Recent Submissions

  • Single-cell phenotypic characterization of Staphylococcus aureus with fluorescent triazole urea activity-based probes.

    Chen, Linhai; Keller, Laura J; Cordasco, Edward A; Bogyo, Matthew; Lentz, Christian S; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (Wiley-Blackwell, 2019-02-15)
    Phenotypically distinct cellular (sub)populations are clinically relevant for virulence and antibiotic resistance of a bacterial pathogen, but functionally different cells are usually indistinguishable from each other. Here, we introduce fluorescent activity-based probes as chemical tools for single-cell phenotypic characterization of enzyme activity levels in Staphylococcus aureus. We screened a 1,2,3-triazole urea library to identify selective inhibitors of fluorophosphonate-binding serine hydrolases and lipases in S. aureus and synthesized target-selective activity-based probes. Molecular imaging and activity-based protein profiling studies with these probes revealed a dynamic network within this enzyme family involving compensatory regulation of specific family members and exposed single-cell phenotypic heterogeneity. We propose chemical probe labeling of enzymatic activities as a generalizable method for phenotyping of bacterial cells at the population and single-cell level.
  • Subcellular Quantification of Uptake in Gram-Negative Bacteria.

    Prochnow, Hans; Fetz, Verena; Hotop, Sven-Kevin; García-Rivera, Mariel A; Heumann, Axel; Brönstrup, Mark; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (ACS Publications, 2019-02-05)
    Infections by Gram-negative pathogens represent a major health care issue of growing concern due to a striking lack of novel antibacterial agents over the course of the last decades. The main scientific problem behind the rational optimization of novel antibiotics is our limited understanding of small molecule translocation into, and their export from, the target compartments of Gram-negative species. To address this issue, a versatile, label-free assay to determine the intracellular localization and concentration of a given compound has been developed for Escherichia coli and its efflux-impaired ΔTolC mutant. The assay applies a fractionation procedure to antibiotic-treated bacterial cells to obtain periplasm, cytoplasm, and membrane fractions of high purity, as demonstrated by Western Blots of compartment-specific marker proteins. This is followed by an LC-MS/MS-based quantification of antibiotic content in each compartment. Antibiotic amounts could be converted to antibiotic concentrations by assuming that an E. coli cell is a cylinder flanked by two half spheres and calculating the volumes of bacterial compartments. The quantification of antibiotics from different classes, namely ciprofloxacin, tetracycline, trimethoprim, and erythromycin, demonstrated pronounced differences in uptake quantities and distribution patterns across the compartments. For example, in the case of ciprofloxacin, a higher amount of compound was located in the cytoplasm than in the periplasm (592 ± 50 pg vs 277 ± 13 pg per 3.9 × 10
  • Metabolome and transcriptome-wide effects of the carbon storage regulator A in enteropathogenic Escherichia coli.

    Berndt, Volker; Beckstette, Michael; Volk, Marcel; Dersch, Petra; Brönstrup, Mark; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer-Nature, 2019-01-15)
    The carbon storage regulator A (CsrA) is a conserved global regulatory system known to control central carbon pathways, biofilm formation, motility, and pathogenicity. The aim of this study was to characterize changes in major metabolic pathways induced by CsrA in human enteropathogenic Escherichia coli (EPEC) grown under virulence factor-inducing conditions. For this purpose, the metabolomes and transcriptomes of EPEC and an isogenic ∆csrA mutant derivative were analyzed by untargeted mass spectrometry and RNA sequencing, respectively. Of the 159 metabolites identified from untargeted GC/MS and LC/MS data, 97 were significantly (fold change ≥ 1.5; corrected p-value ≤ 0.05) regulated between the knockout and the wildtype strain. A lack of csrA led to an accumulation of fructose-6-phosphate (F6P) and glycogen synthesis pathway products, whereas metabolites in lower glycolysis and the citric acid cycle were downregulated. Associated pathways from the citric acid cycle like aromatic amino acid and siderophore biosynthesis were also negatively influenced. The nucleoside salvage pathways were featured by an accumulation of nucleosides and nucleobases, and a downregulation of nucleotides. In addition, a pronounced downregulation of lyso-lipid metabolites was observed. A drastic change in the morphology in the form of vesicle-like structures of the ∆csrA knockout strain was visible by electron microscopy. Colanic acid synthesis genes were strongly (up to 50 fold) upregulated, and the abundance of colanic acid was 3 fold increased according to a colorimetric assay. The findings expand the scope of pathways affected by the csrA regulon and emphasize its importance as a global regulator.
  • Identification and quantification of (t)RNA modifications in Pseudomonas aeruginosa by liquid chromatography-tandem mass spectrometry.

    Grobe, Svenja; Doberenz, Sebastian; Ferreira, Kevin; Krueger, Jonas; Brönstrup, Mark; Kaever, Volkhard; Häußler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley-Blackwell, 2019-01-15)
    Transfer RNA (tRNA) modifications impact the structure and function of tRNAs thus affecting the efficiency and fidelity of translation. In the opportunistic pathogen Pseudomonas aeruginosa translational regulation plays an important but less defined role in the adaptation to changing environments. In this study, we explored tRNA modifications in P. aeruginosa using LC-MS/MS based approaches. Neutral Loss Scan (NLS) demonstrated the potential to identify previously unknown modifications, while Multiple Reaction Monitoring (MRM) can detect modifications with high specificity and sensitivity. In this study, the MRM-based external calibration method allowed for quantification of the 4 canonical and 32 modified ribonucleosides, of which 21 tRNA modifications were quantified in the total tRNA pool of P. aeruginosa PA14. We also purified the single tRNA isoacceptors tRNA-ArgUCU, tRNA-LeuCAA and tRNA-TrpCCA and determined, both qualitatively and quantitatively, their specific modification pattern. Deeper insights into the nature and dynamics of tRNA modifications in P. aeruginosa will pave the way for further studies on posttranscriptional gene regulation as a relatively unexplored molecular mechanism of controlling bacterial pathogenicity and life style.
  • xCELLanalyzer: A Framework for the Analysis of Cellular Impedance Measurements for Mode of Action Discovery

    Franke, Raimo; Hinkelmann, Bettina; Fetz, Verena; Stradal, Theresia; Sasse, Florenz; Klawonn, Frank; Brönstrup, Mark; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Sage, 2019-01-25)
    Mode of action (MoA) identification of bioactive compounds is very often a challenging and time-consuming task. We used a label-free kinetic profiling method based on an impedance readout to monitor the time-dependent cellular response profiles for the interaction of bioactive natural products and other small molecules with mammalian cells. Such approaches have been rarely used so far due to the lack of data mining tools to properly capture the characteristics of the impedance curves. We developed a data analysis pipeline for the xCELLigence Real-Time Cell Analysis detection platform to process the data, assess and score their reproducibility, and provide rank-based MoA predictions for a reference set of 60 bioactive compounds. The method can reveal additional, previously unknown targets, as exemplified by the identification of tubulin-destabilizing activities of the RNA synthesis inhibitor actinomycin D and the effects on DNA replication of vioprolide A. The data analysis pipeline is based on the statistical programming language R and is available to the scientific community through a GitHub repository.
  • Advances and Challenges of Biodegradable Implant Materials with a Focus on Magnesium-Alloys and Bacterial Infections

    Rahim, Muhammad; Ullah, Sami; Mueller, Peter; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2018-07-10)
    Medical implants made of biodegradable materials could be advantageous for temporary applications, such as mechanical support during bone-healing or as vascular stents to keep blood vessels open. After completion of the healing process, the implant would disappear, avoiding long-term side effects or the need for surgical removal. Various corrodible metal alloys based on magnesium, iron or zinc have been proposed as sturdier and potentially less inflammatory alternatives to degradable organic polymers, in particular for load-bearing applications. Despite the recent introduction of magnesium-based screws, the remaining hurdles to routine clinical applications are still challenging. These include limitations such as mechanical material characteristics or unsuitable corrosion characteristics. In this article, the salient features and clinical prospects of currently-investigated biodegradable implant materials are summarized, with a main focus on magnesium alloys. A mechanism of action for the stimulation of bone growth due to the exertion of mechanical force by magnesium corrosion products is discussed. To explain divergent in vitro and in vivo effects of magnesium, a novel model for bacterial biofilm infections is proposed which predicts crucial consequences for antibacterial implant strategies.
  • Investigations on the mode of action of gephyronic acid, an inhibitor of eukaryotic protein translation from myxobacteria.

    Muthukumar, Yazh; Münkemer, Johanna; Mathieu, Daniel; Richter, Christian; Schwalbe, Harald; Steinmetz, Heinrich; Kessler, Wolfgang; Reichelt, Joachim; Beutling, Ulrike; Frank, Ronald; Büssow, Konrad; van den Heuvel, Joop; Brönstrup, Mark; Taylor, Richard E; Laschat, Sabine; Sasse, Florenz (PLOS, 2018-01-01)
    The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.
  • Triazole-based cross-linkers in radical polymerization processes: tuning mechanical properties of poly(acrylamide) and poly( -dimethylacrylamide) hydrogels

    Götz, Tobias; Schädel, Nicole; Petri, Nadja; Kirchhof, Manuel; Bilitewski, Ursula; Tovar, Günter E. M.; Laschat, Sabine; Southan, Alexander; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
    Triazole-based cross-linkers with different spacer lengths and different functional end groups (acrylamides, methacrylamides, maleimides and vinylsulfonamides) were synthesized, investigated for cytotoxic and antibacterial activity, and incorporated into poly(acrylamide) (PAAm) and poly(N,N-dimethylacrylamide) (PDMAAm) hydrogels by free-radical polymerization. Hydrogels prepared with different cross-linkers and cross-linker contents between 0.2% and 1.0% were compared by gel yields, equilibrium degrees of swelling (S) and storage moduli (G0). Generally with increasing cross-linker content, G0 values of the hydrogels increased, while S values decreased. The different polymerizable cross-linker end groups resulted in a decrease of G0 in the following order for cross-linkers with C4 spacers: acrylamide > maleimide > methacrylamide > vinylsulfonamide. Longer cross-linker alkyl spacer lengths caused an increase in G0 and a decrease in S. Independent of the cross-linker used, a universal correlation between G0 and equilibrium polymer volume fraction f was found. For PAAm hydrogels, G0 ranged between 4 kPa and 23 kPa and f between 0.07 and 0.14. For PDMAAm hydrogels, G0 ranged between 0.1 kPa and 4.9 kPa and f between 0.02 and 0.06. The collected data were used to establish an empirical model to predict G0 depending on f. G0 of PAAm and PDMAAm hydrogels is given by G0 ¼ 4034 kPa f2.66 and G0 ¼ 4297 kPa f2.46, respectively.
  • Self-assembly and biological activities of ionic liquid crystals derived from aromatic amino acids.

    Neidhardt, Manuel M; Schmitt, Katharina; Baro, Angelika; Schneider, Carmen; Bilitewski, Ursula; Laschat, Sabine; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-08-08)
    The self-assembly of amino acid-derived ionic liquid crystals (ILCs) into lamellar or micellar-like aggregates suggests that they might interact with biological membranes. To get some insight, guanidinium chlorides derived from the natural l-amino acids phenylalanine (Phe), tyrosine (Tyr) and 3,4-dihydroxyphenylalanine (DOPA) were synthesized and their mesomorphic properties were investigated via polarizing optical microscopy (POM), differential scanning calorimetry (DSC) and X-ray diffraction (SAXS, WAXS). Mesophase types depended on the number of alkoxy side chains. Phe- and Tyr-based ILCs with one and two side chains, respectively, self-assembled into smectic A bilayers (SmA2), while Dopa-derived ILCs with three side chains formed columnar (Colh) mesophases. The mesophase ranges for Phe ILCs increased steadily with side chain length, for Tyr- and Dopa-based ILCs, however, size matching effects were observed. To clarify whether the mesomorphic behaviour has an impact on biological properties, cytotoxic and antibacterial activities of the ILCs were studied. Phe and Tyr ILCs exhibited much higher cytotoxicities (against the L-929 mouse fibroblast cell line) and/or antibacterial activities (against Staphylococcus aureus) than Dopa ILCs, which were mostly inactive. Furthermore, within each series, the side chain length largely influenced the biological activity. Thus, the bulk mesophase behaviour appeared to correlate with the biological properties, in particular, the interactions with membranes, as shown by measuring the intracellular Ca2+ concentration in human monocytic U937 cells after treatment with the amino acid-based ILCs.
  • Detection and Investigation of Eagle Effect Resistance to Vancomycin in With an ATP-Bioluminescence Assay.

    Jarrad, Angie M; Blaskovich, Mark A T; Prasetyoputri, Anggia; Karoli, Tomislav; Hansford, Karl A; Cooper, Matthew A; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-01-01)
    Vancomycin was bactericidal against Clostridium difficile at eightfold the minimum inhibitory concentration (MIC) using a traditional minimum bactericidal concentration (MBC) assay. However, at higher concentrations up to 64 × MIC, vancomycin displayed a paradoxical “more-drug-kills-less” Eagle effect against C. difficile. To overcome challenges associated with performing the labor-intensive agar-based MBC method under anaerobic growth conditions, we investigated an alternative more convenient ATP-bioluminescence assay to assess the Eagle effect in C. difficile. The commercial BacTiter-GloTM assay is a homogenous method to determine bacterial viability based on quantification of bacterial ATP as a marker for metabolic activity. The ATP-bioluminescence assay was advantageous over the traditional MBC-type assay in detecting the Eagle effect because it reduced assay time and was simple to perform; measurement of viability could be performed in less than 10 min outside of the anaerobic chamber. Using this method, we found C. difficile survived clinically relevant, high concentrations of vancomycin (up to 2048 μg/mL). In contrast, C. difficile did not survive high concentrations of metronidazole or fidaxomicin. The Eagle effect was also detected for telavancin, but not for teicoplanin, dalbavancin, oritavancin, or ramoplanin. All four pathogenic strains of C. difficile tested consistently displayed Eagle effect resistance to vancomycin, but not metronidazole or fidaxomicin. These results suggest that Eagle effect resistance to vancomycin in C. difficile could be more prevalent than previously appreciated, with potential clinical implications. The ATP-Bioluminescence assay can thus be used as an alternative to the agar-based MBC assay to characterize the Eagle effect against a variety of antibiotics, at a wide-range of concentrations, with much greater throughput. This may facilitate improved understanding of Eagle effect resistance and promote further research to understand potential clinical relevance.
  • A modular synthesis of tetracyclic meroterpenoid antibiotics

    Wildermuth, Raphael; Speck, Klaus; Haut, Franz-Lucas; Mayer, Peter; Karge, Bianka; Brönstrup, Mark; Magauer, Thomas; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
  • Occupation-Associated Fatal Limbic Encephalitis Caused by Variegated Squirrel Bornavirus 1, Germany, 2013.

    Tappe, Dennis; Schlottau, Kore; Cadar, Daniel; Hoffmann, Bernd; Balke, Lorenz; Bewig, Burkhard; Hoffmann, Donata; Eisermann, Philip; Fickenscher, Helmut; Krumbholz, Andi; Laufs, Helmut; Huhndorf, Monika; Rosenthal, Maria; Schulz-Schaeffer, Walter; Ismer, Gabriele; Hotop, Sven-Kevin; Brönstrup, Mark; Ott, Anthonina; Schmidt-Chanasit, Jonas; Beer, Martin; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-06-01)
    Limbic encephalitis is commonly regarded as an autoimmune-mediated disease. However, after the recent detection of zoonotic variegated squirrel bornavirus 1 in a Prevost's squirrel (Callosciurus prevostii) in a zoo in northern Germany, we retrospectively investigated a fatal case in an autoantibody-seronegative animal caretaker who had worked at that zoo. The virus had been discovered in 2015 as the cause of a cluster of cases of fatal encephalitis among breeders of variegated squirrels (Sciurus variegatoides) in eastern Germany. Molecular assays and immunohistochemistry detected a limbic distribution of the virus in brain tissue of the animal caretaker. Phylogenetic analyses demonstrated a spillover infection from the Prevost's squirrel. Antibodies against bornaviruses were detected in the patient's cerebrospinal fluid by immunofluorescence and newly developed ELISAs and immunoblot. The putative antigenic epitope was identified on the viral nucleoprotein. Other zoo workers were not infected; however, avoidance of direct contact with exotic squirrels and screening of squirrels are recommended.
  • Differential magnesium implant corrosion coat formation and contribution to bone bonding.

    Rahim, Muhammad Imran; Weizbauer, Andreas; Evertz, Florian; Hoffmann, Andrea; Rohde, Manfred; Glasmacher, Birgit; Windhagen, Henning; Gross, Gerhard; Seitz, Jan-Marten; Mueller, Peter P; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017)
    Magnesium alloys are presently under investigation as promising biodegradable implant materials with osteoconductive properties. To study the molecular mechanisms involved, the potential contribution of soluble magnesium corrosion products to the stimulation of osteoblastic cell differentiation was examined. However, no evidence for the stimulation of osteoblast differentiation could be obtained when cultured mesenchymal precursor cells were differentiated in the presence of metallic magnesium or in cell culture medium containing elevated magnesium ion levels. Similarly, in soft tissue no bone induction by metallic magnesium or by the corrosion product magnesium hydroxide could be observed in a mouse model. Motivated by the comparatively rapid accumulation solid corrosion products physicochemical processes were examined as an alternative mechanism to explain the stimulation of bone growth by magnesium-based implants. During exposure to physiological solutions a structured corrosion coat formed on magnesium whereby the elements calcium and phosphate were enriched in the outermost layer which could play a role in the established biocompatible behavior of magnesium implants. When magnesium pins were inserted into avital bones, corrosion lead to increases in the pull out force, suggesting that the expanding corrosion layer was interlocking with the surrounding bone. Since mechanical stress is a well-established inducer of bone growth, volume increases caused by the rapid accumulation of corrosion products and the resulting force development could be a key mechanism and provide an explanation for the observed stimulatory effects of magnesium-based implants in hard tissue. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 697-709, 2017.
  • A multi-target caffeine derived rhodium(i) N-heterocyclic carbene complex: evaluation of the mechanism of action.

    Zhang, Jing-Jing; Muenzner, Julienne K; Abu El Maaty, Mohamed A; Karge, Bianka; Schobert, Rainer; Wölfl, Stefan; Ott, Ingo; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-08-16)
    A rhodium(i) and a ruthenium(ii) complex with a caffeine derived N-heterocyclic carbene (NHC) ligand were biologically investigated as organometallic conjugates consisting of a metal center and a naturally occurring moiety. While the ruthenium(ii) complex was largely inactive, the rhodium(i) NHC complex displayed selective cytotoxicity and significant anti-metastatic and in vivo anti-vascular activities and acted as both a mammalian and an E. coli thioredoxin reductase inhibitor. In HCT-116 cells it increased the reactive oxygen species level, leading to DNA damage, and it induced cell cycle arrest, decreased the mitochondrial membrane potential, and triggered apoptosis. This rhodium(i) NHC derivative thus represents a multi-target compound with promising anti-cancer potential.
  • Synthesis of the AB ring system of clifednamide utilizing Claisen rearrangement and Diels-Alder reaction as key steps.

    Loke, Inga; Bentzinger, Guillaume; Holz, Julia; Raja, Aruna; Bhasin, Aman; Sasse, Florenz; Köhn, Andreas; Schobert, Rainer; Laschat, Sabine; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-01-21)
    In order to construct the functionalized AB ring system of clifednamide, member of the class of macrocyclic tetramic acid lactams, a synthesis was developed which utilized an Ireland-Claisen rearrangement and an intramolecular Diels-Alder reaction. Starting from di-O-isopropylidene-d-mannitol the allyl carboxylate precursor for the sigmatropic rearrangement was prepared. This rearrangement proceeded diastereoselectively only in the presence of an allyl silyl ether instead of the parent enone in the side chain, as suggested by deuteration experiments. A subsequent Diels-Alder reaction yielded the target ethyl hexahydro-1H-indene-carboxylate with high diastereoselectivity. Quantum-chemical investigations of this intramolecular Diels-Alder reaction support the proposed configuration of the final product.
  • Target identification by image analysis.

    Fetz, V; Prochnow, H; Brönstrup, Mark; Sasse, F; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
    Covering: 1997 to the end of 2015Each biologically active compound induces phenotypic changes in target cells that are characteristic for its mode of action. These phenotypic alterations can be directly observed under the microscope or made visible by labelling structural elements or selected proteins of the cells with dyes. A comparison of the cellular phenotype induced by a compound of interest with the phenotypes of reference compounds with known cellular targets allows predicting its mode of action. While this approach has been successfully applied to the characterization of natural products based on a visual inspection of images, recent studies used automated microscopy and analysis software to increase speed and to reduce subjective interpretation. In this review, we give a general outline of the workflow for manual and automated image analysis, and we highlight natural products whose bacterial and eucaryotic targets could be identified through such approaches.
  • The CLU-files: disentanglement of a mystery.

    Rohne, Philipp; Prochnow, Hans; Koch-Brandt, Claudia; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunshweig, Germany. (2016-02)
    The multifaceted protein clusterin (CLU) has been challenging researchers for more than 35 years. The characterization of CLU as a molecular chaperone was one of the major breakthroughs in CLU research. Today, secretory clusterin (sCLU), also known as apolipoprotein J (apoJ), is considered one of the most important extracellular chaperones ever found. It is involved in a broad range of physiological and pathophysiological functions, where it exerts a cytoprotective role. Descriptions of various forms of intracellular CLU have led to further and even contradictory functions. To untangle the current state of knowledge of CLU, this review will combine old views in the field, with new discoveries to highlight the nature and function of this fascinating protein(s). In this review, we further describe the expression and subcellular location of various CLU forms. Moreover, we discuss recent insights into the structure of CLU and assess how structural properties as well as the redox environment determine the chaperone activity of CLU. Eventually, the review connects the biochemistry and molecular cell biology of CLU with medical aspects, to formulate a hypothesis of a CLU function in health and disease.
  • Biosynthesis of methyl-proline containing griselimycins, natural products with anti-tuberculosis activity.

    Lukat, Peer; Katsuyama, Yohei; Wenzel, Silke; Binz, Tina; König, Claudia; Blankenfeldt, Wulf; Brönstrup, Mark; Müller, Rolf; Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-11-01)
    Griselimycins (GMs) are depsidecapeptides with superb anti-tuberculosis activity. They contain up to three (2S,4R)-4-methyl-prolines (4-MePro), of which one blocks oxidative degradation and increases metabolic stability in animal models. The natural congener with this substitution is only a minor component in fermentation cultures. We showed that this product can be significantly increased by feeding the reaction with 4-MePro and we investigated the molecular basis of 4-MePro biosynthesis and incorporation. We identified the GM biosynthetic gene cluster as encoding a nonribosomal peptide synthetase and a sub-operon for 4-MePro formation. Using heterologous expression, gene inactivation, and in vitro experiments, we showed that 4-MePro is generated by leucine hydroxylation, oxidation to an aldehyde, and ring closure with subsequent reduction. The crystal structures of the leucine hydroxylase GriE have been determined in complex with substrates and products, providing insight into the stereospecificity of the reaction.
  • Multivalent Siderophore-DOTAM Conjugates as Theranostics for Imaging and Treatment of Bacterial Infections.

    Ferreira, Kevin; Hu, Hai-Yu; Fetz, Verena; Prochnow, Hans; Rais, Bushra; Müller, Peter P; Brönstrup, Mark; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-07-03)
    There is a strong need to better diagnose infections at deep body sites through noninvasive molecular imaging methods. Herein, we describe the synthesis and characterization of probes based on siderophore conjugates with catechol moieties and a central DOTAM scaffold. The probes can accommodate a metal ion as well as an antibiotic moiety and are therefore suited for theranostic purposes. The translocation of the conjugates across the outer and inner cell membranes of E. coli was confirmed by growth recovery experiments with enterobactin-deficient strains, by the antibacterial activity of ampicillin conjugates, and by confocal imaging using a fluorogen-activating protein-malachite green system adapted to E. coli. The suitability of the probes for in vivo imaging was demonstrated with a Cy5.5 conjugate in mice infected with P. aeruginosa.
  • The Kaposi's sarcoma-associated herpesvirus (KSHV) non-structural membrane protein K15 is required for viral lytic replication and may represent a therapeutic target.

    Abere, Bizunesh; Mamo, Tamrat M; Hartmann, Silke; Samarina, Naira; Hage, Elias; Rückert, Jessica; Hotop, Sven-Kevin; Büsche, Guntram; Schulz, Thomas F; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-09)
    Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of the highly vascularized tumor Kaposi's sarcoma (KS), which is characterized by proliferating spindle cells of endothelial origin, extensive neo-angiogenesis and inflammatory infiltrates. The KSHV K15 protein contributes to the angiogenic and invasive properties of KSHV-infected endothelial cells. Here, we asked whether K15 could also play a role in KSHV lytic replication. Deletion of the K15 gene from the viral genome or its depletion by siRNA lead to reduced virus reactivation, as evidenced by the decreased expression levels of KSHV lytic proteins RTA, K-bZIP, ORF 45 and K8.1 as well as reduced release of infectious virus. Similar results were found for a K1 deletion virus. Deleting either K15 or K1 from the viral genome also compromised the ability of KSHV to activate PLCγ1, Erk1/2 and Akt1. In infected primary lymphatic endothelial (LEC-rKSHV) cells, which have previously been shown to spontaneously display a viral lytic transcription pattern, transfection of siRNA against K15, but not K1, abolished viral lytic replication as well as KSHV-induced spindle cell formation. Using a newly generated monoclonal antibody to K15, we found an abundant K15 protein expression in KS tumor biopsies obtained from HIV positive patients, emphasizing the physiological relevance of our findings. Finally, we used a dominant negative inhibitor of the K15-PLCγ1 interaction to establish proof of principle that pharmacological intervention with K15-dependent pathways may represent a novel approach to block KSHV reactivation and thereby its pathogenesis.

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