Recent Submissions

  • Engineering of Streptomyces lividans for heterologous expression of secondary metabolite gene clusters.

    Ahmed, Yousra; Rebets, Yuriy; Estévez, Marta Rodríguez; Zapp, Josef; Myronovskyi, Maksym; Luzhetskyy, Andriy; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (BioMed Central (BMC), 2020-01-09)
    BACKGROUND: Heterologous expression of secondary metabolite gene clusters is used to achieve increased production of desired compounds, activate cryptic gene clusters, manipulate clusters from genetically unamenable strains, obtain natural products from uncultivable species, create new unnatural pathways, etc. Several Streptomyces species are genetically engineered for use as hosts for heterologous expression of gene clusters. S. lividans TK24 is one of the most studied and genetically tractable actinobacteria, which remain untapped. It was therefore important to generate S. lividans chassis strains with clean metabolic backgrounds. RESULTS: In this study, we generated a set of S. lividans chassis strains by deleting endogenous gene clusters and introducing additional φC31 attB loci for site-specific integration of foreign DNA. In addition to the simplified metabolic background, the engineered S. lividans strains had better growth characteristics than the parental strain in liquid production medium. The utility of the developed strains was validated by expressing four secondary metabolite gene clusters responsible for the production of different classes of natural products. Engineered strains were found to be superior to the parental strain in production of heterologous natural products. Furthermore, S. lividans-based strains were better producers of amino acid-based natural products than other tested common hosts. Expression of a Streptomyces albus subsp. chlorinus NRRL B-24108 genomic library in the modified S. lividans ΔYA9 and S. albus Del14 strains resulted in the production of 7 potentially new compounds, only one of which was produced in both strains. CONCLUSION: The constructed S. lividans-based strains are a great complement to the panel of heterologous hosts for actinobacterial secondary metabolite gene expression. The expansion of the number of such engineered strains will contribute to an increased success rate in isolation of new natural products originating from the expression of genomic and metagenomic libraries, thus raising the chance to obtain novel biologically active compounds.
  • Chemical synthesis of tripeptide thioesters for the biotechnological incorporation into the myxobacterial secondary metabolite argyrin via mutasynthesis.

    Siebert, David C B; Sommer, Roman; Pogorevc, Domen; Hoffmann, Michael; Wenzel, Silke C; Müller, Rolf; Titz, Alexander; BRICS, Braunschweiger Zentrum für Systembiologie, Rebenring 56,38106 Braunschweig, Germany. (Beilstein Institut, 2019-01-01)
    The argyrins are secondary metabolites from myxobacteria with antibiotic activity against Pseudomonas aeruginosa. Studying their structure-activity relationship is hampered by the complexity of the chemical total synthesis. Mutasynthesis is a promising approach where simpler and fully synthetic intermediates of the natural product's biosynthesis can be biotechnologically incorporated. Here, we report the synthesis of a series of tripeptide thioesters as mutasynthons containing the native sequence with a dehydroalanine (Dha) Michael acceptor attached to a sarcosine (Sar) and derivatives. Chemical synthesis of the native sequence ᴅ-Ala-Dha-Sar thioester required revision of the sequential peptide synthesis into a convergent strategy where the thioester with sarcosine was formed before coupling to the Dha-containing dipeptide.
  • Search for hits and early leads from soil bacteria to combat infectious diseases.

    Herrmann, Jennifer; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Open Learning on Enteric Pathogens, 2018-02-22)
  • The nuclear export inhibitor aminoratjadone is a potent effector in extracellular-targeted drug conjugates.

    Klahn, Philipp; Fetz, Verena; Ritter, Antje; Collisi, Wera; Hinkelmann, Bettina; Arnold, Tatjana; Tegge, Werner; Rox, Katharina; Hüttel, Stephan; Mohr, Kathrin I; et al. (Royal Society of Chemistry, 2019-05-28)
    The concept of targeted drug conjugates has been successfully translated to clinical practice in oncology. Whereas the majority of cytotoxic effectors in drug conjugates are directed against either DNA or tubulin, our study aimed to validate nuclear export inhibition as a novel effector principle in drug conjugates. For this purpose, a semisynthetic route starting from the natural product ratjadone A, a potent nuclear export inhibitor, has been developed. The biological evaluation of ratjadones functionalized at the 16-position revealed that oxo- and amino-analogues had very high potencies against cancer cell lines (e.g. 16R-aminoratjadone 16 with IC50 = 260 pM against MCF-7 cells, or 19-oxoratjadone 14 with IC50 = 100 pM against A-549 cells). Mechanistically, the conjugates retained a nuclear export inhibitory activity through binding CRM1. To demonstrate a proof-of-principle for cellular targeting, folate- and luteinizing hormone releasing hormone (LHRH)-based carrier molecules were synthesized and coupled to aminoratjadones as well as fluorescein for cellular efficacy and imaging studies, respectively. The Trojan-Horse conjugates selectively addressed receptor-positive cell lines and were highly potent inhibitors of their proliferation. For example, the folate conjugate FA-7-Val-Cit-pABA-16R-aminoratjadone had an IC50 of 34.3 nM, and the LHRH conjugate d-Orn-Gose-Val-Cit-pABA-16R-aminoratjadone had an IC50 of 12.8 nM. The results demonstrate that nuclear export inhibition is a promising mode-of-action for extracellular-targeted drug conjugate payloads.
  • 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.
  • Labyrinthopeptins exert broad-spectrum antiviral activity through lipid-binding-mediated virolysis.

    Prochnow, Hans; Rox, Katharina; Birudukota, N V Suryanarayana; Weichert, Loreen; Hotop, Sven-Kevin; Klahn, Philipp; Mohr, Kathrin; Franz, Sergej; Banda, Dominic H; Blockus, Sebastian; et al. (ASM, 2019-10-30)
    To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent attention due to their potential in treating (re-)emerging infections, for which direct acting antivirals are not available. We found that labyrinthopeptins A1 and A2, the prototype congeners of carbacyclic lanthipeptides, inhibit the proliferation of diverse enveloped viruses, including Dengue virus, Zika virus, West Nile virus, Hepatitis C virus, Chikungunya virus, Karposi's Sarcoma-associated Herpes virus, Cytomegalovirus, and Herpes Simplex virus, in the low μM to nM range. Mechanistic studies on viral particles revealed that labyrinthopeptins induce a virolytic effect through binding to the viral membrane lipid phosphatidylethanolamine (PE). These effects are enhanced by a combined equimolar application of both labyrinthopeptins, and a clear synergism was observed across a concentration range corresponding to IC10-IC90 values of the compounds. Time-resolved experiments with large unilamellar vesicles (LUVs) reveal that membrane lipid raft compositions (PC/PE/Chol/SM (17:10:33:40)) are particularly sensitive to labyrinthopeptins compared to PC/PE (90:10) LUVs, even though the overall PE-amount remains constant. Labyrinthopeptins exhibited low cytotoxicity and had favorable pharmacokinetic properties in mice (t1/2= 10.0 h), which designates them as promising antiviral compounds acting by an unusual viral lipid targeting mechanism.Importance For many viral infections, current treatment options are insufficient. Because the development of each antiviral drug is time-consuming and expensive, the prospect of finding broad-spectrum antivirals that can fight multiple, diverse viruses - well-known as well as (re-)emerging species - has gained attention, especially for the treatment of viral co-infections. While most known broad spectrum agents address processes in the host cell, we found that targeting lipids of the free virus outside the host cell with the natural products labyrinthopeptin A1 and A2 is a viable strategy to inhibit the proliferation of a broad range of viruses from different families, including Chikungunya virus, Dengue virus, Zika virus, Karposi's Sarcoma-associated Herpes virus, or Cytomegalovirus. Labyrinthopeptins bind to viral phosphatidylethanolamine and induce virolysis without exerting cytotoxicity to host cells. This represents a novel and unusual mechanism to tackle medically relevant viral infections.
  • Biotechnological Potential of Bacteria Isolated from the Sea Cucumber and from Lampung, Indonesia.

    Wibowo, Joko T; Kellermann, Matthias Y; Versluis, Dennis; Putra, Masteria Y; Murniasih, Tutik; Mohr, Kathrin I; Wink, Joachim; Engelmann, Michael; Praditya, Dimas F; Steinmann, Eike; et al. (MPDI, 2019-11-08)
    In order to minimize re-discovery of already known anti-infective compounds, we focused our screening approach on understudied, almost untapped marine environments including marine invertebrates and their associated bacteria. Therefore, two sea cucumber species, Holothuria leucospilota and Stichopus vastus, were collected from Lampung (Indonesia), and 127 bacterial strains were identified by partial 16S rRNA-gene sequencing analysis and compared with the NCBI database. In addition, the overall bacterial diversity from tissue samples of the sea cucumbers H. leucospilota and S. vastus was analyzed using the cultivation-independent Illumina MiSEQ analysis. Selected bacterial isolates were grown to high densities and the extracted biomass was tested against a selection of bacteria and fungi as well as the hepatitis C virus (HCV). Identification of putative bioactive bacterial-derived compounds were performed by analyzing the accurate mass of the precursor/parent ions (MS1) as well as product/daughter ions (MS2) using high resolution mass spectrometry (HRMS) analysis of all active fractions. With this attempt we were able to identify 23 putatively known and two previously unidentified precursor ions. Moreover, through 16S rRNA-gene sequencing we were able to identify putatively novel bacterial species from the phyla Actinobacteria, Proteobacteria and also Firmicutes. Our findings suggest that sea cucumbers like H. leucospilota and S. vastus are promising sources for the isolation of novel bacterial species that produce compounds with potentially high biotechnological potential.
  • 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.
  • Adenosine-to-Inosine RNA Editing in Mouse and Human Brain Proteomes.

    Levitsky, Lev I; Kliuchnikova, Anna A; Kuznetsova, Ksenia G; Karpov, Dmitry S; Ivanov, Mark V; Pyatnitskiy, Mikhail A; Kalinina, Olga V; Gorshkov, Mikhail V; Moshkovskii, Sergei A; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Wiley-Blackwell, 2019-10-01)
    Proteogenomics is based on the use of customized genome or RNA sequencing databases for interrogation of shotgun proteomics data in search for proteome-level evidence of genome variations or RNA editing. In this work, we identified the products of adenosine-to-inosine RNA editing in human and murine brain proteomes using publicly available brain proteome LC-MS/MS datasets and an RNA editome database compiled from several sources. After filtering of false-positive results, 20 and 37 sites of editing in proteins belonging to 14 and 32 genes were identified for murine and human brain proteomes, respectively. Eight sites of editing identified with high spectral counts overlapped between human and mouse brain samples. Some of these sites were previously reported using orthogonal methods, such as NMDA glutamate receptors, CYFIP2, coatomer alpha, etc. Also, differential editing between neurons and microglia was demonstrated in this work for some of the proteins from primary murine brain cell cultures. Because many edited sites are still not characterized functionally at the protein level, our results provide a necessary background for their further analysis in normal and diseased cells and tissues using targeted proteomic approaches.
  • Structurally diverse metabolites from the rare actinobacterium Saccharothrix xinjiangensis.

    Babadi, Zahra Khosravi; Sudarman, Enge; Ebrahimipour, Gholam Hossein; Primahana, Gian; Stadler, Marc; Wink, Joachim; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Japan Antibiotics Research Association, 2019-08-26)
    The bioassay-guided fractionation from cultures of the actinobacterium Saccharothrix xinjiangensis Act24Zk, collected from the Caspian Sea beach in Iran led to the isolation of three new compounds, caerulomycin M (1), saccharopyrone (2), and saccharonoic acid (3), together with the known compound, caerulomycin A (4). Their structures were elucidated from HR-ESIMS and 1D and 2D NMR data. Compound 2 displayed moderate cytotoxic activity against the human cervix carcinoma HeLa cells KB3.1 with an IC50 value of 5.4 µM.
  • Diversity of Bacteria Exhibiting Bile Acid-inducible 7α-dehydroxylation Genes in the Human Gut.

    Vital, Marius; Rud, Tatjana; Rath, Silke; Pieper, Dietmar H; Schlüter, Dirk; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2019-01-01)
    The secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), formed by gut microbiota from primary bile acids via a multi-step 7α-dehydroxylation reaction, have wide-ranging effects on host metabolism and play an important role in health and disease. A few 7α-dehydroxylating strains have been isolated, where bile acid-inducible (bai) genes were organized in a gene cluster and encoded major enzymes involved. However, only little is known on diversity and abundance of intestinal bacteria catalysing DCA/LCA formation in the human gut in situ. In this study, we took the opportunity to screen metagenome-assembled genomes (MAGs) from sequence data of stool samples provided by two recent studies along with newly available gut-derived isolates for the presence of the bai gene cluster. We revealed in total 765 and 620 MAGs encoding the potential to form DCA/LCA that grouped into 21 and 26 metagenomic species, respectively. The majority of MAGs (92.4 and 90.3%) were associated with a Ruminococcaceae clade that still lacks an isolate, whereas less MAGs belonged to Lachnospiraceae along with eight new isolates (n total = 11) that contained the bai genes. Only a few MAGs were linked to Peptostreptococcaceae. Signatures for horizontal transfer of bai genes were observed. This study gives a comprehensive overview of the diversity of bai-exhibiting bacteria in the human gut highlighting the application of metagenomics to unravel potential functions hidden from current isolates. Eventually, isolates of the identified main MAG clade are required in order to prove their capability of 7α-dehydroxylating primary bile acids.
  • 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.
  • 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.
  • 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.
  • Molecular profiling of tissue biopsies reveals unique signatures associated with streptococcal necrotizing soft tissue infections

    Thänert, Robert; Itzek, Andreas; Hoßmann, Jörn; Hamisch, Domenica; Madsen, Martin Bruun; Hyldegaard, Ole; Skrede, Steinar; Bruun, Trond; Norrby-Teglund, Anna; Medina, Eva; et al. (Nature, 2019-08-26)
    Necrotizing soft tissue infections (NSTIs) are devastating infections caused by either a single pathogen, predominantly Streptococcus pyogenes, or by multiple bacterial species. A better understanding of the pathogenic mechanisms underlying these different NSTI types could facilitate faster diagnostic and more effective therapeutic strategies. Here, we integrate microbial community profiling with host and pathogen(s) transcriptional analysis in patient biopsies to dissect the pathophysiology of streptococcal and polymicrobial NSTIs. We observe that the pathogenicity of polymicrobial communities is mediated by synergistic interactions between community members, fueling a cycle of bacterial colonization and inflammatory tissue destruction. In S. pyogenes NSTIs, expression of specialized virulence factors underlies infection pathophysiology. Furthermore, we identify a strong interferon-related response specific to S. pyogenes NSTIs that could be exploited as a potential diagnostic biomarker. Our study provides insights into the pathophysiology of mono- and polymicrobial NSTIs and highlights the potential of host-derived signatures for microbial diagnosis of NSTIs.
  • Clinical Resistome Screening of 1,110 Escherichia coli Isolates Efficiently Recovers Diagnostically Relevant Antibiotic Resistance Biomarkers and Potential Novel Resistance Mechanisms.

    Volz, Carsten; Ramoni, Jonas; Beisken, Stephan; Galata, Valentina; Keller, Andreas; Plum, Achim; Posch, Andreas E; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Frontiers, 2019-01-01)
    Multidrug-resistant pathogens represent one of the biggest global healthcare challenges. Molecular diagnostics can guide effective antibiotics therapy but relies on validated, predictive biomarkers. Here we present a novel, universally applicable workflow for rapid identification of antimicrobial resistance (AMR) biomarkers from clinical Escherichia coli isolates and quantitatively evaluate the potential to recover causal biomarkers for observed resistance phenotypes. For this, a metagenomic plasmid library from 1,110 clinical E. coli isolates was created and used for high-throughput screening to identify biomarker candidates against Tobramycin (TOB), Ciprofloxacin (CIP), and Trimethoprim-Sulfamethoxazole (TMP-SMX). Identified candidates were further validated in vitro and also evaluated in silico for their diagnostic performance based on matched genotype-phenotype data. AMR biomarkers recovered by the metagenomics screening approach mechanistically explained 77% of observed resistance phenotypes for Tobramycin, 76% for Trimethoprim-Sulfamethoxazole, and 20% Ciprofloxacin. Sensitivity for Ciprofloxacin resistance detection could be improved to 97% by complementing results with AMR biomarkers that are undiscoverable due to intrinsic limitations of the workflow. Additionally, when combined in a multiplex diagnostic in silico panel, the identified AMR biomarkers reached promising positive and negative predictive values of up to 97 and 99%, respectively. Finally, we demonstrate that the developed workflow can be used to identify potential novel resistance mechanisms.
  • 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.).

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