisticated mechanisms to adapt to life inside a pathogen-containing vacuole in mammalian host cells. Intracellular Salmonella manipulate the host cell endosomal system resulting in formation of a complex network of tubular vesicles, termed Salmonella-induced filaments (SIFs). We applied quantitative proteomics to intracellular Salmonella in murine macrophages and compared the wild-type strain to mutant strains with aberrant SIF architecture, or no capacity for induction of SIF. We determined that those mutant strains contain higher amounts of transporters for nutrient uptake, and lower amounts of proteins for central carbon metabolism. These observations indicate response to nutrient restriction in absence of fully established SIF. In addition, the mutant strain unable to induce SIF formation showed increased amounts of proteins required for response to antimicrobial factors of the host cells. These data show that the massive remodeling of the endosomal system of host cells by intracellular Salmonella serves to essential needs, i.e. to enable access to nutrients for efficient proliferation of the pathogen, and to withstand hostile conditions within the pathogen-containing vacuole. more...
- Published
- 2019
13. A versatile remote control system for functional expression of bacterial virulence genes based on the tetA promoter
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Torsten Sterzenbach, Nicole Hansmeier, Katarzyna Miskiewicz, Marc Schulte, Michael Hensel, and Laura Elpers
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Microbiology (medical) ,Operon ,Virulence ,Repressor ,Biology ,Microbiology ,Antiporters ,Type three secretion system ,03 medical and health sciences ,chemistry.chemical_compound ,Enteropathogenic Escherichia coli ,Plasmid ,Bacterial Proteins ,Humans ,TetR ,Adhesins, Bacterial ,Promoter Regions, Genetic ,030304 developmental biology ,Yersinia enterocolitica ,0303 health sciences ,030306 microbiology ,Effector ,Tetracycline Resistance ,Salmonella enterica ,General Medicine ,Gene Expression Regulation, Bacterial ,Cell biology ,Bacterial adhesin ,Infectious Diseases ,chemistry ,Flagella ,Tetracyclines ,HeLa Cells - Abstract
The expression of bacterial virulence factors is controlled in response to host or environmental factors and most virulence genes are not expressed under laboratory conditions. Investigations of molecular structures and cellular functions of bacterial virulence factors demand systems for experimentally controlled expression. We describe a simple and robust system that is based on the tetA promoter and the cognate repressor TetR. Expression under control of PtetA can be induced by non-antibiotic derivatives of tetracycline such as anhydrotetracycline (AHT). Tet-on expression cassettes can be used to replace native promoters of chromosomal genes or operons of interest. Tet-on plasmids allow episomal expression in homologous or heterologous host organisms. We demonstrate the application of Tet-on systems for the controlled induction of flagella assembly and motility, and for surface expression of adhesins of the chaperone/usher family of enteropathogenic Escherichia coli and autotransporter adhesins of Yersinia enterocolitica in Salmonella enterica and E. coli. Since inducer AHT can easily cross bacterial envelopes and mammalian cell membranes, the system can also be applied to control virulence genes in intracellular bacteria. We demonstrate the controlled synthesis, translocation and function of effector proteins of the type III secretion system of intracellular S. enterica. more...
- Published
- 2018
14. Cancer evolution, mutations, and clonal selection in relapse neuroblastoma
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Alexander Schramm, Marc Schulte, Johannes Köster, and Sven Rahmann
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0301 basic medicine ,Histology ,Medizin ,Cell Biology ,Biology ,medicine.disease ,Molecular medicine ,Human genetics ,Pathology and Forensic Medicine ,03 medical and health sciences ,030104 developmental biology ,Immune system ,Tumor progression ,Neuroblastoma ,Cancer cell ,medicine ,Cancer research ,Anoikis ,Allele - Abstract
The notion of cancer as a complex evolutionary system has been validated by in-depth molecular analyses of tumor progression over the last years. While a complex interplay of cell-autonomous programs and cell-cell interactions determines proliferation and differentiation during normal development, intrinsic and acquired plasticity of cancer cells allow for evasion of growth factor limitations, apoptotic signals, or attacks from the immune system. Treatment-induced molecular selection processes have been described by a number of studies already, but understanding of those events facilitating metastatic spread, organ-specific homing, and resistance to anoikis is still in its early days. In principle, somatic events giving rise to cancer progression should be easier to follow in childhood tumors bearing fewer mutations and genomic aberrations than their counterparts in adulthood. We have previously reported on the genetic events accompanying relapsing neuroblastoma, a solid tumor of early childhood. Our results indicated significantly higher single nucleotide variants in relapse tumors, gave hints for branched tumor evolution upon treatment and clonal selection as deduced from shifts in allelic frequencies between primary and relapsing neuroblastoma. Here, we will review these findings and give an outlook on dealing with intratumoral heterogeneity and sub-clonal diversity in neuroblastoma for future targeted treatments. more...
- Published
- 2018
15. Cancer evolution, mutations, and clonal selection in relapse neuroblastoma
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Marc, Schulte, Johannes, Köster, Sven, Rahmann, and Alexander, Schramm
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Neuroblastoma ,Recurrence ,Mutation ,Tumor Microenvironment ,Animals ,Humans ,Immunotherapy ,Clone Cells - Abstract
The notion of cancer as a complex evolutionary system has been validated by in-depth molecular analyses of tumor progression over the last years. While a complex interplay of cell-autonomous programs and cell-cell interactions determines proliferation and differentiation during normal development, intrinsic and acquired plasticity of cancer cells allow for evasion of growth factor limitations, apoptotic signals, or attacks from the immune system. Treatment-induced molecular selection processes have been described by a number of studies already, but understanding of those events facilitating metastatic spread, organ-specific homing, and resistance to anoikis is still in its early days. In principle, somatic events giving rise to cancer progression should be easier to follow in childhood tumors bearing fewer mutations and genomic aberrations than their counterparts in adulthood. We have previously reported on the genetic events accompanying relapsing neuroblastoma, a solid tumor of early childhood. Our results indicated significantly higher single nucleotide variants in relapse tumors, gave hints for branched tumor evolution upon treatment and clonal selection as deduced from shifts in allelic frequencies between primary and relapsing neuroblastoma. Here, we will review these findings and give an outlook on dealing with intratumoral heterogeneity and sub-clonal diversity in neuroblastoma for future targeted treatments. more...
- Published
- 2017
16. RITA displays anti-tumor activity in medulloblastomas independent of TP53 status
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Hedwig E. Deubzer, Kristina Althoff, Lukas C. Heukamp, Annette Künkele, Theresa Thor, Alexander Schramm, Marc Schulte, Angelika Eggert, Laura Grunewald, Aline Gottlieb, Johannes H. Schulte, and Andrea Odersky more...
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0301 basic medicine ,Gerontology ,Cancer Research ,endocrine system diseases ,Medizin ,Apoptosis ,Kaplan-Meier Estimate ,Mice ,0302 clinical medicine ,TP53 ,Antitumor activity ,biology ,Proto-Oncogene Proteins c-mdm2 ,Immunohistochemistry ,Oncology ,030220 oncology & carcinogenesis ,Mdm2 ,Functional status ,Female ,Signal Transduction ,Research Paper ,Cyclin-Dependent Kinase Inhibitor p21 ,Cell Survival ,Mice, Nude ,Antineoplastic Agents ,medulloblastoma ,03 medical and health sciences ,MDM2 ,In vivo ,Cell Line, Tumor ,RITA ,medicine ,Animals ,Humans ,Viability assay ,Cerebellar Neoplasms ,Furans ,neoplasms ,Cell Proliferation ,Medulloblastoma ,business.industry ,medicine.disease ,Xenograft Model Antitumor Assays ,In vitro ,nervous system diseases ,CDKN1A ,030104 developmental biology ,Cell culture ,Mutation ,Cancer research ,biology.protein ,Tumor Suppressor Protein p53 ,business - Abstract
Current therapy of medulloblastoma, the most common malignant brain tumor of childhood, achieves 40-70% survival. Secondary chemotherapy resistance contributes to treatment failure, where TP53 pathway dysfunction plays a key role. MDM2 interaction with TP53 leads to its degradation. Reactivating TP53 functionality using small-molecule inhibitors, such as RITA, to disrupt TP53-MDM2 binding may have therapeutic potential. We show here that RITA decreased viability of all 4 analyzed medulloblastoma cell lines, regardless of TP53 functional status. The decrease in cell viability was accompanied in 3 of the 4 medulloblastoma cell lines by accumulation of TP53 protein in the cells and increased CDKN1A expression. RITA treatment in mouse models inhibited medulloblastoma xenograft tumor growth. These data demonstrate that RITA treatment reduces medulloblastoma cell viability in both in vitro and in vivo models, and acts independently of cellular TP53 status, identifying RITA as a potential therapeutic agent to treat medulloblastoma. more...
- Published
- 2016
17. Characterization of pancreatic glucagon-producing tumors and pituitary gland tumors in transgenic mice overexpressing MYCN in hGFAP-positive cells
- Author
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Wolfgang Hartmann, Thomas Knösel, Sandra Elges, Ulrich Schüller, Marc Schulte, Annette Eyking, Daniela Beisser, Jasmin Ohli, Günter Klöppel, Ludger Klein-Hitpass, Alexander Schramm, Kristina Althoff, Henning Reis, Julie Nonnekens, Elke Cario, Kathrin Fielitz, Katleen De Preter, Johannes H. Schulte, and Molecular Genetics more...
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0301 basic medicine ,Pituitary gland ,Pathology ,glucagonoma ,Medizin ,Gene Expression ,PROTEIN ,Glucagonoma ,Neuroendocrine tumors ,BET-BROMODOMAIN INHIBITION ,Mice ,0302 clinical medicine ,TARGETING MYCN ,MYCN ,Medicine and Health Sciences ,N-Myc Proto-Oncogene Protein ,biology ,Chromogranin A ,Immunohistochemistry ,CANCER ,Neuroendocrine Tumors ,medicine.anatomical_structure ,Oncology ,030220 oncology & carcinogenesis ,Pancreas ,AURORA ,EXPRESSION ,medicine.medical_specialty ,Mice, Transgenic ,03 medical and health sciences ,Cell Line, Tumor ,Neuroblastoma ,Glial Fibrillary Acidic Protein ,HUMAN NEUROBLASTOMA ,medicine ,C-MYC ,Animals ,Humans ,Pituitary Neoplasms ,neoplasms ,N-MYC ,CONDITIONAL DELETION ,pancreatic neuroendocrine tumors ,business.industry ,Gene Expression Profiling ,Pituitary tumors ,Biology and Life Sciences ,Glucagon ,medicine.disease ,Pancreatic Neoplasms ,Disease Models, Animal ,030104 developmental biology ,biology.protein ,Transcriptome ,business ,N-Myc ,Priority Research Paper - Abstract
OA gold Amplification or overexpression of MYCN is involved in development and maintenance of multiple malignancies. A subset of these tumors originates from neural precursors, including the most aggressive forms of the childhood tumors, neuroblastoma and medulloblastoma. In order to model the spectrum of MYCN-driven neoplasms in mice, we transgenically overexpressed MYCN under the control of the human GFAP-promoter that, among other targets, drives expression in neural progenitor cells. However, LSL-MYCN;hGFAP-Cre double transgenic mice did neither develop neural crest tumors nor tumors of the central nervous system, but presented with neuroendocrine tumors of the pancreas and, less frequently, the pituitary gland. Pituitary tumors expressed chromogranin A and closely resembled human pituitary adenomas. Pancreatic tumors strongly produced and secreted glucagon, suggesting that they derived from glucagon- and GFAP-positive islet cells. Interestingly, 3 out of 9 human pancreatic neuroendocrine tumors expressed MYCN, supporting the similarity of the mouse tumors to the human system. Serial transplantations of mouse tumor cells into immunocompromised mice confirmed their fully transformed phenotype. MYCN-directed treatment by AuroraA- or Brd4-inhibitors resulted in significantly decreased cell proliferation in vitro and reduced tumor growth in vivo. In summary, we provide a novel mouse model for neuroendocrine tumors of the pancreas and pituitary gland that is dependent on MYCN expression and that may help to evaluate MYCN-directed therapies. more...
- Published
- 2016
18. ADAM10 regulates FasL cell surface expression and modulates FasL-induced cytotoxicity and activation-induced cell death
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Paul Saftig, Dieter Hartmann, B. De Strooper, Andreas Ludwig, Thorsten Maretzky, M Lettau, O Janssen, Marc Schulte, and Karina Reiss
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Programmed cell death ,Fas Ligand Protein ,T-Lymphocytes ,ADAM10 ,chemical and pharmacologic phenomena ,Biology ,Jurkat cells ,Fas ligand ,ADAM10 Protein ,Jurkat Cells ,Mice ,Downregulation and upregulation ,Animals ,Humans ,Enzyme Inhibitors ,Molecular Biology ,Cell Death ,Cell growth ,Membrane Proteins ,hemic and immune systems ,Cell Biology ,Recombinant Proteins ,Cell biology ,ADAM Proteins ,Protein Transport ,Solubility ,Apoptosis ,Tumor necrosis factor alpha ,Amyloid Precursor Protein Secretases ,Protein Processing, Post-Translational - Abstract
The apoptosis-inducing Fas ligand (FasL) is a type II transmembrane protein that is involved in the downregulation of immune reactions by activation-induced cell death (AICD) as well as in T cell-mediated cytotoxicity. Proteolytic cleavage leads to the generation of membrane-bound N-terminal fragments and a soluble FasL (sFasL) ectodomain. sFasL can be detected in the serum of patients with dysregulated inflammatory diseases and is discussed to affect Fas-FasL-mediated apoptosis. Using pharmacological approaches in 293T cells, in vitro cleavage assays as well as loss and gain of function studies in murine embryonic fibroblasts (MEFs), we demonstrate that the disintegrin and metalloprotease ADAM10 is critically involved in the shedding of FasL. In primary human T cells, FasL shedding is significantly reduced after inhibition of ADAM10. The resulting elevated FasL surface expression is associated with increased killing capacity and an increase of T cells undergoing AICD. Overall, our findings suggest that ADAM10 represents an important molecular modulator of FasL-mediated cell death. more...
- Published
- 2007
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19. Regulated ADAM10-dependent Ectodomain Shedding of γ-Protocadherin C3 Modulates Cell-Cell Adhesion
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Paul Saftig, Marcus Frank, Ingrid G. Haas, Andreas Ludwig, Thorsten Maretzky, Karina Reiss, and Marc Schulte
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ADAM10 ,Blotting, Western ,Cadherin Related Proteins ,Glutamic Acid ,Protocadherin ,Biology ,Biochemistry ,Cell Line ,ADAM10 Protein ,Mice ,Cell Adhesion ,Disintegrin ,Animals ,Humans ,Cell adhesion ,Molecular Biology ,Cells, Cultured ,Mice, Knockout ,Neurons ,Cadherin ,Membrane Proteins ,Cell Biology ,Fibroblasts ,Cadherins ,ADAM Proteins ,Cell aggregation ,Cell biology ,Ectodomain ,Synapses ,biology.protein ,Amyloid Precursor Protein Secretases ,K562 Cells - Abstract
Gamma-protocadherins (Pcdh gamma) are type I transmembrane proteins, which are most notably expressed in the nervous system. They are enriched at synapses and involved in synapse formation, specification, and maintenance. In this study, we show that Pcdh gamma C3 and Pcdh gamma B4 are specifically cleaved within their ectodomains by the disintegrin and metalloprotease ADAM10. Analysis of ADAM10-deficient fibroblasts and embryos, inhibitor studies, as well as RNA interference-mediated down-regulation demonstrated that ADAM10 is not only responsible for the constitutive but also for the regulated shedding of these proteins in fibroblasts and in neuronal cells. In contrast to N-cadherin shedding, which was activated by N-methyl-D-aspartic acid receptor activation in neuronal cells, Pcdh gamma shedding was induced by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrate stimulation, suggesting differential regulation mechanisms of cadherin-mediated functions at synapses. Cell aggregation assays in the presence or absence of metalloprotease inhibitors strongly suggest that the ectodomain shedding events modulate the cell adhesion role of Pcdh gamma. The identification of ADAM10 as the protease responsible for constitutive and regulated Pcdh gamma shedding may therefore provide new insight into the regulation of Pcdh gamma functions. more...
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- 2006
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20. Rhodobacter capsulatus XdhC Is Involved in Molybdenum Cofactor Binding and Insertion into Xanthine Dehydrogenase
- Author
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Meina Neumann, Silke Leimkühler, Walter Stöcklein, Nora Jünemann, and Marc Schulte
- Subjects
Xanthine Dehydrogenase ,Protein subunit ,Coenzymes ,Plasma protein binding ,Biology ,Biochemistry ,Chromatography, Affinity ,Rhodobacter capsulatus ,chemistry.chemical_compound ,Metalloproteins ,Escherichia coli ,Xanthine oxidase ,Molecular Biology ,Institut für Biochemie und Biologie ,chemistry.chemical_classification ,Rhodobacter ,Pteridines ,Cell Biology ,Surface Plasmon Resonance ,biology.organism_classification ,Xanthine ,Recombinant Proteins ,Enzyme ,chemistry ,Xanthine dehydrogenase ,Electrophoresis, Polyacrylamide Gel ,Molybdenum cofactor ,Molybdenum Cofactors ,Protein Binding - Abstract
Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a cytoplasmic enzyme with an (alpha beta) 2 heterodimeric structure that is highly identical to homodimeric eukaryotic xanthine oxidoreductases. The crystal structure revealed that the molybdenum cofactor (Moco) is deeply buried within the protein. A protein involved in Moco insertion and XDH maturation has been identified, which was designated XdhC. XdhC was shown to be essential for the production of active XDH but is not a subunit of the purified enzyme. Here we describe the purification of XdhC and the detailed characterization of its role for XDH maturation. We could show that XdhC binds Moco in stoichiometric amounts, which subsequently can be inserted into Moco-free apo-XDH. A specific interaction between XdhC and XdhB was identified. We show that XdhC is required for the stabilization of the sulfurated form of Moco present in enzymes of the xanthine oxidase family. Our findings imply that enzyme-specific proteins exist for the biogenesis of molybdoenzymes, coordinating Moco binding and insertion into their respective target proteins. So far, the requirement of such proteins for molybdoenzyme maturation has been described only for prokaryotes more...
- Published
- 2006
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21. Substrate selectivity of epidermal growth factor-receptor ligand sheddases and their regulation by phorbol esters and calcium influx
- Author
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Dieter Hartmann, Keisuke Horiuchi, Yoshiaki Toyama, Karina Reiss, Gillian Murphy, Carl P. Blobel, Takafumi Yamaguchi, Paul Saftig, Sylvain Le Gall, and Marc Schulte
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TGF alpha ,EGF Family of Proteins ,Heparin-binding EGF-like growth factor ,Recombinant Fusion Proteins ,Biology ,ADAM17 Protein ,Ligands ,Amphiregulin ,Epiregulin ,Substrate Specificity ,chemistry.chemical_compound ,ADAM10 Protein ,Mice ,Calmodulin ,Epidermal growth factor ,Chlorocebus aethiops ,Animals ,Epidermal growth factor receptor ,Betacellulin ,Molecular Biology ,Glycoproteins ,Epidermal Growth Factor ,Ionophores ,Membrane Proteins ,Cell Biology ,Articles ,Sheddase ,Transforming Growth Factor alpha ,Cell biology ,Protein Structure, Tertiary ,ErbB Receptors ,ADAM Proteins ,Biochemistry ,chemistry ,COS Cells ,Phorbol ,biology.protein ,Intercellular Signaling Peptides and Proteins ,Tetradecanoylphorbol Acetate ,Calcium ,Amyloid Precursor Protein Secretases ,Heparin-binding EGF-like Growth Factor - Abstract
Signaling via the epidermal growth factor receptor (EGFR), which has critical roles in development and diseases such as cancer, is regulated by proteolytic shedding of its membrane-tethered ligands. Sheddases for EGFR-ligands are therefore key signaling switches in the EGFR pathway. Here, we determined which ADAMs (a disintegrin and metalloprotease) can shed various EGFR-ligands, and we analyzed the regulation of EGFR-ligand shedding by two commonly used stimuli, phorbol esters and calcium influx. Phorbol esters predominantly activate ADAM17, thereby triggering a burst of shedding of EGFR-ligands from a late secretory pathway compartment. Calcium influx stimulates ADAM10, requiring its cytoplasmic domain. However, calcium influx-stimulated shedding of transforming growth factor α and amphiregulin does not require ADAM17, even though ADAM17 is essential for phorbol ester-stimulated shedding of these EGFR-ligands. This study provides new insight into the machinery responsible for EGFR-ligand release and thus EGFR signaling and demonstrates that dysregulated EGFR-ligand shedding may be caused by increased expression of constitutively active sheddases or activation of different sheddases by distinct stimuli. more...
- Published
- 2006
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