• Rubinisphaera italica sp. nov. isolated from a hydrothermal area in the Tyrrhenian Sea close to the volcanic island Panarea.

      Kallscheuer, Nicolai; Jogler, Mareike; Wiegand, Sandra; Peeters, Stijn H; Heuer, Anja; Boedeker, Christian; Jetten, Mike S M; Rohde, Manfred; Jogler, Christian (Springer, 2019-11-26)
      Planctomycetes is a fascinating phylum of mostly aquatic bacteria, not only due to the environmental importance in global carbon and nitrogen cycles, but also because of a unique cell biology. Their lifestyle and metabolic capabilities are not well explored, which motivated us to study the role of Planctomycetes in biofilms on marine biotic surfaces. Here, we describe the novel strain Pan54T which was isolated from algae in a hydrothermal area close to the volcanic island Panarea in the Tyrrhenian Sea, north of Sicily in Italy. The strain grew best at pH 9.0 and 26 °C and showed typical characteristics of planctomycetal bacteria, e.g. division by polar budding, formation of aggregates and presence of stalks and crateriform structures. Phylogenetically, the strain belongs to the genus Rubinisphaera. Our analysis suggests that Pan54T represents a novel species of this genus, for which we propose the name Rubinisphaera italica sp. nov. We suggest Pan54T (= DSM 29369 = LMG 29789) as the type strain of the novel species.
    • The immunogenic potential of bacterial flagella for Salmonella-mediated tumor therapy.

      Felgner, Sebastian; Spöring, Imke; Pawar, Vinay; Kocijancic, Dino; Preusse, Matthias; Falk, Christine; Rohde, Manfred; Häussler, Susanne; Weiss, Siegfried; Erhardt, Marc; et al. (Wiley-Blackwell, 2019-11-21)
      Genetically engineered Salmonella Typhimurium are potent vectors for prophylactic and therapeutic measures against pathogens as well as cancer. This is based on the potent adjuvanticity that supports strong immune responses. The physiology of Salmonella is well understood. It simplifies engineering of both enhanced immune‐stimulatory properties as well as safety features, thus, resulting in an appropriate balance between attenuation and efficacy for clinical applications. A major virulence factor of Salmonella is the flagellum. It is also a strong pathogen‐associated molecular pattern recognized by extra‐ and intracellular receptors of immune cells of the host. At the same time, it represents a serious metabolic burden. Accordingly, the bacteria evolved tight regulatory mechanisms that control flagella synthesis in vivo. Here, we systematically investigated the immunogenicity and adjuvant properties of various flagella mutants of Salmonella in vitro and in a mouse cancer model in vivo. We found that mutants lacking the flagellum‐specific ATPase FliHIJ or the inner membrane ring FliF displayed the greatest stimulatory capacity and strongest anti‐tumor effects, while remaining safe in vivo. Scanning electron microscopy revealed the presence of outer membrane vesicles in the ΔfliF and ΔfliHIJ mutants. Finally, the combination of the ΔfliF and ΔfliHIJ mutations with our previously described attenuated and immunogenic background strain SF102 displayed strong efficacy against the highly resistant cancer cell line RenCa. We thus conclude that manipulating flagella biosynthesis has great potential for the construction of highly efficacious and versatile Salmonella vector strains.
    • Prothrombotic and Proinflammatory Activities of the β-Hemolytic Group B Streptococcal Pigment.

      Siemens, Nikolai; Oehmcke-Hecht, Sonja; Hoßmann, Jörn; Skorka, Sebastian B; Nijhuis, Roel H T; Ruppen, Corinne; Skrede, Steinar; Rohde, Manfred; Schultz, Daniel; Lalk, Michael; et al. (Karger, 2019-11-19)
      A prominent feature of severe streptococcal infections is the profound inflammatory response that contributes to systemic toxicity. In sepsis the dysregulated host response involves both immunological and nonimmunological pathways. Here, we report a fatal case of an immunocompetent healthy female presenting with toxic shock and purpura fulminans caused by group B streptococcus (GBS; serotype III, CC19). The strain (LUMC16) was pigmented and hyperhemolytic. Stimulation of human primary cells with hyperhemolytic LUMC16 and STSS/NF-HH strains and pigment toxin resulted in a release of proinflammatory mediators, including tumor necrosis factor, interleukin (IL)-1β, and IL-6. In addition, LUMC16 induced blood clotting and showed factor XII activity on its surface, which was linked to the presence of the pigment. The expression of pigment was not linked to a mutation within the CovR/S region. In conclusion, our study shows that the hemolytic lipid toxin contributes to the ability of GBS to cause systemic hyperinflammation and interferes with the coagulation system.
    • N-WASP Guides Cancer Cells toward LPA.

      Rottner, Klemens; Schaks, Matthias; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2019-11-18)
      The actin remodeling factor N-WASP is best known as an Arp2/3 complex activator in processes like endocytosis, extracellular matrix degradation, and host-pathogen interaction. In this issue of Developmental Cell, Juin et al. establish a novel trafficking function for N-WASP in driving lysophosphatidic acid-dependent chemotaxis and metastasis of pancreatic cancer cells.
    • Non-Invasive Approach for Evaluation of Pulmonary Hypertension Using Extracellular Vesicle-Associated Small Non-Coding RNA.

      Lipps, Christoph; Northe, Philipp; Figueiredo, Ricardo; Rohde, Manfred; Brahmer, Alexandra; Krämer-Albers, Eva-Maria; Liebetrau, Christoph; Wiedenroth, Christoph B; Mayer, Eckhard; Kriechbaum, Steffen D; et al. (MDPI, 2019-10-29)
      Extracellular vesicles are released by numerous cell types of the human body under physiological but also under pathophysiological conditions. They are important for cell-cell communication and carry specific signatures of peptides and RNAs. In this study, we aimed to determine whether extracellular vesicles isolated from patients with pulmonary hypertension show a disease specific signature of small non-coding RNAs and thus have the potential to serve as diagnostic and prognostic biomarkers. Extracellular vesicles were isolated from the serum of 23 patients with chronic thromboembolic pulmonary hypertension (CTEPH) and 23 controls using two individual methods: a column-based method or by precipitation. Extracellular vesicle- associated RNAs were analyzed by next-generation sequencing applying molecular barcoding, and differentially expressed small non-coding RNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). We identified 18 microRNAs and 21 P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) or piRNA clusters that were differentially expressed in CTEPH patients compared with controls. Bioinformatic analysis predicted a contribution of these piRNAs to the progression of cardiac and vascular remodeling. Expression levels of DQ593039 correlated with clinically meaningful parameters such as mean pulmonary arterial pressure, pulmonary vascular resistance, right ventricular systolic pressure, and levels of N-terminal pro-brain natriuretic peptide. Thus, we identified the extracellular vesicle- derived piRNA, DQ593039, as a potential biomarker for pulmonary hypertension and right heart disease.
    • R18C is a new viable P2-like bacteriophage of rabbit origin infecting Citrobacter rodentium and Shigella sonnei strains.

      Sváb, Domonkos; Horváth, Balázs; Rohde, Manfred; Maróti, Gergely; Tóth, István; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer-Nature, 2019-10-23)
      Here, we report a novel virulent P2-like bacteriophage, R18C, isolated from rabbit faeces, which, in addition to Escherichia coli K-12 strains, was able to be propagated on Citrobacter rodentium strain ICC169 and a range of Shigella sonnei strains with high efficiency of plating (EOP). It represents the first lytic bacteriophage originating from rabbit and the first infectious P2-like phage of animal origin. In the three characteristic moron-containing regions of P2-like phages, R18C contains genes with unknown function that have so far only been found in cryptic P2-like prophages.
    • EPLIN-α and -β Isoforms Modulate Endothelial Cell Dynamics through a Spatiotemporally Differentiated Interaction with Actin.

      Taha, Muna; Aldirawi, Mohammed; März, Sigrid; Seebach, Jochen; Odenthal-Schnittler, Maria; Bondareva, Olga; Bojovic, Vesna; Schmandra, Thomas; Wirth, Benedikt; Mietkowska, Magdalena; et al. (Elsevier, 2019-10-22)
      Actin-binding proteins are essential for linear and branched actin filament dynamics that control shape change, cell migration, and cell junction remodeling in vascular endothelium (endothelial cells [ECs]). The epithelial protein lost in neoplasm (EPLIN) is an actin-binding protein, expressed as EPLIN-α and EPLIN-β by alternative promoters; however, the isoform-specific functions are not yet understood. Aortic compared to cava vein ECs and shear stress-exposed cultured ECs express increased EPLIN-β levels that stabilize stress fibers. In contrast, EPLIN-α expression is increased in growing and migrating ECs, is targeted to membrane protrusions, and terminates their growth via interaction with the Arp2/3 complex. The data indicate that EPLIN-α controls protrusion dynamics while EPLIN-β has an actin filament stabilizing role, which is consistent with FRAP analyses demonstrating a lower EPLIN-β turnover rate compared to EPLIN-α. Together, EPLIN isoforms differentially control actin dynamics in ECs, essential in shear stress responses, cell migration, and barrier function.
    • The secRNome of Listeria monocytogenes Harbors Small Noncoding RNAs That Are Potent Inducers of Beta Interferon.

      Frantz, Renate; Teubner, Lisa; Schultze, Tilman; La Pietra, Luigi; Müller, Christin; Gwozdzinski, Konrad; Pillich, Helena; Hain, Torsten; Weber-Gerlach, Michaela; Panagiotidis, Georgios-Dimitrios; et al. (ASM, 2019-10-08)
      Cellular sensing of bacterial RNA is increasingly recognized as a determinant of host-pathogen interactions. The intracellular pathogen Listeria monocytogenes induces high levels of type I interferons (alpha/beta interferons [IFN-α/β]) to create a growth-permissive microenvironment during infection. We previously demonstrated that RNAs secreted by L. monocytogenes (comprising the secRNome) are potent inducers of IFN-β. We determined the composition and diversity of the members of the secRNome and found that they are uniquely enriched for noncoding small RNAs (sRNAs). Testing of individual sRNAs for their ability to induce IFN revealed several sRNAs with this property. We examined ril32, an intracellularly expressed sRNA that is highly conserved for the species L. monocytogenes and that was the most potent inducer of IFN-β expression of all the sRNAs tested in this study, in more detail. The rli32-induced IFN-β response is RIG-I (retinoic acid inducible gene I) dependent, and cells primed with rli32 inhibit influenza virus replication. We determined the rli32 motif required for IFN induction. rli32 overproduction promotes intracellular bacterial growth, and a mutant lacking rli32 is restricted for intracellular growth in macrophages. rli32-overproducing bacteria are resistant to H2O2 and exhibit both increased catalase activity and changes in the cell envelope. Comparative transcriptome sequencing (RNA-Seq) analysis indicated that ril32 regulates expression of the lhrC locus, previously shown to be involved in cell envelope stress. Inhibition of IFN-β signaling by ruxolitinib reduced rli32-dependent intracellular bacterial growth, indicating a link between induction of the interferon system and bacterial physiology. rli32 is, to the best of our knowledge, the first secreted individual bacterial sRNA known to trigger the induction of the type I IFN response.IMPORTANCE Interferons are potent and broadly acting cytokines that stimulate cellular responses to nucleic acids of unusual structures or locations. While protective when induced following viral infections, the induction of interferons is detrimental to the host during L. monocytogenes infection. Here, we identify specific sRNAs, secreted by the bacterium, with the capacity to induce type I IFN. Further analysis of the most potent sRNA, rli32, links the ability to induce RIG-I-dependent induction of the type I IFN response to the intracellular growth properties of the bacterium. Our findings emphasize the significance of released RNA for Listeria infection and shed light on a compartmental strategy used by an intracellular pathogen to modulate host responses to its advantage.
    • Actin dynamics in cell migration

      Schaks, Matthias; Giannone, Grégory; Rottner, Klemens; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Portland Press Ltd., 2019-09-24)
      Cell migration is an essential process, both in unicellular organisms such as amoeba and as individual or collective motility in highly developed multicellular organisms like mammals. It is controlled by a variety of activities combining protrusive and contractile forces, normally generated by actin filaments. Here, we summarize actin filament assembly and turnover processes, and how respective biochemical activities translate into different protrusion types engaged in migration. These actin-based plasma membrane protrusions include actin-related protein 2/3 complex-dependent structures such as lamellipodia and membrane ruffles, filopodia as well as plasma membrane blebs. We also address observed antagonisms between these protrusion types, and propose a model – also inspired by previous literature – in which a complex balance between specific Rho GTPase signaling pathways dictates the protrusion mechanism employed by cells. Furthermore, we revisit published work regarding the fascinating antagonism between Rac and Rho GTPases, and how this intricate signaling network can define cell behavior and modes of migration. Finally, we discuss how the assembly of actin filament networks can feed back onto their regulators, as exemplified for the lamellipodial factor WAVE regulatory complex, tightly controlling accumulation of this complex at specific subcellular locations as well as its turnover.
    • RhoG and Cdc42 can contribute to Rac-dependent lamellipodia formation through WAVE regulatory complex-binding.

      Schaks, Matthias; Döring, Hermann; Kage, Frieda; Steffen, Anika; Klünemann, Thomas; Blankenfeldt, Wulf; Stradal, Theresia; Rottner, Klemens; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Taylor and Francis, 2019-08-26)
      Cell migration frequently involves the formation of lamellipodial protrusions, the initiation of which requires Rac GTPases signalling to heteropentameric WAVE regulatory complex (WRC). While Rac-related RhoG and Cdc42 can potently stimulate lamellipodium formation, so far presumed to occur by upstream signalling to Rac activation, we show here that the latter can be bypassed by RhoG and Cdc42 given that WRC has been artificially activated. This evidence arises from generation of B16-F1 cells simultaneously lacking both Rac GTPases and WRC, followed by reconstitution of lamellipodia formation with specific Rho-GTPase and differentially active WRC variant combinations. We conclude that formation of canonical lamellipodia requires WRC activation through Rac, but can possibly be tuned, in addition, by WRC interactions with RhoG and Cdc42.
    • Mitochondria Are a Subset of Extracellular Vesicles Released by Activated Monocytes and Induce Type I IFN and TNF Responses in Endothelial Cells.

      Puhm, Florian; Afonyushkin, Taras; Resch, Ulrike; Obermayer, Georg; Rohde, Manfred; Penz, Thomas; Schuster, Michael; Wagner, Gabriel; Rendeiro, Andre F; Melki, Imene; et al. (Lippincott,Williams & Wilkins, 2019-06-21)
      Extracellular vesicles, including microvesicles, are increasingly recognized as important mediators in cardiovascular disease. The cargo and surface proteins they carry are considered to define their biological activity, including their inflammatory properties. Monocyte to endothelial cell signaling is a prerequisite for the propagation of inflammatory responses. However, the contribution of microvesicles in this process is poorly understood. OBJECTIVE: To elucidate the mechanisms by which microvesicles derived from activated monocytic cells exert inflammatory effects on endothelial cells. METHODS AND RESULTS: LPS (lipopolysaccharide)-stimulated monocytic cells release free mitochondria and microvesicles with mitochondrial content as demonstrated by flow cytometry, quantitative polymerase chain reaction, Western Blot, and transmission electron microscopy. Using RNAseq analysis and quantitative reverse transcription-polymerase chain reaction, we demonstrated that both mitochondria directly isolated from and microvesicles released by LPS-activated monocytic cells, as well as circulating microvesicles isolated from volunteers receiving low-dose LPS-injections, induce type I IFN (interferon), and TNF (tumor necrosis factor) responses in endothelial cells. Depletion of free mitochondria significantly reduced the ability of these microvesicles to induce type I IFN and TNF-dependent genes. We identified mitochondria-associated TNFα and RNA from stressed mitochondria as major inducers of these responses. Finally, we demonstrated that the proinflammatory potential of microvesicles and directly isolated mitochondria were drastically reduced when they were derived from monocytic cells with nonrespiring mitochondria or monocytic cells cultured in the presence of pyruvate or the mitochondrial reactive oxygen species scavenger MitoTEMPO. CONCLUSIONS: Mitochondria and mitochondria embedded in microvesicles constitute a major subset of extracellular vesicles released by activated monocytes, and their proinflammatory activity on endothelial cells is determined by the activation status of their parental cells. Thus, mitochondria may represent critical intercellular mediators in cardiovascular disease and other inflammatory settings associated with type I IFN and TNF signaling.
    • The Gram-Positive Bacterial Cell Wall

      Rohde, Manfred; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (American Society for Microbiology, 2019-05-24)
      The chapter about the Gram-positive bacterial cell wall gives a brief historical background on the discovery of Gram-positive cell walls and their constituents and microscopic methods applied for studying the Gram-positive cell envelope. Followed by the description of the different chemical building blocks of peptidoglycan and the biosynthesis of the peptidoglycan layers and high turnover of peptidoglycan during bacterial growth. Lipoteichoic acids and wall teichoic acids are highlighted as major components of the cell wall. Characterization of capsules and the formation of extracellular vesicles by Gram-positive bacteria close the section on cell envelopes which have a high impact on bacterial pathogenesis. In addition, the specialized complex and unusual cell wall of mycobacteria is introduced thereafter. Next a short back view is given on the development of electron microscopic examinations for studying bacterial cell walls. Different electron microscopic techniques and methods applied to examine bacterial cell envelopes are discussed in the view that most of the illustrated methods should be available in a well-equipped life sciences orientated electron microscopic laboratory. In addition, newly developed and mostly well-established cryo-methods like high-pressure freezing and freeze-substitution (HPF-FS) and cryo-sections of hydrated vitrified bacteria (CEMOVIS, Cryo-electron microscopy of vitreous sections) are described. At last, modern cryo-methods like cryo-electron tomography (CET) and cryo-FIB-SEM milling (focus ion beamscanning electron microscopy) are introduced which are available only in specialized institutions, but at present represent the best available methods and techniques to study Gram-positive cell walls under close-to-nature conditions in great detail and at high resolution.
    • Still Something to Discover: Novel Insights into Phage Diversity and Taxonomy.

      Korf, Imke H E; Meier-Kolthoff, Jan P; Adriaenssens, Evelien M; Kropinski, Andrew M; Nimtz, Manfred; Rohde, Manfred; van Raaij, Mark J; Wittmann, Johannes; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2019-05-17)
      The aim of this study was to gain further insight into the diversity of Escherichia coli phagesfollowed by enhanced work on taxonomic issues in that field. Therefore, we present the genomiccharacterization and taxonomic classification of 50 bacteriophages against E. coli isolated fromvarious sources, such as manure or sewage. All phages were examined for their host range on a setof different E. coli strains, originating, e.g., from human diagnostic laboratories or poultry farms.Transmission electron microscopy revealed a diversity of morphotypes (70% Myo-, 22% Sipho-, and8% Podoviruses), and genome sequencing resulted in genomes sizes from ~44 to ~370 kb.Annotation and comparison with databases showed similarities in particular to T4- and T5-likephages, but also to less-known groups. Though various phages against E. coli are already describedin literature and databases, we still isolated phages that showed no or only few similarities to otherphages, namely phages Goslar, PTXU04, and KWBSE43-6. Genome-based phylogeny andclassification of the newly isolated phages using VICTOR resulted in the proposal of new generaand led to an enhanced taxonomic classification of E. coli phages.
    • Spatiotemporal control of FlgZ activity impacts Pseudomonas aeruginosa flagellar motility.

      Bense, Sarina; Bruchmann, Sebastian; Steffen, Anika; Stradal, Theresia E B; Häussler, Susanne; Düvel, Juliane; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (Wiley-Blackwell, 2019-03-12)
      The c-di-GMP-binding effector protein FlgZ has been demonstrated to control motility in the opportunistic pathogen Pseudomonas aeruginosa and it was suggested that c-di-GMP-bound FlgZ impedes motility via its interaction with the MotCD stator. To further understand how motility is downregulated in P. aeruginosa and to elucidate the general control mechanisms operating during bacterial growth, we examined the spatiotemporal activity of FlgZ. We re-annotated the P. aeruginosaflgZ open reading frame and demonstrated that FlgZ-mediated downregulation of motility is fine-tuned via three independent mechanisms. First, we found that flgZ gene is transcribed independently from flgMN in stationary growth phase to increase FlgZ protein levels in the cell. Second, FlgZ localizes to the cell pole upon c-di-GMP binding and third, we describe that FimV, a cell pole anchor protein, is involved in increasing the polar localized c-di-GMP bound FlgZ to inhibit both, swimming and swarming motility. Our results shed light on the complex dynamics and spatiotemporal control of c-di-GMP-dependent bacterial motility phenotypes and on how the polar anchor protein FimV, the motor brake FlgZ and the stator proteins function to repress flagella-driven swimming and swarming motility.
    • DncV Synthesizes Cyclic GMP-AMP and Regulates Biofilm Formation and Motility in ECOR31.

      Li, Fengyang; Cimdins, Annika; Rohde, Manfred; Jänsch, Lothar; Kaever, Volkhard; Nimtz, Manfred; Römling, Ute; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (ASM, 2019-03-05)
      Cyclic dinucleotides (cDNs) act as intracellular second messengers, modulating bacterial physiology to regulate the fundamental life style transition between motility and sessility commonly known as biofilm formation. Cyclic GMP-AMP (cGAMP), synthesized by the dinucleotide cyclase DncV, is a newly discovered cDN second messenger involved in virulence and chemotaxis in Vibrio cholerae O1 biovar El Tor. Here we report a novel role for horizontally transferred DncV in cGAMP production and regulation of biofilm formation and motility in the animal commensal strain Escherichia coli ECOR31. ECOR31 expresses a semiconstitutive temperature-independent rdar (red, dry, and rough) morphotype on Congo red agar plates characterized by the extracellular matrix components cellulose and curli fimbriae which requires activation by the major biofilm regulator CsgD and cyclic di-GMP signaling. In contrast, C-terminal His-tagged DncV negatively regulates the rdar biofilm morphotype and cell aggregation via downregulation of csgD mRNA steady-state level. Furthermore, DncV sequentially promotes and inhibits adhesion to the abiotic surface after 24 h and 48 h of growth, respectively. DncV also suppresses swimming and swarming motility posttranscriptional of the class 1 flagellum regulon gene flhD Purified DncV produced different cDNs, cyclic di-GMP, cyclic di-AMP, an unknown product(s), and the dominant species 3'3'-cGAMP. In vivo, only the 3'3'-cGAMP concentration was elevated upon short-term overexpression of dncV, making this work a first report on cGAMP production in E. coli Regulation of rdar biofilm formation and motility upon overexpression of untagged DncV in combination with three adjacent cotransferred gene products suggests a novel temperature-dependent cGAMP signaling module in E. coli ECOR31.IMPORTANCE The ability of bacteria to sense and respond to environmental signals is critical for survival. Bacteria use cyclic dinucleotides as second messengers to regulate a number of physiological processes, such as the fundamental life style transition between motility and sessility (biofilm formation). cGAMP, which is synthesized by a dinucleotide cyclase called DncV, is a newly discovered second messenger involved in virulence and chemotaxis in the Vibrio cholerae biovar El Tor causing the current 7th cholera pandemic. However, to what extent cGAMP exists and participates in physiological processes in other bacteria is still unknown. In this study, we found an elevated cGAMP level to possibly regulate biofilm formation and motility in the animal commensal E. coli strain ECOR31. Thus, we detected a novel role for cGAMP signaling in regulation of physiological processes other than those previously reported in proteobacterial species.
    • Microbiome yarns: the Global Phenotype-Genotype Survey: Episode II: laryngeal microbiota and vocal phenotypes (or diction and addiction).

      Timmis, Kenneth; Jebok, Franziska; Rohde, M; Molinari, Gabriella; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (Wiley-Blackwell, 2019-03-01)
    • The Effect of Cytochalasans on the Actin Cytoskeleton of Eukaryotic Cells and Preliminary Structure⁻Activity Relationships.

      Kretz, Robin; Wendt, Lucile; Wongkanoun, Sarunyou; Luangsa-Ard, J Jennifer; Surup, Frank; Helaly, Soleiman E; Noumeur, Sara R; Stadler, Marc; Stradal, Theresia E B; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (MDPI, 2019-02-19)
      In our ongoing search for new bioactive fungal metabolites, two new cytochalasans were isolated from stromata of the hypoxylaceous ascomycete Hypoxylon fragiforme. Their structures were elucidated via high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR) spectroscopy. Together with 23 additional cytochalasans isolated from ascomata and mycelial cultures of different Ascomycota, they were tested on their ability to disrupt the actin cytoskeleton of mammal cells in a preliminary structure⁻activity relationship study. Out of all structural features, the presence of hydroxyl group at the C7 and C18 residues, as well as their stereochemistry, were determined as important factors affecting the potential to disrupt the actin cytoskeleton. Moreover, reversibility of the actin disrupting effects was tested, revealing no direct correlations between potency and reversibility in the tested compound group. Since the diverse bioactivity of cytochalasans is interesting for various applications in eukaryotes, the exact effect on eukaryotic cells will need to be determined, e.g., by follow-up studies involving medicinal chemistry and by inclusion of additional natural cytochalasans. The results are also discussed in relation to previous studies in the literature, including a recent report on the anti-Biofilm activities of essentially the same panel of compounds against the pathogenic bacterium, Staphylococcus aureus.
    • The Effect of Cytochalasans on the Actin Cytoskeleton of Eukaryotic Cells and Preliminary Structure⁻Activity Relationships.

      Kretz, Robin; Wendt, Lucile; Wongkanoun, Sarunyou; Luangsa-Ard, J Jennifer; Surup, Frank; Helaly, Soleiman E; Noumeur, Sara R; Stadler, Marc; Stradal, Theresia E B; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (MPDI, 2019-02-19)
      In our ongoing search for new bioactive fungal metabolites, two new cytochalasans were isolated from stromata of the hypoxylaceous ascomycete Hypoxylon fragiforme. Their structures were elucidated via high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR) spectroscopy. Together with 23 additional cytochalasans isolated from ascomata and mycelial cultures of different Ascomycota, they were tested on their ability to disrupt the actin cytoskeleton of mammal cells in a preliminary structure–activity relationship study. Out of all structural features, the presence of hydroxyl group at the C7 and C18 residues, as well as their stereochemistry, were determined as important factors affecting the potential to disrupt the actin cytoskeleton. Moreover, reversibility of the actin disrupting effects was tested, revealing no direct correlations between potency and reversibility in the tested compound group. Since the diverse bioactivity of cytochalasans is interesting for various applications in eukaryotes, the exact effect on eukaryotic cells will need to be determined, e.g., by follow-up studies involving medicinal chemistry and by inclusion of additional natural cytochalasans. The results are also discussed in relation to previous studies in the literature, including a recent report on the anti-Biofilm activities of essentially the same panel of compounds against the pathogenic bacterium, Staphylococcus aureus. View Full-Text
    • The herpesviral antagonist m152 reveals differential activation of STING-dependent IRF and NF-κB signaling and STING's dual role during MCMV infection.

      Stempel, Markus; Chan, Baca; Juranić Lisnić, Vanda; Krmpotić, Astrid; Hartung, Josephine; Paludan, Søren R; Füllbrunn, Nadia; Lemmermann, Niels Aw; Brinkmann, Melanie M; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley-Blackwell, 2019-01-29)
      Cytomegaloviruses (CMVs) are master manipulators of the host immune response. Here, we reveal that the murine CMV (MCMV) protein m152 specifically targets the type I interferon (IFN) response by binding to stimulator of interferon genes (STING), thereby delaying its trafficking to the Golgi compartment from where STING initiates type I IFN signaling. Infection with an MCMV lacking m152 induced elevated type I IFN responses and this leads to reduced viral transcript levels both in vitro and in vivo. This effect is ameliorated in the absence of STING. Interestingly, while m152 inhibits STING‐mediated IRF signaling, it did not affect STING‐mediated NF‐κB signaling. Analysis of how m152 targets STING translocation reveals that STING activates NF‐κB signaling already from the ER prior to its trafficking to the Golgi. Strikingly, this response is important to promote early MCMV replication. Our results show that MCMV has evolved a mechanism to specifically antagonize the STING‐mediated antiviral IFN response, while preserving its pro‐viral NF‐κB response, providing an advantage in the establishment of an infection.
    • xCELLanalyzer: A Framework for the Analysis of Cellular Impedance Measurements for Mode of Action Discovery

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