Recent Submissions

  • 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.
  • 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.
  • Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors.

    Zhang, Linlin; Lin, Daizong; Sun, Xinyuanyuan; Curth, Ute; Drosten, Christian; Sauerhering, Lucie; Becker, Stephan; Rox, Katharina; Hilgenfeld, Rolf; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (AAAS, 2020-03-20)
    The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a global health emergency. An attractive drug target among coronaviruses is the main protease (Mpro, also called 3CLpro) because of its essential role in processing the polyproteins that are translated from the viral RNA. We report the x-ray structures of the unliganded SARS-CoV-2 Mpro and its complex with an α-ketoamide inhibitor. This was derived from a previously designed inhibitor but with the P3-P2 amide bond incorporated into a pyridone ring to enhance the half-life of the compound in plasma. On the basis of the unliganded structure, we developed the lead compound into a potent inhibitor of the SARS-CoV-2 Mpro The pharmacokinetic characterization of the optimized inhibitor reveals a pronounced lung tropism and suitability for administration by the inhalative route.
  • Labyrinthopeptins as virolytic inhibitors of respiratory syncytial virus cell entry.

    Blockus, Sebastian; Sake, Svenja M; Wetzke, Martin; Grethe, Christina; Graalmann, Theresa; Pils, Marina; Le Goffic, Ronan; Galloux, Marie; Prochnow, Hans; Rox, Katharina; et al. (Elsevier, 2020-03-18)
    Acute lower respiratory tract infections (ALRI) caused by respiratory syncytial virus (RSV) are associated with a severe disease burden among infants and elderly patients. Treatment options are limited. While numerous drug candidates with different viral targets are under development, the utility of RSV entry inhibitors is challenged by a low resistance barrier and by single mutations causing cross-resistance against a wide spectrum of fusion inhibitor chemotypes. We developed a cell-based screening assay for discovery of compounds inhibiting infection with primary RSV isolates. Using this system, we identified labyrinthopeptin A1 and A2 (Laby A1/A2), lantibiotics isolated from Actinomadura namibiensis, as effective RSV cell entry inhibitors with IC50s of 0.39 μM and 4.97 μM, respectively, and with favourable therapeutic index (>200 and > 20, respectively). Both molecules were active against multiple RSV strains including primary isolates and their antiviral activity against RSV was confirmed in primary human airway cells ex vivo and a murine model in vivo. Laby A1/A2 were antiviral in prophylactic and therapeutic treatment regimens and displayed synergistic activity when applied in combination with each other. Mechanistic studies showed that Laby A1/A2 exert virolytic activity likely by binding to phosphatidylethanolamine moieties within the viral membrane and by disrupting virus particle membrane integrity. Probably due to its specific mode of action, Laby A1/A2 antiviral activity was not affected by common resistance mutations to known RSV entry inhibitors. Taken together, Laby A1/A2 represent promising candidates for development as RSV inhibitors. Moreover, the cell-based screening system with primary RSV isolates described here should be useful to identify further antiviral agents.
  • Inhibition of Respiration of Candida albicans by Small Molecules Increases Phagocytosis Efficacy by Macrophages.

    Cui, Shuna; Li, Minghui; Hassan, Rabeay Y A; Heintz-Buschart, Anna; Wang, Junsong; Bilitewski, Ursula; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstraße 7, 38124 Braunschweig, Germany. (American Society of Microbiology, 2020-04-15)
    Candida albicans adapts to various conditions in different body niches by regulating gene expression, protein synthesis, and metabolic pathways. These adaptive reactions not only allow survival but also influence the interaction with host cells, which is governed by the composition and structure of the fungal cell wall. Numerous studies had shown linkages between mitochondrial functionality, cell wall integrity and structure, and pathogenicity. Thus, we decided to inhibit single complexes of the respiratory chain of C. albicans and to analyze the resultant interaction with macrophages via their phagocytic activity. Remarkably, inhibition of the fungal bc1 complex by antimycin A increased phagocytosis, which correlated with an increased accessibility of β-glucans. To contribute to mechanistic insights, we performed metabolic studies, which highlighted significant changes in the abundance of constituents of the plasma membrane. Collectively, our results reinforce the strong linkage between fungal energy metabolism and other components of fungal physiology, which also determine the vulnerability to immune defense reactions.IMPORTANCE The yeast Candida albicans is one of the major fungal human pathogens, for which new therapeutic approaches are required. We aimed at enhancements of the phagocytosis efficacy of macrophages by targeting the cell wall structure of C. albicans, as the coverage of the β-glucan layer by mannans is one of the immune escape mechanisms of the fungus. We unambiguously show that inhibition of the fungal bc1 complex correlates with increased accessibilities of β-glucans and improved phagocytosis efficiency. Metabolic studies proved not only the known direct effects on reactive oxygen species (ROS) production and fermentative pathways but also the clear downregulation of the ergosterol pathway and upregulation of unsaturated fatty acids. The changed composition of the plasma membrane could also influence the interaction with the overlying cell wall. Thus, our work highlights the far-reaching relevance of energy metabolism, indirectly also for host-pathogen interactions, without affecting viability.
  • A NanoLuc luciferase-based assay enabling the real-time analysis of protein secretion and injection by bacterial type III secretion systems.

    Westerhausen, Sibel; Nowak, Melanie; Torres-Vargas, Claudia E; Bilitewski, Ursula; Bohn, Erwin; Grin, Iwan; Wagner, Samuel; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2020-02-18)
    The elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a shortage of assays to quantitatively assess secretion kinetics. Also the analysis of the biological role of these secretion systems as well as the identification of inhibitors targeting these systems would greatly benefit from the availability of a simple, quick and quantitative assay to monitor principle secretion and injection into host cells. Here, we present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase to assess the function of the type III secretion system encoded by Salmonella pathogenicity island 1. Type III secretion substrate-NanoLuc fusions are readily secreted into the culture supernatant, where they can be quantified by luminometry after removal of bacteria. The NanoLuc-based secretion assay features a very high signal-to-noise ratio and sensitivity down to the nanolitre scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384-well microplates. We further developed a split NanoLuc-based assay that enables the real-time monitoring of type III secretion-dependent injection of effector-HiBiT fusions into host cells stably expressing the complementing NanoLuc-LgBiT.
  • Potential TMA-Producing Bacteria Are Ubiquitously Found in Mammalia.

    Rath, Silke; Rud, Tatjana; Pieper, Dietmar H; Vital, Marius; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2019-01-01)
    Human gut bacteria metabolize dietary components such as choline and carnitine to trimethylamine (TMA) that is subsequently oxidized to trimethylamine-N-oxide (TMAO) by hepatic enzymes. Increased plasma levels of TMAO are associated with the development of cardiovascular and renal disease. In this study, we applied gene-targeted assays in order to quantify (qPCR) and characterize (MiSeq) bacterial genes encoding enzymes responsible for TMA production, namely choline-TMA lyase (CutC), carnitine oxygenase (CntA) and betaine reductase (GrdH) in 89 fecal samples derived from various mammals spanning three dietary groups (carnivores, omnivores and herbivores) and four host orders (Carnivora, Primates, Artiodactyla and Perissodactyla). All samples contained potential TMA-producing bacteria, however, at low abundances (<1.2% of total community). The cutC gene was more abundant in omnivores and carnivores compared with herbivores. CntA was almost absent from herbivores and grdH showed lowest average abundance of all three genes. Bacteria harboring cutC and grdH displayed high diversities where sequence types affiliated with various taxa within Firmicutes dominated, whereas cntA comprised sequences primarily linked to Escherichia. Composition of TMA-forming communities was strongly influenced by diet and host taxonomy and despite their high correlation, both factors contributed uniquely to community structure. Furthermore, Random Forest (RF) models could differentiate between groups at high accuracies. This study gives a comprehensive overview of potential TMA-producing bacteria in the mammalian gut demonstrating that both diet and host taxonomy govern their abundance and composition. It highlights the role of functional redundancy sustaining potential TMA formation in distinct gut environments.
  • Discovery of Novel Latency-Associated Nuclear Antigen Inhibitors as Antiviral Agents Against Kaposi's Sarcoma-Associated Herpesvirus.

    Kirsch, Philine; Jakob, Valentin; Elgaher, Walid A M; Walt, Christine; Oberhausen, Kevin; Schulz, Thomas F; Empting, Martin; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany.;HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (American Chemical Society (ACS), 2020-01-24)
    With the aim to develop novel antiviral agents against Kaposi's Sarcoma Herpesvirus (KSHV), we are targeting the latency-associated nuclear antigen (LANA). This protein plays an important role in viral genome maintenance during latent infection. LANA has the ability to tether the viral genome to the host nucleosomes and, thus, ensures latent persistence of the viral genome in the host cells. By inhibition of the LANA-DNA interaction, we seek to eliminate or reduce the load of the viral DNA in the host. To achieve this goal, we screened our in-house library using a dedicated fluorescence polarization (FP)-based competition assay, which allows for the quantification of LANA-DNA-interaction inhibition by small organic molecules. We successfully identified three different compound classes capable of disrupting this protein-nucleic acid interaction. We characterized these compounds by IC50 dose-response evaluation and confirmed the compound-LANA interaction using surface plasmon resonance (SPR) spectroscopy. Furthermore, two of the three hit scaffolds showed only marginal cytotoxicity in two human cell lines. Finally, we conducted STD-NMR competition experiments with our new hit compounds and a previously described fragment-sized inhibitor. Based on these results, future compound linking approaches could serve as a promising strategy for further optimization studies in order to generate highly potent KSHV inhibitors.
  • Cystobactamid 507: Concise Synthesis, Mode of Action and Optimization toward More Potent Antibiotics.

    Elgaher, Walid A M; Hamed, Mostafa M; Baumann, Sascha; Herrmann, Jennifer; Siebenbürger, Lorenz; Krull, Jana; Cirnski, Katarina; Kirschning, Andreas; Brönstrup, Mark; Müller, Rolf; et al. (Wiley-VCH, 2020-01-26)
    Lack of new antibiotics and increasing antimicrobial resistance are the main concerns of healthcare community nowadays, which necessitate the search for novel antibacterial agents. Recently, we discovered the cystobactamids - a novel natural class of antibiotics with broad-spectrum antibacterial activity. In this work, we describe a concise total synthesis of cystobactamid 507, the identification of the bioactive conformation using non-covalently bonded rigid analogs, the first structure–activity relationship (SAR) study for cystobactamid 507 leading to new analogs with high metabolic stability, superior topoisomerase IIA inhibition, antibacterial activity and, importantly, stability toward the resistant factor AlbD. Deeper insight into the mode of action revealed that the cystobactamids employ DNA minor groove binding as part of the drug–target interaction without showing significant intercalation. By designing a new analog of cystobactamid 919-2 we finally demonstrated that these findings could be further exploited to obtain more potent hexapeptides against Gram-negative bacteria.
  • Identification of benzene-degrading Proteobacteria in a constructed wetland by employing in situ microcosms and RNA-stable isotope probing.

    Nitz, Henrike; Duarte, Márcia; Jauregui, Ruy; Pieper, Dietmar H; Müller, Jochen A; Kästner, Matthias; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer, 2019-12-23)
    Constructed wetlands (CWs) are effective ecological remediation technologies for various contaminated water bodies. Here, we queried for benzene-degrading microbes in a horizontal subsurface flow CW with reducing conditions in the pore water and fed with benzene-contaminated groundwater. For identification of relevant microbes, we employed in situ microcosms (BACTRAPs, which are made from granulated activated carbon) coupled with 13C-stable isotope probing and Illumina sequencing of 16S rRNA amplicons. A significant incorporation of 13C was detected in RNA isolated from BACTRAPs loaded with 13C-benzene and exposed in the CW for 28 days. A shorter incubation time did not result in detectable 13C incorporation. After 28 days, members from four genera, namely Dechloromonas, Hydrogenophaga, and Zoogloea from the Betaproteobacteria and Arcobacter from the Epsilonproteobacteria were significantly labeled with 13C and were abundant in the bacterial community on the BACTRAPs. Sequences affiliated to Geobacter were also numerous on the BACTRAPs but apparently those microbes did not metabolize benzene as no 13C label incorporation was detected. Instead, they may have metabolized plant-derived organic compounds while using the BACTRAPs as electron sink. In representative wetland samples, sequences affiliated with Dechloromonas, Zoogloea, and Hydrogenophaga were present at relative proportions of up to a few percent. Sequences affiliated with Arcobacter were present at < 0.01% in wetland samples. In conclusion, we identified microbes of likely significance for benzene degradation in a CW used for remediation of contaminated water.
  • T4SS-dependent TLR5 activation by Helicobacter pylori infection.

    Pachathundikandi, Suneesh Kumar; Tegtmeyer, Nicole; Arnold, Isabelle Catherine; Lind, Judith; Neddermann, Matthias; Falkeis-Veits, Christina; Chattopadhyay, Sujay; Brönstrup, Mark; Tegge, Werner; Hong, Minsun; et al. (Nature publishing group, 2019-12-16)
    Toll-like receptor TLR5 recognizes a conserved domain, termed D1, that is present in flagellins of several pathogenic bacteria but not in Helicobacter pylori. Highly virulent H. pylori strains possess a type IV secretion system (T4SS) for delivery of virulence factors into gastric epithelial cells. Here, we show that one of the H. pylori T4SS components, protein CagL, can act as a flagellin-independent TLR5 activator. CagL contains a D1-like motif that mediates adherence to TLR5+ epithelial cells, TLR5 activation, and downstream signaling in vitro. TLR5 expression is associated with H. pylori infection and gastric lesions in human biopsies. Using Tlr5-knockout and wild-type mice, we show that TLR5 is important for efficient control of H. pylori infection. Our results indicate that CagL, by activating TLR5, may modulate immune responses to H. pylori.
  • 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.
  • Risk factors and Predictors of Mortality in Streptococcal Necrotizing Soft-Tissue Infections: A Multicenter Prospective Study.

    Bruun, Trond; Rath, Eivind; Bruun Madsen, Martin; Oppegaard, Oddvar; Nekludov, Michael; Arnell, Per; Karlsson, Ylva; Babbar, Anshu; Bergey, Francois; Itzek, Andreas; et al. (Oxford University Press (OUP), 2020-01-10)
    Background Necrotizing soft-tissue infections (NSTI) are life-threatening conditions often caused by β-hemolytic streptococci, group A streptococcus (GAS) in particular. Optimal treatment is contentious. The INFECT cohort includes the largest set of prospectively enrolled streptococcal NSTI cases to date. Methods From the INFECT cohort of 409 adults admitted with NSTI to five clinical centers in Scandinavia, patients culture-positive for GAS or Streptococcus dysgalactiae (SD) were selected. Risk factors were identified by comparison with a cohort of non-necrotizing streptococcal cellulitis. The impact of baseline factors and treatment on 90-day mortality was explored using Lasso regression. Whole-genome sequencing of bacterial isolates was used for emm typing and virulence gene profiling. Results The 126 GAS NSTI cases and 27 cases caused by SD constituted 31% and 7% of the whole NSTI cohort, respectively. When comparing to non-necrotizing streptococcal cellulitis, streptococcal NSTI was associated to blunt trauma, absence of pre-existing skin lesions, and a lower BMI. Septic shock was significantly more frequent in GAS (65%) compared to SD (41%) and polymicrobial, non-streptococcal NSTI (46%). Age, male sex, septic shock, and no administration of intravenous immunoglobulin (IVIG) were among factors associated with 90-day mortality. Predominant emm types were emm1, emm3 and emm28 in GAS and stG62647 in SD.
  • Correlation between immunoglobulin dose administered and plasma neutralization of streptococcal superantigens in patients with necrotizing soft tissue infections.

    Bergsten, Helena; Madsen, Martin Bruun; Bergey, Francois; Hyldegaard, Ole; Skrede, Steinar; Arnell, Per; Oppegaard, Oddvar; Itzek, Andreas; Perner, Anders; Svensson, Mattias; et al. (Oxford University Press, 2020-01-09)
    Analyses of plasma collected pre- and post-administration of intravenous immunoglobulin G (IVIG) from patients with Group A Streptococcus necrotizing soft tissue infections demonstrated a negative correlation between IVIG dose and toxin-triggered T-cell proliferation (r=-0.67, p<0.0001). One 25g-dose IVIG was sufficient to yield patient plasma neutralizing activity against streptococcal superantigens.
  • Importance of superoxide dismutase A and M for protection of Staphylococcus aureus in the oxidative stressful environment of cystic fibrosis airways.

    Treffon, Janina; Chaves-Moreno, Diego; Niemann, Silke; Pieper, Dietmar Helmut; Vogl, Thomas; Roth, Johannes; Kahl, Barbara C; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2020-01-02)
    Staphylococcus aureus is one of the earliest pathogens that persists the airways of cystic fibrosis (CF) patients and contributes to increased inflammation and decreased lung function. In contrast to other staphylococci, S. aureus possesses two superoxide dismutases (SODs), SodA and SodM, with SodM being unique to S. aureus. Both SODs arm S. aureus for its fight against oxidative stress, a byproduct of inflammatory reactions. Despite complex investigations it is still unclear, if both enzymes are crucial for the special pathogenicity of S. aureus. To investigate the role of both SODs during staphylococcal persistence in CF airways, we analyzed survival and gene expression of S. aureus CF isolates and laboratory strains in different CF-related in vitro and ex vivo settings. Bacteria located in inflammatory and oxidized CF sputum transcribed high levels of sodA and sodM. Especially expression values of sodM were remarkably higher in CF sputum than in bacterial in vitro cultures. Interestingly, also S. aureus located in airway epithelial cells expressed elevated transcript numbers of both SODs, indicating that S. aureus is exposed to oxidative stress at various sites within CF airways. Both enzymes promoted survival of S. aureus during PMN killing and seem to act compensatory, thereby giving evidence that the interwoven interaction of SodA and SodM contributes to S. aureus virulence and facilitates S. aureus persistence within CF airways.
  • 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.
  • 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
  • High Nuclease Activity of Long Persisting Isolates Within the Airways of Cystic Fibrosis Patients Protects Against NET-Mediated Killing.

    Herzog, Susann; Dach, Felix; de Buhr, Nicole; Niemann, Silke; Schlagowski, Jannik; Chaves-Moreno, Diego; Neumann, Claudia; Goretzko, Jonas; Schwierzeck, Vera; Mellmann, Alexander; et al. (Frontiers, 2019-01-01)
    Staphylococcus aureus is one of the first and most prevalent pathogens cultured from the airways of cystic fibrosis (CF) patients, which can persist there for extended periods. Airway infections in CF patients are characterized by a strong inflammatory response of highly recruited neutrophils. One killing mechanism of neutrophils is the formation of neutrophil extracellular traps (NETs), which capture and eradicate bacteria by extracellular fibers of neutrophil chromatin decorated with antimicrobial granule proteins. S. aureus secretes nuclease, which can degrade NETs. We hypothesized, that S. aureus adapts to the airways of CF patients during persistent infection by escaping from NET-mediated killing via an increase of nuclease activity. Sputum samples of CF patients with chronic S. aureus infection were visualized by confocal microscopy after immuno-fluorescence staining for NET-specific markers, S. aureus bacteria and overall DNA structures. Nuclease activity was analyzed in sequential isogenic long persisting S. aureus isolates, as confirmed by whole genome sequencing, from an individual CF patient using a FRET-based nuclease activity assay. Additionally, some of these isolates were selected and analyzed by qRT-PCR to determine the expression of nuc1 and regulators of interest. NET-killing assays were performed with clinical S. aureus isolates to evaluate killing and bacterial survival depending on nuclease activity. To confirm the role of nuclease during NET-mediated killing, a clinical isolate with low nuclease activity was transformed with a nuclease expression vector (pCM28nuc). Furthermore, two sputa from an individual CF patient were subjected to RNA-sequence analysis to evaluate the activity of nuclease in vivo. In sputa, S. aureus was associated to extracellular DNA structures. Nuclease activity in clinical S. aureus isolates increased in a time-and phenotype-dependent manner. In the clinical isolates, the expression of nuc1 was inversely correlated to the activity of agr and was independent of saeS. NET-mediated killing was significantly higher in S. aureus isolates with low compared to isolates with high nuclease activity. Importantly, transformation of the clinical isolate with low nuclease activity with pCM28nuc conferred protection against NET-mediated killing confirming the beneficial role of nuclease for protection against NETs. Also, nuclease expression in in vivo sputa was high, which underlines the important role of nuclease within the highly inflamed CF airways. In conclusion, our data show that S. aureus adapts to the neutrophil-rich environment of CF airways with increasing nuclease expression most likely to avoid NET-killing during long-term persistence.
  • MAIT Cells Are Major Contributors to the Cytokine Response in Group A Streptococcal Toxic Shock Syndrome.

    Emgård, Johanna; Bergsten, Helena; McCormick, John K; Barrantes, Israel; Skrede, Steinar; Sandberg, Johan K; Norrby-Teglund, Anna; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (National Academy of Sciences, 2019-11-26)
    Streptococcal toxic shock syndrome (STSS) is a rapidly progressing, life-threatening, systemic reaction to invasive infection caused by group A streptococci (GAS). GAS superantigens are key mediators of STSS through their potent activation of T cells leading to a cytokine storm and consequently vascular leakage, shock, and multiorgan failure. Mucosal-associated invariant T (MAIT) cells recognize MR1-presented antigens derived from microbial riboflavin biosynthesis and mount protective innate-like immune responses against the microbes producing such metabolites. GAS lack de novo riboflavin synthesis, and the role of MAIT cells in STSS has therefore so far been overlooked. Here we have conducted a comprehensive analysis of human MAIT cell responses to GAS, aiming to understand the contribution of MAIT cells to the pathogenesis of STSS. We show that MAIT cells are strongly activated and represent the major T cell source of IFNγ and TNF in the early stages of response to GAS. MAIT cell activation is biphasic with a rapid TCR Vβ2-specific, TNF-dominated response to superantigens and a later IL-12- and IL-18-dependent, IFNγ-dominated response to both bacterial cells and secreted factors. Depletion of MAIT cells from PBMC resulted in decreased total production of IFNγ, IL-1β, IL-2, and TNFβ. Peripheral blood MAIT cells in patients with STSS expressed elevated levels of the activation markers CD69, CD25, CD38, and HLA-DR during the acute compared with the convalescent phase. Our data demonstrate that MAIT cells are major contributors to the early cytokine response to GAS, and are therefore likely to contribute to the pathological cytokine storm underlying STSS.
  • 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.

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