• EU-OPENSCREEN-chemical tools for the study of plant biology and resistance mechanisms.

      Meiners, Torsten; Stechmann, Bahne; Frank, Ronald; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2014-10)
      EU-OPENSCREEN is an academic research infrastructure initiative in Europe for enabling researchers in all life sciences to take advantage of chemical biology approaches to their projects. In a collaborative effort of national networks in 16 European countries, EU-OPENSCREEN will develop novel chemical compounds with external users to address questions in, among other fields, systems and network biology (directed and selective perturbation of signalling pathways), structural biology (compound-target interactions at atomic resolution), pharmacology (early drug discovery and toxicology) and plant biology (response of wild or crop plants to environmental and agricultural substances). EU-OPENSCREEN supports all stages of a tool development project, including assay adaptation, high-throughput screening and chemical optimisation of the 'hit' compounds. All tool compounds and data will be made available to the scientific community. EU-OPENSCREEN integrates high-capacity screening platforms throughout Europe, which share a rationally selected compound collection comprising up to 300,000 (commercial and proprietary compounds collected from European chemists). By testing systematically this chemical collection in hundreds of assays originating from very different biological themes, the screening process generates enormous amounts of information about the biological activities of the substances and thereby steadily enriches our understanding of how and where they act.
    • Evaluation of the inflammatory potential of implant materials in a mouse model by bioluminescent imaging of intravenously injected bone marrow cells.

      Rais, Bushra; Köster, Mario; Rahim, Muhammad Imran; Pils, Marina; Seitz, Jan-Marten; Hauser, Hansjörg; Wirth, Dagmar; Mueller, Peter P; Helmholtz Centre for infection research, Inhoffenstr. 7,38124 Braunschweig, Germany. (2016-09)
      To evaluate the inflammatory potential of implants a bioluminescent imaging assay was developed using luciferase-expressing bone marrow cells that were injected into the blood circulation of wild-type mice. After subcutaneous implantation of titanium discs as an example for a clinically established biocompatible material, the luminosity was modest. Similarly, low luminosity signals were generated by pure magnesium implants that were used to represent metallic alloys that are presently under investigation as novel degradable implant materials. Increased luminosity was observed in response to degradable polymeric PLGA implants. Surgical wounds induced a basic luminescent response even in the absence of an implant. However, the material-independent response to injury could be minimized using injectable microparticle suspensions. In parallel with the resorption of biodegradable microparticles, the signal induced by PLGA declined faster when compared to non-degradable polystyrene suspensions. By using an interferon type I inducible Mx2 promoter construct to drive luciferase gene expression, the highest luminosity was observed in response to bacteria, indicating that the system could also be employed to monitor implant infections. Overall, labeled bone marrow cells yielded specific, well-defined localized signals that correlated with the inflammatory responses to implants. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2149-2158, 2016.
    • Evidence for inoculum size and gas interfaces as critical factors in bacterial biofilm formation on magnesium implants in an animal model.

      Rahim, Muhammad Imran; Szafrański, Szymon P; Ingendoh-Tsakmakidis, Alexandra; Stiesch, Meike; Mueller, Peter P; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2019-11-28)
      Infections of medical implants caused by bacterial biofilms are a major clinical problem. Bacterial colonization is predicted to be prevented by alkaline magnesium surfaces. However, in experimental animal studies, magnesium implants prolonged infections. The reason for this peculiarity likely lies within the ‒still largely hypothetical‒ mechanism by which infection arises. Investigating subcutaneous magnesium implants infected with bioluminescent Pseudomonas aeruginosa via in vivo imaging, we found that the rate of implant infections was critically dependent on a surprisingly high quantity of injected bacteria. At high inocula, bacteria were antibiotic-refractory immediately after infection. High cell densities are known to limit nutrient availability, restricting proliferation and trigger quorum sensing which could both contribute to the rapid initial resistance. We propose that gas bubbles such as those formed during magnesium corrosion, can then act as interfaces that support biofilm formation and permit long-term survival. This model could provide an explanation for the apparent ineffectiveness of innovative contact-dependent bactericidal implant surfaces in patients. In addition, the model points toward air bubbles in tissue, either by inclusion during surgery or by spontaneous gas bubble formation later on, could constitute a key risk factor for clinical implant infections
    • Expansion of functional personalized cells with specific transgene combinations.

      Lipps, Christoph; Klein, Franziska; Wahlicht, Tom; Seiffert, Virginia; Butueva, Milada; Zauers, Jeannette; Truschel, Theresa; Luckner, Martin; Köster, Mario; MacLeod, Roderick; et al. (Springer Nature, 2018-03-08)
      Fundamental research and drug development for personalized medicine necessitates cell cultures from defined genetic backgrounds. However, providing sufficient numbers of authentic cells from individuals poses a challenge. Here, we present a new strategy for rapid cell expansion that overcomes current limitations. Using a small gene library, we expanded primary cells from different tissues, donors, and species. Cell-type-specific regimens that allow the reproducible creation of cell lines were identified. In depth characterization of a series of endothelial and hepatocytic cell lines confirmed phenotypic stability and functionality. Applying this technology enables rapid, efficient, and reliable production of unlimited numbers of personalized cells. As such, these cell systems support mechanistic studies, epidemiological research, and tailored drug development.
    • Filovirus antiviral activity of cationic amphiphilic drugs is associated with lipophilicity and ability to induce phospholipidosis.

      Gunesch, Antonia P; Zapatero-Belinchon, Francisco J; Pinkert, Lukas; Steinmann, Eike; Manns, Michael P; Schneider, Gisbert; Pietschmann, Thomas; Brönstrup, Mark; von Hahn, Thomas; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (ASM, 2020-06-08)
      Several cationic amphiphilic drugs (CADs) have been found to inhibit cell entry of filoviruses and other enveloped viruses. Structurally unrelated CADs may have antiviral activity, yet the underlying common mechanism and structure-activity relationship are incompletely understood.We aimed to understand how widespread antiviral activity is among CADs and which structural and physico-chemical properties are linked to entry inhibition.We measured inhibition of Marburg virus pseudoparticle (MARVpp) cell entry by 45 heterogeneous and mostly FDA-approved CADs and cytotoxicity in EA.hy926 cells. We analysed correlation of antiviral activity with four chemical properties: pKa, ClogP, molecular weight and distance between the basic group and hydrophobic ring structures. Additionally, we quantified drug-induced phospholipidosis (DIPL) of a CAD subset by flow cytometry. Structurally similar compounds (derivatives) and those with similar chemical properties but unrelated structure (analogues) to strong inhibitors were obtained by two in silico similarity search approaches and tested for antiviral activity. Overall 11 out of 45 (24 %) CADs inhibited MARVpp by 40 % or more. The strongest antiviral compounds were dronedarone, triparanol and quinacrine. Structure-activity relationship studies revealed highly significant correlations between antiviral activity, hydrophobicity (ClogP>4), and DIPL. Moreover, pKa and intra-molecular distance between hydrophobic and hydrophilic moieties correlated with antiviral activity, but to a lesser extent. We also showed that in contrast to analogues, derivatives had similar antiviral activity as the seed compound dronedarone. Overall, one quarter of CADs inhibits MARVpp entry in vitro and antiviral activity of CADs mostly relies on their hydrophobicity, yet is promoted by the individual structure.
    • Firefly Bioluminescence-Based Detection of ATP

      Jarrad, Angie M,; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (CSIRO Publishing, 2019-06-04)
      Adenosine triphosphate (ATP) bioluminescence is a powerful light-producing phenomenon that occurs in nature in a variety of organisms, with ATP bioluminescence of fireflies one of the most well-known examples. The firefly ATP bioluminescence reaction has been adapted to the laboratory with a wide range of applications that include monitoring cellular processes, antimicrobial susceptibility testing, and the detection of bacterial contamination of environmental surfaces. ATP bioluminescence occurs through a multistep reaction between firefly luciferase, ATP, magnesium salt, and oxygen (Scheme 1).[1] As a simplified overview, luciferyl adenylate 2 is first formed from luciferin 1 and Mg2+-ATP. The luciferyl adenylate 2 is then oxidised with molecular oxygen to form a dioxetanone cyclic peroxide intermediate 3. Following intramolecular conversion to produce electronically excited states of oxyluciferin, the dioxetanone is decarboxylated. Finally, the return of excited oxyluciferin to the ground state 5 results in emission of visible light. For more detailed insights into the reaction mechanism, including alternative reactions and different tautomers of oxyluciferin at varying pH values, readers are referred to additional literature.
    • First Syntheses of Melophlins P, Q, and R, and Effects of Melophlins on the Growth of Microorganisms and Tumor Cells

      Biersack, Bernhard; Diestel, Randi; Jagusch, Carsten; Rapp, Georg; Sasse, Florenz; Schobert, Rainer; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2008-11)
    • From binary to multivalued to continuous models: the lac operon as a case study.

      Franke, Raimo; Theis, Fabian J; Klamt, Steffen; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2010-12-14)
      Using the lac operon as a paradigmatic example for a gene regulatory system in prokaryotes, we demonstrate how qualitative knowledge can be initially captured using simple discrete (Boolean) models and then stepwise refined to multivalued logical models and finally to continuous (ODE) models. At all stages, signal transduction and transcriptional regulation is integrated in the model description. We first show the potential benefit of a discrete binary approach and discuss then problems and limitations due to indeterminacy arising in cyclic networks. These limitations can be partially circumvented by using multilevel logic as generalization of the Boolean framework enabling one to formulate a more realistic model of the lac operon. Ultimately a dynamic description is needed to fully appreciate the potential dynamic behavior that can be induced by regulatory feedback loops. As a very promising method we show how the use of multivariate polynomial interpolation allows transformation of the logical network into a system of ordinary differential equations (ODEs), which then enables the analysis of key features of the dynamic behavior.
    • The full-ORF clone resource of the German cDNA Consortium

      Bechtel, Stephanie; Rosenfelder, Heiko; Duda, Anny; Peter Schmidt, Christian; Ernst, Ute; Wellenreuther, Ruth; Mehrle, Alexander; Schuster, Claudia; Bahr, Andre; Blöcker, Helmut; et al. (2007-10-31)
      Abstract Background With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis. Gene and ORF coverage with full-length ORF clones thus needs to be extended. In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes. Results Here we describe the generation of a full-ORF clone resource of human genes applying the Gateway cloning technology (Invitrogen). A pipeline for efficient cloning and sequencing was developed and a sample tracking database was implemented to streamline the clone production process targeting more than 2,200 different ORFs. In addition, a robust cloning strategy was established, permitting the simultaneous generation of two clone variants that contain a particular ORF with as well as without a stop codon by the implementation of only one additional working step into the cloning procedure. Up to 92 % of the targeted ORFs were successfully amplified by PCR and more than 93 % of the amplicons successfully cloned. Conclusion The German cDNA Consortium ORFeome resource currently consists of more than 3,800 sequence-verified entry clones representing ORFs, cloned with and without stop codon, for about 1,700 different gene loci. 177 splice variants were cloned representing 121 of these genes. The entry clones have been used to generate over 5,000 different expression constructs, providing the basis for functional profiling applications. As a member of the recently formed international ORFeome collaboration we substantially contribute to generating and providing a whole genome human ORFeome collection in a unique cloning system that is made freely available in the community.
    • Generation of novel-substrate-accepting biphenyl dioxygenases through segmental random mutagenesis and identification of residues involved in enzyme specificity.

      Zielinski, Marco; Kahl, Silke; Standfuss-Gabisch, Christine; Cámara, Beatriz; Seeger, Michael; Hofer, Bernd; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2006-03)
      Aryl-hydroxylating dioxygenases are of interest for the degradation of persistant aromatic pollutants, such as polychlorobiphenyls (PCBs), or as catalysts for the functionalization of aromatic scaffolds. In order to achieve dioxygenation of technical mixtures of PCBs, enzymes with broadened or altered substrate ranges are essential. To alter the substrate specificity of the biphenyl dioxygenase (BphA) of Burkholderia xenovorans LB400, we applied a directed evolution approach that used structure-function relationship data to target random mutageneses to specific segments of the enzyme. The limitation of random amino acid (AA) substitutions to regions that are critical for substrate binding and the exclusion of AA exchanges from positions that are essential for catalytic activity yielded enzyme variants of interest at comparatively high frequencies. After only a single mutagenic cycle, 10 beneficial variants were detected in a library of fewer than 1,000 active enzymes. Compared to the parental BphA, they showed between 5- and 200-fold increased turnover of chlorinated biphenyls, with substituent patterns that rendered them largely recalcitrant to attack by BphA-LB400. Determination of their sequences identified AAs that prevent the acceptance of specific PCBs by the wild-type enzyme, such as Pro334 and Phe384. The results suggest prime targets for subsequent cycles of BphA modification. Correlations with a three-dimensional model of the enzyme indicated that most of the exchanges with major influence on substrate turnover do not involve pocket-lining residues and had not been predictable through structural modeling.
    • High immune response rates and decreased frequencies of regulatory T cells in metastatic renal cell carcinoma patients after tumor cell vaccination.

      Pohla, Heike; Buchner, Alexander; Stadlbauer, Birgit; Frankenberger, Bernhard; Stevanovic, Stefan; Walter, Steffen; Frank, Ronald; Schwachula, Tim; Olek, Sven; Kopp, Joachim; et al. (2013-02-08)
      Our previously reported phase I clinical trial with the allogeneic gene-modified tumor cell line RCC-26/CD80/IL-2 showed that vaccination was well tolerated and feasible in metastatic renal cell carcinoma (RCC) patients. Substantial disease stabilization was observed in most patients despite a high tumor burden at study entry. To investigate alterations in immune responses that might contribute to this effect, we performed an extended immune monitoring that included analysis of reactivity against multiple antigens, cytokine/chemokine changes in serum and determination of the frequencies of immune suppressor cell populations, including natural regulatory T cells (nTregs) and myeloid-derived suppressor cell subsets (MDSCs). An overall immune response capacity to virus-derived control peptides was present in 100% of patients before vaccination. Vaccine-induced immune responses to tumor-associated antigens occurred in 75% of patients, demonstrating the potent immune stimulatory capacity of this generic vaccine. Furthermore, some patients reacted to peptide epitopes of antigens not expressed by the vaccine, showing that epitope-spreading occurred in vivo. Frequencies of nTregs and MDSCs were comparable to healthy donors at the beginning of study. A significant decrease of nTregs was detected after vaccination (p = 0.012). High immune response rates, decreased frequencies of nTregs and a mixed T helper 1/T helper 2 (T(H)1/T(H)2)-like cytokine pattern support the applicability of this RCC generic vaccine for use in combination therapies.
    • High level expression of a recombinant amylosucrase gene and selected properties of the enzyme.

      Schneider, Jens; Fricke, Christin; Overwin, Heike; Hofer, Bernd; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2011-03)
      Two high-level heterologous expression systems for amylosucrase genes have been constructed. One depends on sigma-70 bacterial RNA polymerase, the other on phage T7 RNA polymerase. Translational fusions were formed between slightly truncated versions of the gene from Neisseria polysaccharea and sequences of expression vectors pQE-81L or pET33b(+), respectively. These constructs were introduced into different Escherichia coli strains. The resulting recombinants yielded up to 170 mg of dissolved enzyme per litre of culture at a moderate cell density of five OD(600). To our knowledge, this is the highest yield per cell described so far for amylosucrases. The recombinant enzymes could rapidly be purified through the use of histidine tags in the N-terminally attached sequences. These segments did not alter catalytic properties and therefore need not be removed for most applications. Investigations with glucose and malto-oligosaccharides of different lengths identified rate-limiting steps in the elongation (acceptor reaction) and truncation (donor reaction) of these substrates. The elongation of maltotriose and its reversal, the truncation of maltotetraose, were found to be particularly slow reactions. Potential reasons are discussed, based on the crystal structure of the enzyme. It is furthermore shown that amylosucrase is able to synthesise mixed disaccharides. All of the glucose epimers mannose, allose, and galactose served as acceptors, yielding between one and three main products. We also demonstrate that, as an alternative to the use of purified amylosucrase, cells of the constructed recombinant strains can be used to carry out glucosylations of acceptors.
    • High-throughput screening and whole genome sequencing identifies an antimicrobially active inhibitor of Vibrio cholerae.

      Sergeev, Galina; Roy, Sambit; Jarek, Michael; Zapolskii, Viktor; Kaufmann, Dieter E; Nandy, Ranjan K; Tegge, Werner (2014)
      Pathogenic serotypes of Vibrio cholerae cause the life-threatening diarrheal disease cholera. The increasing development of bacterial resistances against the known antibiotics necessitates the search for new antimicrobial compounds and targets for this pathogen.
    • Host-induced spermidine production in motile triggers phagocytic uptake.

      Felgner, Sebastian; Preusse, Matthias; Beutling, Ulrike; Stahnke, Stephanie; Pawar, Vinay; Rohde, Manfred; Brönstrup, Mark; Stradal, Theresia; Häussler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (elifeSciences, 2020-09-22)
      Exploring the complexity of host-pathogen communication is vital to understand why microbes persist within a host, while others are cleared. Here, we employed a dual-sequencing approach to unravel conversational turn-taking of dynamic host-pathogen communications. We demonstrate that upon hitting a host cell, motile Pseudomonas aeruginosa induce a specific gene expression program. This results in the expression of spermidine on the surface, which specifically activates the PIP3-pathway to induce phagocytic uptake into primary or immortalized murine cells. Non-motile bacteria are more immunogenic due to a lower expression of arnT upon host-cell contact, but do not produce spermidine and are phagocytosed less. We demonstrate that not only the presence of pathogen inherent molecular patterns induces immune responses, but that bacterial motility is linked to a host-cell-induced expression of additional immune modulators. Our results emphasize on the value of integrating microbiological and immunological findings to unravel complex and dynamic host-pathogen interactions.
    • Hydantoin analogs inhibit the fully assembled ClpXP protease without affecting the individual peptidase and chaperone domains.

      Fetzer, Christian; Korotkov, Vadim S; Sieber, Stephan A; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Royal Society of Chemistry, 2019-08-14)
      Proteolysis mediated by ClpXP is a crucial cellular process linked to bacterial pathogenesis. The development of specific inhibitors has largely focused on ClpP. However, this focus was challenged by a recent finding showing that conformational control by ClpX leads to a rejection of ClpP binders. Thus, we here follow up on a hit molecule from a high throughput screen performed against the whole ClpXP complex and demonstrate that stable inhibition with high potency is possible. Further investigations revealed that the small molecule binds to ClpP without affecting its activity. Likewise, the molecule does not inhibit ClpX and retains the overall oligomeric state of ClpXP upon binding. Structure activity relationship studies confirmed structural constraints in all three parts of the molecule suggesting binding into a defined stereospecific pocket. Overall, the inhibition of ClpXP without affecting the individual components represents a novel mechanism with perspectives for further optimization for in situ applications.
    • 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.
    • Identification of a PA-binding peptide with inhibitory activity against influenza A and B virus replication.

      Wunderlich, Kerstin; Mayer, Daniel; Ranadheera, Charlene; Holler, Anne-Sophie; Mänz, Benjamin; Martin, Arnold; Chase, Geoffrey; Tegge, Werner; Frank, Ronald; Kessler, Ulrich; et al. (2009-10-20)
      There is an urgent need for new drugs against influenza type A and B viruses due to incomplete protection by vaccines and the emergence of resistance to current antivirals. The influenza virus polymerase complex, consisting of the PB1, PB2 and PA subunits, represents a promising target for the development of new drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between the PB1 and PA subunits of the polymerase complex of influenza A virus using a small peptide derived from the PA-binding domain of PB1. However, this influenza A virus-derived peptide did not affect influenza B virus polymerase activity. Here we report that the PA-binding domain of the polymerase subunit PB1 of influenza A and B viruses is highly conserved and that mutual amino acid exchange shows that they cannot be functionally exchanged with each other. Based on phylogenetic analysis and a novel biochemical ELISA-based screening approach, we were able to identify an influenza A-derived peptide with a single influenza B-specific amino acid substitution which efficiently binds to PA of both virus types. This dual-binding peptide blocked the viral polymerase activity and growth of both virus types. Our findings provide proof of principle that protein-protein interaction inhibitors can be generated against influenza A and B viruses. Furthermore, this dual-binding peptide, combined with our novel screening method, is a promising platform to identify new antiviral lead compounds.
    • Identification of high-affinity PB1-derived peptides with enhanced affinity to the PA protein of influenza A virus polymerase.

      Wunderlich, Kerstin; Juozapaitis, Mindaugas; Ranadheera, Charlene; Kessler, Ulrich; Martin, Arnold; Eisel, Jessica; Beutling, Ulrike; Frank, Ronald; Schwemmle, Martin; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2011-02)
      The influenza A virus polymerase complex, consisting of the subunits PB1, PB2, and PA, represents a promising target for the development of new antiviral drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between PA and PB1 using peptides derived from the extreme N terminus of PB1 (amino acids [aa] 1 to 15), comprising the PA-binding domain of PB1. To increase the binding affinity of these peptides, we performed a systematic structure-affinity relationship analysis. Alanine and aspartic acid scans revealed that almost all amino acids in the core binding region (aa 5 to 11) are indispensable for PA binding. Using a library of immobilized peptides representing all possible single amino acid substitutions, we were able to identify amino acid positions outside the core PA-binding region (aa 1, 3, 12, 14, and 15) that are variable and can be replaced by affinity-enhancing residues. Surface plasmon resonance binding studies revealed that combination of several affinity-enhancing mutations led to an additive effect. Thus, the feasibility to enhance the PA-binding affinity presents an intriguing possibility to increase antiviral activity of the PB1-derived peptide and one step forward in the development of an antiviral drug against influenza A viruses.
    • Identification of myxobacteria-derived HIV inhibitors by a high-throughput two-step infectivity assay

      Martinez, Javier P; Hinkelmann, Bettina; Fleta-Soriano, Eric; Steinmetz, Heinrich; Jansen, Rolf; Diez, Juana; Frank, Ronald; Sasse, Florenz; Meyerhans, Andreas (2013-09-24)
      Abstract Background Drug-resistance and therapy failure due to drug-drug interactions are the main challenges in current treatment against Human Immunodeficiency Virus (HIV) infection. As such, there is a continuous need for the development of new and more potent anti-HIV drugs. Here we established a high-throughput screen based on the highly permissive TZM-bl cell line to identify novel HIV inhibitors. The assay allows discriminating compounds acting on early and/or late steps of the HIV replication cycle. Results The platform was used to screen a unique library of secondary metabolites derived from myxobacteria. Several hits with good anti-HIV profiles were identified. Five of the initial hits were tested for their antiviral potency. Four myxobacterial compounds, sulfangolid C, soraphen F, epothilon D and spirangien B, showed EC50 values in the nM range with SI > 15. Interestingly, we found a high amount of overlapping hits compared with a previous screen for Hepatitis C Virus (HCV) using the same library. Conclusion The unique structures and mode-of-actions of these natural compounds make myxobacteria an attractive source of chemicals for the development of broad-spectrum antivirals. Further biological and structural studies of our initial hits might help recognize smaller drug-like derivatives that in turn could be synthesized and further optimized.
    • Immunization of pigs to prevent disease in humans: construction and protective efficacy of a Salmonella enterica serovar Typhimurium live negative-marker vaccine.

      Selke, Martin; Meens, Jochen; Springer, Sven; Frank, Ronald; Gerlach, Gerald-F; nstitute for Microbiology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Hannover, Germany. (2007-05)
      Zoonotic infections caused by Salmonella enterica serovar Typhimurium pose a constant threat to consumer health, with the pig being a particularly major source of multidrug-resistant isolates. Vaccination, as a promising approach to reduce colonization and shedding, has been scarcely used, as it interferes with current control programs relying on serology as a means of herd classification. In order to overcome this problem, we set out to develop a negative-marker vaccine allowing the differentiation of infected from vaccinated animals (DIVA). Applying an immunoproteomic approach with two-dimensional gel electrophoresis, Western blot, and quadrupole time-of-flight tandem mass spectrometry, we identified the OmpD protein as a suitable negative marker. Using allelic exchange, we generated an isogenic mutant of the licensed live vaccine strain Salmoporc and showed that virulence of Salmoporc and that of the mutant strain, SalmoporcDeltaompD, were indistinguishable in BALB/c mice. In a pig infection experiment including two oral immunizations with SalmoporcDeltaompD and challenge with a multiresistant S. enterica serovar Typhimurium DT104 clinical isolate, we confirmed the protective efficacy of SalmoporcDeltaompD in pigs, showing a significant reduction of both clinical symptoms and colonization of lymph nodes and intestinal tract. OmpD immunogenic epitopes were determined by peptide spot array analyses. Upon testing of several 9-mer peptides, each including an immunogenic epitope, one peptide (positions F(100) to Y(108)) that facilitated the detection of infected animals independent of their vaccination status (DIVA function) was identified. The approach described overcomes the problems currently limiting the use of bacterial live vaccines and holds considerable potential for future developments in the field.