• SAR Studies of the Leupyrrins: Design and Total Synthesis of Highly Potent Simplified Leupylogs.

      Wosniok, Paul R; Knopf, Christopher; Dreisigacker, Sandra; Orozco-Rodriguez, J Manuel; Hinkelmann, Bettina; Mueller, Peter P; Brönstrup, Mark; Menche, Dirk; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2020-11-11)
      Invited for the cover of this issue is the group of Dirk Menche at the University of Bonn. The image depicts the natural product leupyrrin A1 and a synthetic leupylog in balance on an IC50 weighing scale. Read the full text of the article at 10.1002/chem.202002622.
    • SAR studies on hydropentalene derivatives--Important core units of biologically active tetramic acid macrolactams and ptychanolides.

      Lutz, Vanessa; Mannchen, Fabian; Krebs, Michael; Park, Natja; Krüger, Claudia; Raja, Aruna; Sasse, Florenz; Baro, Angelika; Laschat, Sabine; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2014-07-01)
      Structurally diverse bicyclo[3.3.0]octanes were prepared and tested for their biological activity. Both the antiproliferative activity and the results of phenotypic characterization varied with the substitution patterns. Two derivatives displayed high inhibitory (IC50 ≤3μM) activity against the L-929 cell line, but differed in their mode of action. A cluster analysis with impedance profiling data showed the two compounds in relationship to microtubule interfering compounds. In PtK2 cells treated with both derivatives a perturbing effect on the microtubular network was observed, whereas the actin cytoskeleton in incubated PtK2 cells was disturbed only by one compound. The effects on tubulin and actin polymerization could be confirmed by in vitro polymerization experiments.
    • Screening and characterization of molecules that modulate the biological activity of IFNs-I.

      Bürgi, Milagros; Zapol'skii, Viktor A; Hinkelmann, Bettina; Köster, Mario; Kaufmann, Dieter E; Sasse, Florenz; Hauser, Hansjörg; Etcheverrigaray, Marina; Kratje, Ricardo; Bollati-Fogolín, Mariela; et al. (2016-09-10)
      Type I Interferons (IFNs-I) are species-specific glycoproteins which play an important role as primary defence against viral infections and that can also modulate the adaptive immune system. In some autoimmune diseases, interferons (IFNs) are over-produced. IFNs are widely used as biopharmaceuticals for a variety of cancer indications, chronic viral diseases, and for their immuno-modulatory action in patients with multiple sclerosis; therefore, increasing their therapeutic efficiency and decreasing their side effects is of high clinical value. In this sense, it is interesting to find molecules that can modulate the activity of IFNs. In order to achieve that, it was necessary to establish a simple, fast and robust assay to analyze numerous compounds simultaneously. We developed four reporter gene assays (RGAs) to identify IFN activity modulator compounds by using WISH-Mx2/EGFP, HeLa-Mx2/EGFP, A549-Mx2/EGFP, and HEp2-Mx2/EGFP reporter cell lines (RCLs). All of them present a Z' factor higher than 0.7. By using these RGAs, natural and synthetic compounds were analyzed simultaneously. A total of 442 compounds were studied by the Low Throughput Screening (LTS) assay using the four RCLs to discriminate between their inhibitory or enhancing effects on IFN activity. Some of them were characterized and 15 leads were identified. Finally, one promising candidate with enhancing effect on IFN-α/-β activity and five compounds with inhibitory effect were described.
    • A selective 3-acylation of tetramic acids and the first synthesis of ravenic acid.

      Schlenk, Andrea; Diestel, Randi; Sasse, Florenz; Schobert, Rainer; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2010-02-22)
      3-Acyltetramic acids, including delicate 3-oligoenoyl derivatives, such as the Penicillium metabolite ravenic acid, were prepared in two high-yielding steps. Reaction of tetramic acids with the ylide Ph(3)PCCO afforded exclusively the corresponding 3-acylylidenetetramic acids. These were amenable to Wittig olefinations with aliphatic, aromatic, saturated and unsaturated aldehydes after deprotonation with KOtBu. Due to its simplicity, selectivity and tolerance of pH-sensitive groups this method is superior to the established acylation protocols by Jones and Yoshii. It is also applicable to the synthesis of 3-acyltetronic acids. The new 3-oligoenoyl tetramic acids exhibited structure-dependent antimicrobial and cytotoxic activity.
    • 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.
    • Single-cell phenotypic characterization of Staphylococcus aureus with fluorescent triazole urea activity-based probes.

      Chen, Linhai; Keller, Laura J; Cordasco, Edward A; Bogyo, Matthew; Lentz, Christian S; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (Wiley-Blackwell, 2019-02-15)
      Phenotypically distinct cellular (sub)populations are clinically relevant for virulence and antibiotic resistance of a bacterial pathogen, but functionally different cells are usually indistinguishable from each other. Here, we introduce fluorescent activity-based probes as chemical tools for single-cell phenotypic characterization of enzyme activity levels in Staphylococcus aureus. We screened a 1,2,3-triazole urea library to identify selective inhibitors of fluorophosphonate-binding serine hydrolases and lipases in S. aureus and synthesized target-selective activity-based probes. Molecular imaging and activity-based protein profiling studies with these probes revealed a dynamic network within this enzyme family involving compensatory regulation of specific family members and exposed single-cell phenotypic heterogeneity. We propose chemical probe labeling of enzymatic activities as a generalizable method for phenotyping of bacterial cells at the population and single-cell level.
    • Soraphen A: A broad-spectrum antiviral natural product with potent anti-hepatitis C virus activity.

      Koutsoudakis, George; Romero-Brey, Inés; Berger, Carola; Pérez-Vilaró, Gemma; Monteiro Perin, Paula; Vondran, Florian Wolfgang Rudolf; Kalesse, Markus; Harmrolfs, Kirsten; Müller, Rolf; Martinez, Javier P; et al. (2015-06-10)
      Soraphen A (SorA) is a myxobacterial metabolite that inhibits the acetyl-CoA carboxylase, a key enzyme in lipid biosynthesis. We have previously identified SorA to efficiently inhibit the human immunodeficiency virus (HIV). The aim of the present study was to evaluate the capacity of SorA and analogues to inhibit hepatitis C virus (HCV) infection.
    • Species-Specific Conservation of Linear Antigenic Sites on Vaccinia Virus A27 Protein Homologs of Orthopoxviruses.

      Ahsendorf, Henrike P; Gan, Li L; Eltom, Kamal H; Abd El Wahed, Ahmed; Hotop, Sven-Kevin; Roper, Rachel L; Beutling, Ulrike; Broenstrup, Mark; Stahl-Hennig, Christiane; Hoelzle, Ludwig E; et al. (MPDI, 2019-05-29)
      The vaccinia virus (VACV) A27 protein and its homologs, which are found in a large number of members of the genus Orthopoxvirus (OPXV), are targets of viral neutralization by host antibodies. We have mapped six binding sites (epitopes #1A: aa 32-39, #1B: aa 28-33, #1C: aa 26-31, #1D: 28-34, #4: aa 9-14, and #5: aa 68-71) of A27 specific monoclonal antibodies (mAbs) using peptide arrays. MAbs recognizing epitopes #1A-D and #4 neutralized VACV Elstree in a complement dependent way (50% plaque-reduction: 12.5-200 µg/mL). Fusion of VACV at low pH was blocked through inhibition of epitope #1A. To determine the sequence variability of the six antigenic sites, 391 sequences of A27 protein homologs available were compared. Epitopes #4 and #5 were conserved among most of the OPXVs, while the sequential epitope complex #1A-D was more variable and, therefore, responsible for species-specific epitope characteristics. The accurate and reliable mapping of defined epitopes on immuno-protective proteins such as the A27 of VACV enables phylogenetic studies and insights into OPXV evolution as well as to pave the way to the development of safer vaccines and chemical or biological antivirals.
    • Subcellular Quantification of Uptake in Gram-Negative Bacteria.

      Prochnow, Hans; Fetz, Verena; Hotop, Sven-Kevin; García-Rivera, Mariel A; Heumann, Axel; Brönstrup, Mark; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (ACS Publications, 2019-02-05)
      Infections by Gram-negative pathogens represent a major health care issue of growing concern due to a striking lack of novel antibacterial agents over the course of the last decades. The main scientific problem behind the rational optimization of novel antibiotics is our limited understanding of small molecule translocation into, and their export from, the target compartments of Gram-negative species. To address this issue, a versatile, label-free assay to determine the intracellular localization and concentration of a given compound has been developed for Escherichia coli and its efflux-impaired ΔTolC mutant. The assay applies a fractionation procedure to antibiotic-treated bacterial cells to obtain periplasm, cytoplasm, and membrane fractions of high purity, as demonstrated by Western Blots of compartment-specific marker proteins. This is followed by an LC-MS/MS-based quantification of antibiotic content in each compartment. Antibiotic amounts could be converted to antibiotic concentrations by assuming that an E. coli cell is a cylinder flanked by two half spheres and calculating the volumes of bacterial compartments. The quantification of antibiotics from different classes, namely ciprofloxacin, tetracycline, trimethoprim, and erythromycin, demonstrated pronounced differences in uptake quantities and distribution patterns across the compartments. For example, in the case of ciprofloxacin, a higher amount of compound was located in the cytoplasm than in the periplasm (592 ± 50 pg vs 277 ± 13 pg per 3.9 × 10
    • Sulfur, selenium and tellurium pseudopeptides: synthesis and biological evaluation.

      Shaaban, Saad; Sasse, Florenz; Burkholz, Torsten; Jacob, Claus; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2014-07-15)
      A new series of sulfur, selenium and tellurium peptidomimetic compounds was prepared employing the Passerini and Ugi isocyanide based multicomponent reactions (IMCRs). These reactions were clearly superior to conventional methods traditionally used for organoselenium and organotellurium synthesis, such as classical nucleophilic substitution and coupling methods. From the biological point of view, these compounds are of considerable interest because of suspected anticancer and antimicrobial activities. While the sulfur and selenium containing compounds generally did not show either anticancer or antimicrobial activities, their tellurium based counterparts frequently exhibited antimicrobial activity and were also cytotoxic. Some of the compounds synthesized even showed selective activity against certain cancer cells in cell culture. These compounds induced a cell cycle delay in the G0/G1 phase. At closer inspection, the ER and the actin cytoskeleton appeared to be the primary cellular targets of these tellurium compounds, in line with some of our previous studies. As most of these peptidomimetic compounds also comply with Lipinski's Rule of Five, they promise good bioavailability, which needs to be studied as part of future investigations.
    • Synthesis of the AB ring system of clifednamide utilizing Claisen rearrangement and Diels-Alder reaction as key steps.

      Loke, Inga; Bentzinger, Guillaume; Holz, Julia; Raja, Aruna; Bhasin, Aman; Sasse, Florenz; Köhn, Andreas; Schobert, Rainer; Laschat, Sabine; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-01-21)
      In order to construct the functionalized AB ring system of clifednamide, member of the class of macrocyclic tetramic acid lactams, a synthesis was developed which utilized an Ireland-Claisen rearrangement and an intramolecular Diels-Alder reaction. Starting from di-O-isopropylidene-d-mannitol the allyl carboxylate precursor for the sigmatropic rearrangement was prepared. This rearrangement proceeded diastereoselectively only in the presence of an allyl silyl ether instead of the parent enone in the side chain, as suggested by deuteration experiments. A subsequent Diels-Alder reaction yielded the target ethyl hexahydro-1H-indene-carboxylate with high diastereoselectivity. Quantum-chemical investigations of this intramolecular Diels-Alder reaction support the proposed configuration of the final product.
    • Synthetic studies of cystobactamids as antibiotics and bacterial imaging carriers lead to compounds with high: In vivo efficacy

      Testolin, Giambattista; Cirnski, Katarina; Rox, Katharina; Prochnow, Hans; Fetz, Verena; Grandclaudon, Charlotte; Mollner, Tim; Baiyoumy, Alain; Ritter, Antje; Leitner, Christian; et al. (RSC, 2020-01-01)
      There is an alarming scarcity of novel chemical matter with bioactivity against multidrug-resistant Gram-negative bacterial pathogens. Cystobactamids, recently discovered natural products from myxobacteria, are an exception to this trend. Their unusual chemical structure, composed of oligomeric para-aminobenzoic acid moieties, is associated with a high antibiotic activity through the inhibition of gyrase. In this study, structural determinants of cystobactamid's antibacterial potency were defined at five positions, which were varied using three different synthetic routes to the cystobactamid scaffold. The potency against Acinetobacter baumannii could be increased ten-fold to an MIC (minimum inhibitory concentration) of 0.06 μg mL−1, and the previously identified spectrum gap of Klebsiella pneumoniae could be closed compared to the natural products (MIC of 0.5 μg mL−1). Proteolytic degradation of cystobactamids by the resistance factor AlbD was prevented by an amide-triazole replacement. Conjugation of cystobactamid's N-terminal tetrapeptide to a Bodipy moiety induced the selective localization of the fluorophore for bacterial imaging purposes. Finally, a first in vivo proof of concept was obtained in an E. coli infection mouse model, where derivative 22 led to the reduction of bacterial loads (cfu, colony-forming units) in muscle, lung and kidneys by five orders of magnitude compared to vehicle-treated mice. These findings qualify cystobactamids as highly promising lead structures against infections caused by Gram-positive and Gram-negative bacterial pathogens.
    • 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.
    • Tailored Cofactor Traps for the Detection of Hemithioacetal-Forming Pyridoxal Kinases.

      Hübner, Ines; Dienemann, Jan-Niklas; Friederich, Julia; Schneider, Sabine; Sieber, Stephan A; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (American Society for Chemistry (ACS), 2020-12-03)
      Pyridoxal kinases (PLK) are crucial enzymes for the biosynthesis of pyridoxal phosphate, an important cofactor in a plethora of enzymatic reactions. The evolution of these enzymes resulted in different catalytic designs. In addition to the active site, the importance of a cysteine, embedded within a distant flexible lid region, was recently demonstrated. This cysteine forms a hemithioacetal with the pyridoxal aldehyde and is essential for catalysis. Despite the prevalence of these enzymes in various organisms, no tools were yet available to study the relevance of this lid residue. Here, we introduce pyridoxal probes, each equipped with an electrophilic trapping group in place of the aldehyde to target PLK reactive lid cysteines as a mimic of hemithioacetal formation. The addition of alkyne handles placed at two different positions within the pyridoxal structure facilitates enrichment of PLKs from living cells. Interestingly, depending on the position, the probes displayed a preference for either Gram-positive or Gram-negative PLK enrichment. By applying the cofactor traps, we were able to validate not only previously investigated Staphylococcus aureus and Enterococcus faecalis PLKs but also Escherichia coli and Pseudomonas aeruginosa PLKs, unravelling a crucial role of the lid cysteine for catalysis. Overall, our tailored probes facilitated a reliable readout of lid cysteine containing PLKs, qualifying them as chemical tools for mining further diverse proteomes for this important enzyme class.
    • Target identification by image analysis.

      Fetz, V; Prochnow, H; Brönstrup, Mark; Sasse, F; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
      Covering: 1997 to the end of 2015Each biologically active compound induces phenotypic changes in target cells that are characteristic for its mode of action. These phenotypic alterations can be directly observed under the microscope or made visible by labelling structural elements or selected proteins of the cells with dyes. A comparison of the cellular phenotype induced by a compound of interest with the phenotypes of reference compounds with known cellular targets allows predicting its mode of action. While this approach has been successfully applied to the characterization of natural products based on a visual inspection of images, recent studies used automated microscopy and analysis software to increase speed and to reduce subjective interpretation. In this review, we give a general outline of the workflow for manual and automated image analysis, and we highlight natural products whose bacterial and eucaryotic targets could be identified through such approaches.
    • Toll-like Receptor 5 Activation by the CagY Repeat Domains of Helicobacter pylori.

      Tegtmeyer, Nicole; Neddermann, Matthias; Lind, Judith; Pachathundikandi, Suneesh Kumar; Sharafutdinov, Irshad; Gutiérrez-Escobar, Andrés Julián; Brönstrup, Mark; Tegge, Werner; Hong, Minsun; Rohde, Manfred; et al. (Cell Press, 2020-11-15)
      Helicobacter pylori (Hp) is an important human pathogen associated with gastric inflammation and neoplasia. It is commonly believed that this bacterium avoids major immune recognition by Toll-like receptors (TLRs) because of low intrinsic activity of its flagellin and lipopolysaccharides (LPS). In particular, TLR5 specifically detects flagellins in various bacterial pathogens, while Hp evolved mutations in flagellin to evade detection through TLR5. Cancerogenic Hp strains encode a type IV secretion system (T4SS). The T4SS core component and pilus-associated protein CagY, a large VirB10 ortholog, drives effector molecule translocation. Here, we identify CagY as a flagellin-independent TLR5 agonist. We detect five TLR5 interaction sites, promoting binding of CagY-positive Hp to TLR5-expressing cells, TLR5 stimulation, and intracellular signal transduction. Consequently, CagY constitutes a remarkable VirB10 member detected by TLR5, driving crucial innate immune responses by this human pathogen.
    • Unprecedented deoxygenation at C-7 of the ansamitocin core during mutasynthetic biotransformations.

      Knobloch, Tobias; Dräger, Gerald; Collisi, Wera; Sasse, Florenz; Kirschning, Andreas; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2012)
      We describe the unprecedented formation of six ansamitocin derivatives that are deoxygenated at C-7 of the ansamitocin core, obtained during fermentation experiments by employing a variety of Actinosynnema pretiosum mutants and mutasynthetic approaches. We suggest that the formation of these derivatives is based on elimination at C-7/C-8 followed by reduction(s) of the intermediate enone. In bioactivity tests, only ansamitocin derivatives bearing an ester side chain at C-3 showed strong antiproliferative activity.
    • Untargeted LC-MS Metabolomics Differentiates Between Virulent and Avirulent Clinical Strains of Pseudomonas aeruginosa

      Depke, Tobias; Thöming, Janne Gesine; Kordes, Adrian; Häussler, Susanne; Brönstrup, Mark; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-07-13)
      Pseudomonas aeruginosa is a facultative pathogen that can cause, inter alia, acute or chronic pneumonia in predisposed individuals. The gram-negative bacterium displays considerable genomic and phenotypic diversity that is also shaped by small molecule secondary metabolites. The discrimination of virulence phenotypes is highly relevant to the diagnosis and prognosis of P. aeruginosa infections. In order to discover small molecule metabolites that distinguish different virulence phenotypes of P. aeruginosa, 35 clinical strains were cultivated under standard conditions, characterized in terms of virulence and biofilm phenotype, and their metabolomes were investigated by untargeted liquid chromatography-mass spectrometry. The data was both mined for individual candidate markers as well as used to construct statistical models to infer the virulence phenotype from metabolomics data. We found that clinical strains that differed in their virulence and biofilm phenotype also had pronounced divergence in their metabolomes, as underlined by 332 features that were significantly differentially abundant with fold changes greater than 1.5 in both directions. Important virulence-associated secondary metabolites like rhamnolipids, alkyl quinolones or phenazines were found to be strongly upregulated in virulent strains. In contrast, we observed little change in primary metabolism. A hitherto novel cationic metabolite with a sum formula of C12H15N2 could be identified as a candidate biomarker. A random forest model was able to classify strains according to their virulence and biofilm phenotype with an area under the Receiver Operation Characteristics curve of 0.84. These findings demonstrate that untargeted metabolomics is a valuable tool to characterize P. aeruginosa virulence, and to explore interrelations between clinically important phenotypic traits and the bacterial metabolome.
    • Use of Single-Frequency Impedance Spectroscopy to Characterize the Growth Dynamics of Biofilm Formation in Pseudomonas aeruginosa.

      van Duuren, Jozef B J H; Müsken, Mathias; Karge, Bianka; Tomasch, Jürgen; Wittmann, Christoph; Häussler, Susanne; Brönstrup, Mark (2017-07-12)
      Impedance spectroscopy has been applied in prokaryotic and eukaryotic cytometry as a label-free method for the investigation of adherent cells. In this paper, its use for characterizing the growth dynamics of P. aeruginosa biofilms is described and compared to crystal violet staining and confocal microscopy. The method allows monitoring the growth of biofilm-forming P. aeruginosa in a continuous and label-free manner over a period of 72 h in a 96 well plate format. Impedance curves obtained for P. aeruginosa PA14 wild type and mutant strains with a transposon insertion in pqsA and pelA genes exhibited distinct phases. We propose that the slope of the declining curve following a maximum at ca. 35-40 h is a measure of biofilm formation. Transplant experiments with P. aeruginosa biofilms and paraffin suggest that the impedance also reflects pellicle formation at the liquid-air interface, a barely considered contributor to impedance. Finally, the impairment of biofilm formation upon treatment of cultures with L-arginine and with ciprofloxacin, tobramycin and meropenem was studied by single frequency impedance spectroscopy. We suggest that these findings qualify impedance spectroscopy as an additional technique to characterize biofilm formation and its modulation by small molecule drugs.
    • Von Willebrand Factor Mediates Pneumococcal Aggregation and Adhesion in Blood Flow.

      Jagau, Hilger; Behrens, Ina-Kristin; Lahme, Karen; Lorz, Georgina; Köster, Reinhard W; Schneppenheim, Reinhard; Obser, Tobias; Brehm, Maria A; König, Gesa; Kohler, Thomas P; et al. (Frontiers, 2019-01-01)
      Streptococcus pneumoniae is a major cause of community acquired pneumonia and septicaemia in humans. These diseases are frequently associated with thromboembolic cardiovascular complications. Pneumococci induce the exocytosis of endothelial Weibel-Palade Bodies and thereby actively stimulate the release of von Willebrand factor (VWF), which is an essential glycoprotein of the vascular hemostasis. Both, the pneumococcus induced pulmonary inflammation and the thromboembolytic complications are characterized by a dysbalanced hemostasis including a marked increase in VWF plasma concentrations. Here, we describe for the first time VWF as a novel interaction partner of capsulated and non-encapsulated pneumococci. Moreover, cell culture infection analyses with primary endothelial cells characterized VWF as bridging molecule that mediates bacterial adherence to endothelial cells in a heparin-sensitive manner. Due to the mechanoresponsive changes of the VWF protein conformation and multimerization status, which occur in the blood stream, we used a microfluidic pump system to generate shear flow-induced multimeric VWF strings on endothelial cell surfaces and analyzed attachment of RFP-expressing pneumococci in flow. By applying immunofluorescence visualization and additional electron microscopy, we detected a frequent and enduring bacterial attachment to the VWF strings. Bacterial attachment to the endothelium was confirmed in vivo using a zebrafish infection model, which is described in many reports and acknowledged as suitable model to study hemostasis mechanisms and protein interactions of coagulation factors. Notably, we visualized the recruitment of zebrafish-derived VWF to the surface of pneumococci circulating in the blood stream and detected a VWF-dependent formation of bacterial aggregates within the vasculature of infected zebrafish larvae. Furthermore, we identified the surface-exposed bacterial enolase as pneumococcal VWF binding protein, which interacts with the VWF domain A1 and determined the binding kinetics by surface plasmon resonance. Subsequent epitope mapping using an enolase peptide array indicates that the peptide 181YGAEIFHALKKILKS195 might serve as a possible core sequence of the VWF interaction site. In conclusion, we describe a VWF-mediated mechanism for pneumococcal anchoring within the bloodstream via surface-displayed enolase, which promotes intravascular bacterial aggregation.