• The Cytotoxic Natural Product Vioprolide A Targets Nucleolar Protein 14, Which Is Essential for Ribosome Biogenesis.

      Kirsch, Volker C; Orgler, Christina; Braig, Simone; Jeremias, Irmela; Auerbach, David; Müller, Rolf; Vollmar, Angelika M; Sieber, Stephan A; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Wiley-Blackwell, 2020-01-20)
      Novel targets are needed for treatment of devastating diseases such as cancer. For decades, natural products have guided innovative therapies by addressing diverse pathways. Inspired by the potent cytotoxic bioactivity of myxobacterial vioprolides A-D, we performed in-depth studies on their mode of action. Based on its prominent potency against human acute lymphoblastic leukemia (ALL) cells, we conducted thermal proteome profiling (TPP) and deciphered the target proteins of the most active derivative vioprolide A (VioA) in Jurkat cells. Nucleolar protein 14 (NOP14), which is essential in ribosome biogenesis, was confirmed as a specific target of VioA by a suite of proteomic and biological follow-up experiments. Given its activity against ALL cells compared to healthy lymphocytes, VioA exhibits unique therapeutic potential for anticancer therapy through a novel mode of action
    • Structures of lipoprotein signal peptidase II from Staphylococcus aureus complexed with antibiotics globomycin and myxovirescin.

      Olatunji, Samir; Yu, Xiaoxiao; Bailey, Jonathan; Huang, Chia-Ying; Zapotoczna, Marta; Bowen, Katherine; Remškar, Maja; Müller, Rolf; Scanlan, Eoin M; Geoghegan, Joan A; et al. (Nature publishing group, 2020-01-09)
      Antimicrobial resistance is a major global threat that calls for new antibiotics. Globomycin and myxovirescin are two natural antibiotics that target the lipoprotein-processing enzyme, LspA, thereby compromising the integrity of the bacterial cell envelope. As part of a project aimed at understanding their mechanism of action and for drug development, we provide high-resolution crystal structures of the enzyme from the human pathogen methicillin-resistant Staphylococcus aureus (MRSA) complexed with globomycin and with myxovirescin. Our results reveal an instance of convergent evolution. The two antibiotics possess different molecular structures. Yet, they appear to inhibit identically as non-cleavable tetrahedral intermediate analogs. Remarkably, the two antibiotics superpose along nineteen contiguous atoms that interact similarly with LspA. This 19-atom motif recapitulates a part of the substrate lipoprotein in its proposed binding mode. Incorporating this motif into a scaffold with suitable pharmacokinetic properties should enable the development of effective antibiotics with built-in resistance hardiness.
    • Semisynthesis and biological evaluation of amidochelocardin derivatives as broad-spectrum antibiotics.

      Grandclaudon, Charlotte; Birudukota, N V Suryanarayana; Elgaher, Walid A M; Jumde, Ravindra P; Yahiaoui, Samir; Arisetti, Nanaji; Hennessen, Fabienne; Hüttel, Stephan; Stadler, Marc; Herrmann, Jennifer; et al. (Elsevier, 2019-12-20)
      To address the global challenge of emerging antimicrobial resistance, the hitherto most successful strategy to new antibiotics has been the optimization of validated natural products; most of these efforts rely on semisynthesis. Herein, we report the semisynthetic modification of amidochelocardin, an atypical tetracycline obtained via genetic engineering of the chelocardin producer strain. We report modifications at C4, C7, C10 and C11 by the application of methylation, acylation, electrophilic substitution, and oxidative C-C coupling reactions. The antibacterial activity of the reaction products was tested against a panel of Gram-positive and Gram-negative pathogens. The emerging structure-activity relationships (SARs) revealed that positions C7 and C10 are favorable anchor points for the semisynthesis of optimized derivatives. The observed SAR was different from that known for tetracyclines, which underlines the pronounced differences between the two compound classes.
    • Setting Our Sights on Infectious Diseases.

      De Rycker, Manu; Horn, David; Aldridge, Bree; Amewu, Richard K; Barry, Clifton E; Buckner, Frederick S; Cook, Sarah; Ferguson, Michael A J; Gobeau, Nathalie; Herrmann, Jennifer; et al. (American Society for Chemistry, 2019-12-06)
      In May 2019, the Wellcome Centre for Anti-Infectives Research (WCAIR) at the University of Dundee, UK, held an international conference with the aim of discussing some key questions around discovering new medicines for infectious diseases and a particular focus on diseases affecting Low and Middle Income Countries. There is an urgent need for new drugs to treat most infectious diseases. We were keen to see if there were lessons that we could learn across different disease areas and between the preclinical and clinical phases with the aim of exploring how we can improve and speed up the drug discovery, translational, and clinical development processes. We started with an introductory session on the current situation and then worked backward from clinical development to combination therapy, pharmacokinetic/pharmacodynamic (PK/PD) studies, drug discovery pathways, and new starting points and targets. This Viewpoint aims to capture some of the learnings.
    • PLGA nanocapsules improve the delivery of clarithromycin to kill intracellular Staphylococcus aureus and Mycobacterium abscessus.

      Anversa Dimer, Frantiescoli; de Souza Carvalho-Wodarz, Cristiane; Goes, Adriely; Cirnski, Katarina; Herrmann, Jennifer; Schmitt, Viktoria; Pätzold, Linda; Abed, Nadia; de Rossi, Chiara; Bischoff, Markus; et al. (Elsevier, 2019-11-18)
      Drug delivery systems are promising for targeting antibiotics directly to infected tissues. To reach intracellular Staphylococcus aureus and Mycobacterium abscessus, we encapsulated clarithromycin in PLGA nanocapsules, suitable for aerosol delivery by nebulization of an aqueous dispersion. Compared to the same dose of free clarithromycin, nanoencapsulation reduced 1000 times the number of intracellular S. aureus in vitro. In RAW cells, while untreated S. aureus was located in acidic compartments, the treated ones were mostly situated in non-acidic compartments. Clarithromycin-nanocapsules were also effective against M. abscessus (70-80% killing efficacy). The activity of clarithromycin-nanocapsules against S. aureus was also confirmed in vivo, using a murine wound model as well as in zebrafish. The permeability of clarithromycin-nanocapsules across Calu-3 monolayers increased in comparison to the free drug, suggesting an improved delivery to sub-epithelial tissues. Thus, clarithromycin-nanocapsules are a promising strategy to target intracellular S. aureus and M. abscessus.
    • Homologous bd oxidases share the same architecture but differ in mechanism.

      Theßeling, Alexander; Rasmussen, Tim; Burschel, Sabrina; Wohlwend, Daniel; Kägi, Jan; Müller, Rolf; Böttcher, Bettina; Friedrich, Thorsten; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Nature Publication group, 2019-11-13)
      Cytochrome bd oxidases are terminal reductases of bacterial and archaeal respiratory chains. The enzyme couples the oxidation of ubiquinol or menaquinol with the reduction of dioxygen to water, thus contributing to the generation of the protonmotive force. Here, we determine the structure of the Escherichia coli bd oxidase treated with the specific inhibitor aurachin by cryo-electron microscopy (cryo-EM). The major subunits CydA and CydB are related by a pseudo two fold symmetry. The heme b and d cofactors are found in CydA, while ubiquinone-8 is bound at the homologous positions in CydB to stabilize its structure. The architecture of the E. coli enzyme is highly similar to that of Geobacillus thermodenitrificans, however, the positions of heme b595 and d are interchanged, and a common oxygen channel is blocked by a fourth subunit and substituted by a more narrow, alternative channel. Thus, with the same overall fold, the homologous enzymes exhibit a different mechanism.
    • The mRNA-binding Protein TTP/ZFP36 in Hepatocarcinogenesis and Hepatocellular Carcinoma.

      Kröhler, Tarek; Kessler, Sonja M; Hosseini, Kevan; List, Markus; Barghash, Ahmad; Patial, Sonika; Laggai, Stephan; Gemperlein, Katja; Haybaeck, Johannes; Müller, Rolf; et al. (MDPI, 2019-11-08)
      Hepatic lipid deposition and inflammation represent risk factors for hepatocellular carcinoma (HCC). The mRNA-binding protein tristetraprolin (TTP, gene name ZFP36) has been suggested as a tumor suppressor in several malignancies, but it increases insulin resistance. The aim of this study was to elucidate the role of TTP in hepatocarcinogenesis and HCC progression. Employing liver-specific TTP-knockout (lsTtp-KO) mice in the diethylnitrosamine (DEN) hepatocarcinogenesis model, we observed a significantly reduced tumor burden compared to wild-type animals. Upon short-term DEN treatment, modelling early inflammatory processes in hepatocarcinogenesis, lsTtp-KO mice exhibited a reduced monocyte/macrophage ratio as compared to wild-type mice. While short-term DEN strongly induced an abundance of saturated and poly-unsaturated hepatic fatty acids, lsTtp-KO mice did not show these changes. These findings suggested anti-carcinogenic actions of TTP deletion due to effects on inflammation and metabolism. Interestingly, though, investigating effects of TTP on different hallmarks of cancer suggested tumor-suppressing actions: TTP inhibited proliferation, attenuated migration, and slightly increased chemosensitivity. In line with a tumor-suppressing activity, we observed a reduced expression of several oncogenes in TTP-overexpressing cells. Accordingly, ZFP36 expression was downregulated in tumor tissues in three large human data sets. Taken together, this study suggests that hepatocytic TTP promotes hepatocarcinogenesis, while it shows tumor-suppressive actions during hepatic tumor progression.
    • Differential regulation of AMP-activated protein kinase in healthy and cancer cells explains why V-ATPase inhibition selectively kills cancer cells.

      Bartel, Karin; Müller, Rolf; von Schwarzenberg, Karin; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (American Society for biochemistry and Molecular Biology, 2019-10-11)
      The cellular energy sensor AMP-activated protein kinase (AMPK) is a metabolic hub regulating various pathways involved in tumor metabolism. Here, we report that vacuolar H+-ATPase (V-ATPase) inhibition differentially affects regulation of AMPK in tumor and non-tumor cells and that this differential regulation contributes to the selectivity of V-ATPase inhibitors for tumor cells. In non-malignant cells, the V-ATPase inhibitor archazolid increased phosphorylation and lysosomal localization of AMPK. We noted that AMPK localization has a pro-survival role, as AMPK silencing decreased cellular growth rates. In contrast, in cancer cells, we found that AMPK is constitutively active and that archazolid does not affect its phosphorylation and localization. Moreover, V-ATPase-independent AMPK induction in the tumor cells protected them from archazolid-induced cytotoxicity, further underlining the role of AMPK as a pro-survival mediator. These observations indicate that AMPK regulation is uncoupled from V-ATPase activity in cancer cells and that this makes them more susceptible to cell death induction by V-ATPase inhibitors. In both tumor and healthy cells, V-ATPase inhibition induced a distinct metabolic regulatory cascade downstream of AMPK, affecting ATP and NADPH levels, glucose uptake, and reactive oxygen species (ROS) production. We could attribute the pro-survival effects to AMPK's ability to maintain redox homeostasis by inhibiting ROS production and maintaining NADPH levels. In summary, the results of our work indicate that V-ATPase inhibition has differential effects on AMPK-mediated metabolic regulation in cancer and healthy cells and explain the tumor-specific cytotoxicity of V-ATPase inhibition.
    • Polyunsaturated fatty acid production by Yarrowia lipolytica employing designed myxobacterial PUFA synthases.

      Gemperlein, Katja; Dietrich, Demian; Kohlstedt, Michael; Zipf, Gregor; Bernauer, Hubert S; Wittmann, Christoph; Wenzel, Silke C; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Nature publishing Group, 2019-09-06)
      Long-chain polyunsaturated fatty acids (LC-PUFAs), particularly the omega-3 LC-PUFAs eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA), have been associated with beneficial health effects. Consequently, sustainable sources have to be developed to meet the increasing demand for these PUFAs. Here, we demonstrate the design and construction of artificial PUFA biosynthetic gene clusters (BGCs) encoding polyketide synthase-like PUFA synthases from myxobacteria adapted for the oleaginous yeast Yarrowia lipolytica. Genomic integration and heterologous expression of unmodified or hybrid PUFA BGCs yielded different yeast strains with specific LC-PUFA production profiles at promising yield and thus valuable for the biotechnological production of distinct PUFAs. Nutrient screening revealed a strong enhancement of PUFA production, when cells were phosphate limited. This represents, to the best of our knowledge, highest concentration of DHA (16.8 %) in total fatty acids among all published PUFA-producing Y. lipolytica strains.
    • Acetyl-CoA carboxylase 1-dependent lipogenesis promotes autophagy downstream of AMPK.

      Gross, Angelina S; Zimmermann, Andreas; Pendl, Tobias; Schroeder, Sabrina; Schoenlechner, Hannes; Knittelfelder, Oskar; Lamplmayr, Laura; Santiso, Ana; Aufschnaiter, Andreas; Waltenstorfer, Daniel; et al. (American Society for biochemistry and molecular biology, 2019-08-09)
      Autophagy, a membrane-dependent catabolic process, ensures survival of aging cells and depends on the cellular energetic status. Acetyl-CoA carboxylase 1 (Acc1) connects central energy metabolism to lipid biosynthesis and is rate-limiting for the de novo synthesis of lipids. However, it is unclear how de novo lipogenesis and its metabolic consequences affect autophagic activity. Here, we show that in aging yeast, autophagy levels highly depend on the activity of Acc1. Constitutively active Acc1 (acc1S/A ) or a deletion of the Acc1 negative regulator, Snf1 (yeast AMPK), shows elevated autophagy levels, which can be reversed by the Acc1 inhibitor soraphen A. Vice versa, pharmacological inhibition of Acc1 drastically reduces cell survival and results in the accumulation of Atg8-positive structures at the vacuolar membrane, suggesting late defects in the autophagic cascade. As expected, acc1S/A cells exhibit a reduction in acetate/acetyl-CoA availability along with elevated cellular lipid content. However, concomitant administration of acetate fails to fully revert the increase in autophagy exerted by acc1S/A Instead, administration of oleate, while mimicking constitutively active Acc1 in WT cells, alleviates the vacuolar fusion defects induced by Acc1 inhibition. Our results argue for a largely lipid-dependent process of autophagy regulation downstream of Acc1. We present a versatile genetic model to investigate the complex relationship between acetate metabolism, lipid homeostasis, and autophagy and propose Acc1-dependent lipogenesis as a fundamental metabolic path downstream of Snf1 to maintain autophagy and survival during cellular aging.
    • Watching DNA replication inhibitors in action: Exploiting time-lapse microfluidic microscopy as a tool for target-drug interaction studies in Mycobacterium .

      Trojanowski, Damian; Kołodziej, Marta; Hołówka, Joanna; Müller, Rolf; Zakrzewska-Czerwińska, Jolanta; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (American Society of microbiology, 2019-08-05)
      Spreading resistance to antibiotics and the emergence of multidrug-resistant strains have become frequent in many bacterial species, including mycobacteria - a causative agents of severe diseases and have profound impacts on global health. Here, we used a system of microfluidics, fluorescence microscopy and target-tagged fluorescent reporter strains of Mycobacterium smegmatis to perform real-time monitoring of replisome and chromosome dynamics following the addition of replication-altering drugs (novobiocin, nalidixic acid and griselimycin) at the single-cell level. We found that novobiocin stalled replication forks and caused relaxation of the nucleoid, nalidixic acid triggered rapid replisome collapse and compaction of the nucleoid, while griselimycin caused replisome instability with subsequent over-initiation of chromosome replication and over-relaxation of the nucleoid. In addition to study target-drug interactions, our system also enabled to observe how the tested antibiotics affected the physiology of mycobacterial cells (i.e., growth, chromosome segregation, etc.).
    • Connecting lysosomes and mitochondria - a novel role for lipid metabolism in cancer cell death.

      Bartel, Karin; Pein, Helmut; Popper, Bastian; Schmitt, Sabine; Janaki-Raman, Sudha; Schulze, Almut; Lengauer, Florian; Koeberle, Andreas; Werz, Oliver; Zischka, Hans; et al. (BMC, 2019-07-29)
      BACKGROUND: The understanding of lysosomes has been expanded in recent research way beyond their view as cellular trash can. Lysosomes are pivotal in regulating metabolism, endocytosis and autophagy and are implicated in cancer. Recently it was discovered that the lysosomal V-ATPase, which is known to induce apoptosis, interferes with lipid metabolism in cancer, yet the interplay between these organelles is poorly understood. METHODS: LC-MS/MS analysis was performed to investigate lipid distribution in cells. Cell survival and signaling pathways were analyzed by means of cell biological methods (qPCR, Western Blot, flow cytometry, CellTiter-Blue). Mitochondrial structure was analyzed by confocal imaging and electron microscopy, their function was determined by flow cytometry and seahorse measurements. RESULTS: Our data reveal that interfering with lysosomal function changes composition and subcellular localization of triacylglycerids accompanied by an upregulation of PGC1α and PPARα expression, master regulators of energy and lipid metabolism. Furthermore, cardiolipin content is reduced driving mitochondria into fission, accompanied by a loss of membrane potential and reduction in oxidative capacity, which leads to a deregulation in cellular ROS and induction of mitochondria-driven apoptosis. Additionally, cells undergo a metabolic shift to glutamine dependency, correlated with the fission phenotype and sensitivity to lysosomal inhibition, most prominent in Ras mutated cells. CONCLUSION: This study sheds mechanistic light on a largely uninvestigated triangle between lysosomes, lipid metabolism and mitochondrial function. Insight into this organelle crosstalk increases our understanding of mitochondria-driven cell death. Our findings furthermore provide a first hint on a connection of Ras pathway mutations and sensitivity towards lysosomal inhibitors.
    • A central hydrophobic E1 region controls the pH range of hepatitis C virus membrane fusion and susceptibility to fusion inhibitors.

      Banda, Dominic H; Perin, Paula M; Brown, Richard J P; Todt, Daniel; Solodenko, Wladimir; Hoffmeyer, Patrick; Kumar Sahu, Kamlesh; Houghton, Michael; Meuleman, Philip; Müller, Rolf; et al. (Elsevier, 2019-06-01)
    • Integrating Culture-based Antibiotic Resistance Profiles with Whole-genome Sequencing Data for 11,087 Clinical Isolates.

      Galata, Valentina; Laczny, Cédric C; Backes, Christina; Hemmrich-Stanisak, Georg; Schmolke, Susanne; Franke, Andre; Meese, Eckart; Herrmann, Mathias; von Müller, Lutz; Plum, Achim; et al. (Elsevier, 2019-05-14)
      Emergingantibiotic resistanceis a major global health threat. The analysis of nucleic acidsequences linked to susceptibility phenotypes facilitates the study of genetic antibiotic resistancedeterminants to inform molecular diagnostics and drug development. We collected genetic data(11,087 newly-sequenced whole genomes) and culture-based resistance profiles (10,991 out of the11,087 isolates comprehensively tested against 22 antibiotics in total) of clinical isolates including18 main species spanning a time period of 30 years. Species and drug specific resistance patternswere observed including increased resistance rates forAcinetobacter baumanniito carbapenemsand forEscherichia colito fluoroquinolones. Species-levelpan-genomeswere constructed to reflectthe genetic repertoire of the respective species, including conserved essential genes and known resis-tance factors. Integrating phenotypes and genotypes through species-level pan-genomes allowed toinfer gene–drug resistance associations using statistical testing. The isolate collection and the analysis results have been integrated into GEAR-base, a resource available for academic research use free of charge athttps://gear-base.com
    • Tools for studying the metabolism of new psychoactive substances for toxicological screening purposes - A comparative study using pooled human liver S9, HepaRG cells, and zebrafish larvae.

      Richter, Lilian H J; Herrmann, Jennifer; Andreas, Anastasia; Park, Yu Mi; Wagmann, Lea; Flockerzi, Veit; Müller, Rolf; Meyer, Markus R; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (Elsevier, 2019-05-01)
      New psychoactive substances (NPS) are an emerging topic amongst abused compounds. New varieties appear constantly on the market, without any knowledge about their toxicodynamic and/or -kinetic properties and knowledge of their metabolism is crucial for the development of analytical methods employed for their detection. Controlled human studies would of course be best suited but due to ethical reasons and lack of preclinical safety data, they are usually not available. Often, in vitro models are used to evaluate similarities to human in vivo hepatic phase I and II metabolism and systems explored include primary human hepatocytes, pooled human S9 fraction, and HepaRG, a human hepatic cell line. All these in vitro models have considerable limitations and drug distribution, reabsorption, enterohepatic circulation, and renal elimination cannot be studied. In the recent years, zebrafish (Danio rerio) larvae (embryos) were discussed as a potential in vivo model to overcome these limitations. To date, no studies demonstrating its suitability for studying NPS metabolism in the context of analytical toxicology are available. The aim of this study was to elucidate whether zebrafish larvae can serve as a surrogate for human hepatic metabolism of NPS to develop toxicological screening procedures. Here, we used methyl 2-(1-(5-fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamido)-3,3-dimethylbutanoate (7'N-5F-ADB), a new synthetic cannabinoid, whose human metabolism was recently described in the literature, as a model compound to evaluate zebrafish larvae as a new tool for metabolism studies. Different conditions for zebrafish larvae and HepaRG protocols were tested. As zebrafish larvae and HepaRG cell incubations provided the highest number of metabolites and the most authentic spectrum of human metabolites. The most suitable larvae protocol was the incubation via medium and the analysis of the extracted zebrafish larvae. The zebrafish larvae model might be a promising preclinical surrogate for human hepatic metabolism of NPS.
    • A high-affinity fluorescence probe for copper(II) ions and its application in fluorescence lifetime correlation spectroscopy.

      Grüter, Andreas; Hoffmann, Michael; Müller, Rolf; Wohland, Thorsten; Jung, Gregor; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer, 2019-04-26)
      Copper is one of the most important transition metals in many organisms where it catalyzes a manifold of different processes. As a result of copper's redox activity, organisms have to avoid unbound ions, and a dysfunctional copper homeostasis may lead to multifarious pathological processes in cells with very severe ramifications for the affected organisms. In many neurodegenerative diseases, however, the exact role of copper ions is still not completely clarified. In this work, a high-affinity and highly selective copper probe molecule, based on the naturally occurring tetrapeptide DAHK is synthesized. The sensor (log KD = - 12.8 ± 0.1) is tagged with a fluorescent BODIPY dye whose fluorescence lifetime distinctly decreases from 5.8 ns ± 0.2 ns to 0.4 ns ± 0.1 ns on binding to copper(II) cations. It is shown by using fluorescence lifetime correlation spectroscopy that the concentration of both probe and probe-copper complex can be simultaneously measured even at nanomolar concentration levels. This work presents a possible starting point for a new type of probe and method for future in vivo studies to further reveal the exact role of copper ions in organisms. Graphical abstract.
    • Targeting actin inhibits repair of doxorubicin-induced DNA damage: a novel therapeutic approach for combination therapy.

      Pfitzer, Lisa; Moser, Christina; Gegenfurtner, Florian; Arner, Anja; Foerster, Florian; Atzberger, Carina; Zisis, Themistoklis; Kubisch-Dohmen, Rebekka; Busse, Johanna; Smith, Rebecca; et al. (Springer-Nature, 2019-04-03)
      Severe side effects often restrict clinical application of the widely used chemotherapeutic drug doxorubicin. In order to decrease required substance concentrations, new concepts for successful combination therapy are needed. Since doxorubicin causes DNA damage, combination with compounds that modulate DNA repair could be a promising strategy. Very recently, a role of nuclear actin for DNA damage repair has been proposed, making actin a potential target for cancer therapy in combination with DNA-damaging therapeutics. This is of special interest, since actin-binding compounds have not yet found their way into clinics. We find that low-dose combination treatment of doxorubicin with the actin polymerizer chondramide B (ChB) synergistically inhibits tumor growth in vivo. On the cellular level we demonstrate that actin binders inhibit distinctive double strand break (DSB) repair pathways. Actin manipulation impairs the recruitment of replication factor A (RPA) to the site of damage, a process crucial for homologous recombination. In addition, actin binders reduce autophosphorylation of DNA-dependent protein kinase (DNA-PK) during nonhomologous end joining. Our findings substantiate a direct involvement of actin in nuclear DSB repair pathways, and propose actin as a therapeutic target for combination therapy with DNA-damaging agents such as doxorubicin.
    • Cystobactamids 920-1 and 920-2: Assignment of the Constitution and Relative Configuration by Total Synthesis.

      Planke, Therese; Moreno, María; Hüttel, Stephan; Fohrer, Jörg; Gille, Franziska; Norris, Matthew D; Siebke, Maik; Wang, Liangliang; Müller, Rolf; Kirschning, Andreas; et al. (ACS Publications, 2019-03-01)
      Total synthesis of cystobactamid 920-1 and its epimer has allowed an unambiguous assignment of the relative and absolute configuration of the natural product. A careful structural analysis of each isomer using both NMR and computational techniques also prompted a constitutional revision of the structures originally reported for cystobactamids 920-1 and 920-2, and has provided further insight into the unique conformational preferences of the cystobactamid family
    • A Highly Polymorphic Receptor Governs Many Distinct Self-Recognition Types within the Myxococcales Order.

      Cao, Pengbo; Wei, Xueming; Awal, Ram Prasad; Müller, Rolf; Wall, Daniel; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (American Society of Microbiology, 2019-02-12)
      Self-recognition underlies sociality in many group-living organisms. In bacteria, cells use various strategies to recognize kin to form social groups and, in some cases, to transition into multicellular life. One strategy relies on a single genetic locus that encodes a variable phenotypic tag (“greenbeard”) for recognizing other tag bearers. Previously, we discovered a polymorphic cell surface receptor called TraA that directs self-identification through homotypic interactions in the social bacterium Myxococcus xanthus. Recognition by TraA leads to cellular resource sharing in a process called outer membrane exchange (OME). A second gene in the traA operon, traB, is also required for OME but is not involved in recognition. Our prior studies of TraA identified only six recognition groups among closely related M. xanthus isolates. Here we hypothesize that the number of traA polymorphisms and, consequently, the diversity of recognition in wild isolates are much greater. To test this hypothesis, we expand the scope of TraA characterization to the order Myxococcales. From genomic sequences within the three suborders of Myxococcales, we identified 90 traA orthologs. Sequence analyses and functional characterization of traAB loci suggest that OME is well maintained among diverse myxobacterial taxonomic groups. Importantly, TraA orthologs are highly polymorphic within their variable domain, the region that confers selectivity in self-recognition. We experimentally defined 10 distinct recognition groups and, based on phylogenetic and experimental analyses, predicted >60 recognition groups among the 90 traA alleles. Taken together, our findings revealed a widespread greenbeard locus that mediates the diversity of self-recognition across the order Myxococcales.
    • Aurantimycin resistance genes contribute to survival of Listeria monocytogenes during life in the environment.

      Hauf, Samuel; Herrmann, Jennifer; Miethke, Marcus; Gibhardt, Johannes; Commichau, Fabian M; Müller, Rolf; Fuchs, Stephan; Halbedel, Sven; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Wiley-Blackwell, 2019-01-15)
      Bacteria can cope with toxic compounds such as antibiotics by inducing genes for their detoxification. A common detoxification strategy is compound excretion by ATP-binding cassette (ABC) transporters, which are synthesized upon compound contact. We previously identified the multi drug resistance ABC transporter LieAB in Listeria monocytogenes, a Gram-positive bacterium that occurs ubiquitously in the environment, but also causes severe infections in humans upon ingestion. Expression of the lieAB genes is strongly induced in cells lacking the PadR-type transcriptional repressor LftR, but compounds leading to relief of this repression in wild type cells were not known. Using RNA-Seq and promoter-lacZ fusions we demonstrate highly specific repression of the lieAB and lftRS promoters through LftR. Screening of a natural compound library yielded the depsipeptide aurantimycin A - synthesized by the soil-dwelling Streptomyces aurantiacus - as the first known naturally occurring inducer of lieAB expression. Genetic and phenotypic experiments concordantly show that aurantimycin A is a substrate of the LieAB transporter and thus, lftRS and lieAB represent the first known genetic module conferring and regulating aurantimycin A resistance. Collectively, these genes may support the survival of L. monocytogenes when it comes into contact with antibiotic-producing bacteria in the soil. This article is protected by copyright. All rights reserved.