TY - JOUR A1 - Hertlein, Tobias A1 - Sturm, Volker A1 - Kircher, Stefan A1 - Basse-Lüsebrink, Thomas A1 - Haddad, Daniel A1 - Ohlsen, Knut A1 - Jakob, Peter T1 - Visualization of Abscess Formation in a Murine Thigh Infection Model of \(Staphylococcus\) \(aureus\) by (19)F-Magnetic Resonance Imaging (MRI) JF - PLoS ONE N2 - Background: During the last years, (19)F-MRI and perfluorocarbon nanoemulsion (PFC) emerged as a powerful contrast agent methodology to track cells and to visualize inflammation. We applied this new modality to visualize deep tissue abscesses during acute and chronic phase of inflammation caused by Staphylococcus aureus infection. Methodology and Principal Findings: In this study, a murine thigh infection model was used to induce abscess formation and PFC or CLIO (cross linked ironoxides) was administered during acute or chronic phase of inflammation. 24 h after inoculation, the contrast agent accumulation was imaged at the site of infection by MRI. Measurements revealed a strong accumulation of PFC at the abscess rim at acute and chronic phase of infection. The pattern was similar to CLIO accumulation at chronic phase and formed a hollow sphere around the edema area. Histology revealed strong influx of neutrophils at the site of infection and to a smaller extend macrophages during acute phase and strong influx of macrophages at chronic phase of inflammation. Conclusion and Significance: We introduce (19)F-MRI in combination with PFC nanoemulsions as a new platform to visualize abscess formation in a murine thigh infection model of S. aureus. The possibility to track immune cells in vivo by this modality offers new opportunities to investigate host immune response, the efficacy of antibacterial therapies and the influence of virulence factors for pathogenesis. KW - Soft-tissue infection KW - In-vivo KW - Iron-oxide KW - F-19 MRI KW - Inflammation KW - Particles KW - Tracking KW - Lesions KW - Images KW - Rats Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-142846 VL - 6 IS - 3 ER - TY - JOUR A1 - Cecil, Alexander A1 - Rikanovic, Carina A1 - Ohlsen, Knut A1 - Liang, Chunguang A1 - Bernhardt, Jorg A1 - Oelschlaeger, Tobias A. A1 - Gulder, Tanja A1 - Bringmann, Gerd A1 - Holzgrabe, Ulrike A1 - Unger, Matthias A1 - Dandekar, Thomas T1 - Modeling antibiotic and cytotoxic effects of the dimeric isoquinoline IQ-143 on metabolism and its regulation in Staphylococcus aureus, Staphylococcus epidermidis and human cells N2 - Background: Xenobiotics represent an environmental stress and as such are a source for antibiotics, including the isoquinoline (IQ) compound IQ-143. Here, we demonstrate the utility of complementary analysis of both host and pathogen datasets in assessing bacterial adaptation to IQ-143, a synthetic analog of the novel type N,C-coupled naphthyl-isoquinoline alkaloid ancisheynine. Results: Metabolite measurements, gene expression data and functional assays were combined with metabolic modeling to assess the effects of IQ-143 on Staphylococcus aureus, Staphylococcus epidermidis and human cell lines, as a potential paradigm for novel antibiotics. Genome annotation and PCR validation identified novel enzymes in the primary metabolism of staphylococci. Gene expression response analysis and metabolic modeling demonstrated the adaptation of enzymes to IQ-143, including those not affected by significant gene expression changes. At lower concentrations, IQ-143 was bacteriostatic, and at higher concentrations bactericidal, while the analysis suggested that the mode of action was a direct interference in nucleotide and energy metabolism. Experiments in human cell lines supported the conclusions from pathway modeling and found that IQ-143 had low cytotoxicity. Conclusions: The data suggest that IQ-143 is a promising lead compound for antibiotic therapy against staphylococci. The combination of gene expression and metabolite analyses with in silico modeling of metabolite pathways allowed us to study metabolic adaptations in detail and can be used for the evaluation of metabolic effects of other xenobiotics. KW - Staphylococcus aureus Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68802 ER - TY - JOUR A1 - Jakob, Peter A1 - Hertlein, Tobias A1 - Sturm, Volker A1 - Kircher, Stefan A1 - Basse-Lüsebrink, Thomas A1 - Haddad, Daniel A1 - Ohlsen, Knut T1 - Visualization of Abscess Formation in a Murine Thigh Infection Model of Staphylococcus aureus by 19F-Magnetic Resonance Imaging (MRI) N2 - Background: During the last years, 19F-MRI and perfluorocarbon nanoemulsion (PFC) emerged as a powerful contrast agent based MRI methodology to track cells and to visualize inflammation. We applied this new modality to visualize deep tissue abscesses during acute and chronic phase of inflammation caused by Staphylococcus aureus infection. Methodology and Principal Findings: In this study, a murine thigh infection model was used to induce abscess formation and PFC or CLIO (cross linked ironoxides) was administered during acute or chronic phase of inflammation. 24 h after inoculation, the contrast agent accumulation was imaged at the site of infection by MRI. Measurements revealed a strong accumulation of PFC at the abscess rim at acute and chronic phase of infection. The pattern was similar to CLIO accumulation at chronic phase and formed a hollow sphere around the edema area. Histology revealed strong influx of neutrophils at the site of infection and to a smaller extend macrophages during acute phase and strong influx of macrophages at chronic phase of inflammation. Conclusion and Significance: We introduce 19F-MRI in combination with PFC nanoemulsions as a new platform to visualize abscess formation in a murine thigh infection model of S. aureus. The possibility to track immune cells in vivo by this modality offers new opportunities to investigate host immune response, the efficacy of antibacterial therapies and the influence of virulence factors for pathogenesis. KW - Staphylococcus aureus Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-74994 ER - TY - JOUR A1 - Rakette, Sonja A1 - Donat, Stefanie A1 - Ohlsen, Knut A1 - Stehle, Thilo T1 - Structural Analysis of Staphylococcus aureus Serine/Threonine Kinase PknB JF - PLoS One N2 - Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 angstrom resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation. KW - SER/THR kinase KW - domain KW - subunit KW - dependent protein-kinase KW - mycobacterium-tuberculosis KW - activation mechanism KW - crystal structure KW - antibiotic resistance KW - catalytic KW - methicillin KW - inhibitor Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-135369 VL - 7 IS - 6 ER - TY - JOUR A1 - Hertlein, Tobias A1 - Sturm, Volker A1 - Jakob, Peter A1 - Ohlsen, Knut T1 - \(^{19}\)F Magnetic Resonance Imaging of Perfluorocarbons for the Evaluation of Response to Antibiotic Therapy in a Staphylococcus aureus Infection Model JF - PLoS ONE N2 - Background The emergence of antibiotic resistant bacteria in recent decades has highlighted the importance of developing new drugs to treat infections. However, in addition to the design of new drugs, the development of accurate preclinical testing methods is essential. In vivo imaging technologies such as bioluminescence imaging (BLI) or magnetic resonance imaging (MRI) are promising approaches. In a previous study, we showed the effectiveness of \(^{19}\)F MRI using perfluorocarbon (PFC) emulsions for detecting the site of Staphylococcus aureus infection. In the present follow-up study, we investigated the use of this method for in vivo visualization of the effects of antibiotic therapy. Methods/Principal findings Mice were infected with S. aureus Xen29 and treated with 0.9% NaCl solution, vancomycin or linezolid. Mock treatment led to the highest bioluminescence values during infection followed by vancomycin treatment. Counting the number of colony-forming units (cfu) at 7 days post-infection (p.i.) showed the highest bacterial burden for the mock group and the lowest for the linezolid group. Administration of PFCs at day 2 p.i. led to the accumulation of \(^{19}\)F at the rim of the abscess in all mice (in the shape of a hollow sphere), and antibiotic treatment decreased the \(^{19}\)F signal intensity and volume. Linezolid showed the strongest effect. The BLI, cfu, and MRI results were comparable. Conclusions \(^{19}\)F-MRI with PFCs is an effective non-invasive method for assessing the effects of antibiotic therapy in vivo. This method does not depend on pathogen specific markers and can therefore be used to estimate the efficacy of antibacterial therapy against a broad range of clinically relevant pathogens, and to localize sites of infection. KW - staphylococcus aureus KW - abscesses KW - vancomycin KW - antibiotics KW - magnetic resonance imaging KW - emulsions KW - bioluminescence imaging KW - in vivo imaging Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130113 VL - 8 IS - 5 ER - TY - JOUR A1 - Neumann, Yvonne A1 - Ohlsen, Knut A1 - Donat, Stefanie A1 - Engelmann, Susanne A1 - Kusch, Harald A1 - Albrecht, Dirk A1 - Cartron, Michael A1 - Hurd, Alexander A1 - Foster, Simon J. T1 - The effect of skin fatty acids on Staphylococcus aureus JF - Archives of Microbiology N2 - Staphylococcus aureus is a commensal of the human nose and skin. Human skin fatty acids, in particular cis-6-hexadecenoic acid (C-6-H), have high antistaphylococcal activity and can inhibit virulence determinant production. Here, we show that sub-MIC levels of C-6-H result in induction of increased resistance. The mechanism(s) of C-6-H activity was investigated by combined transcriptome and proteome analyses. Proteome analysis demonstrated a pleiotropic effect of C-6-H on virulence determinant production. In response to C-6-H, transcriptomics revealed altered expression of over 500 genes, involved in many aspects of virulence and cellular physiology. The expression of toxins (hla, hlb, hlgBC) was reduced, whereas that of host defence evasion components (cap, sspAB, katA) was increased. In particular, members of the SaeRS regulon had highly reduced expression, and the use of specific mutants revealed that the effect on toxin production is likely mediated via SaeRS KW - S. aureus KW - skin fatty acid KW - C-6-H KW - resistance Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121657 VL - 197 ER - TY - JOUR A1 - Nguyen, Minh Thu A1 - Kraft, Beatrice A1 - Yu, Wenqi A1 - Demicrioglu, Dogan Doruk A1 - Hertlein, Tobias A1 - Burian, Marc A1 - Schmaler, Mathias A1 - Boller, Klaus A1 - Bekeredjian-Ding, Isabelle A1 - Ohlsen, Knut A1 - Schittek, Birgit A1 - Götz, Friedrich T1 - The vSa\(\alpha\) Specific Lipoprotein Like Cluster (lpl) of S. aureus USA300 Contributes to Immune Stimulation and Invasion in Human Cells JF - PLoS Pathogens N2 - All Staphylococcus aureus genomes contain a genomic island, which is termed vSa\(\alpha\) and characterized by two clusters of tandem repeat sequences, i.e. the exotoxin (set) and 'lipoprotein-like' genes (lpl). Based on their structural similarities the vSa\(\alpha\) islands have been classified as type I to IV. The genomes of highly pathogenic and particularly epidemic S. aureus strains (USA300, N315, Mu50, NCTC8325, Newman, COL, JH1 or JH9) belonging to the clonal complexes CC5 and CC8 bear a type I vSa\(\alpha\) island. Since the contribution of the lpl gene cluster encoded in the vSa\(\alpha\) island to virulence is unclear to date, we deleted the entire lpl gene cluster in S. aureus USA300. The results showed that the mutant was deficient in the stimulation of pro-inflammatory cytokines in human monocytes, macrophages and keratinocytes. Purified lipoprotein Lpl1 was further shown to elicit a TLR2-dependent response. Furthermore, heterologous expression of the USA300 lpl cluster in other S. aureus strains enhanced their immune stimulatory activity. Most importantly, the lpl cluster contributed to invasion of S. aureus into human keratinocytes and mouse skin and the non-invasive S. carnosus expressing the lpl gene cluster became invasive. Additionally, in a murine kidney abscess model the bacterial burden in the kidneys was higher in wild type than in mutant mice. In this infection model the lpl cluster, thus, contributes to virulence. The present report is one of the first studies addressing the role of the vSa\(\alpha\) encoded lpl gene cluster in staphylococcal virulence. The finding that the lpl gene cluster contributes to internalization into non-professional antigen presenting cells such as keratinocytes high-lights the lpl as a new cell surface component that triggers host cell invasion by S. aureus. Increased invasion in murine skin and an increased bacterial burden in a murine kidney abscess model suggest that the lpl gene cluster serves as an important virulence factor. KW - resistant Staphylococcus-aureus KW - bacterial lipoproteins KW - internalization KW - evolution KW - fibronectin-binding protein KW - toll-like receptor 2 KW - epithelial cells KW - genome sequence KW - activation KW - mechanisms Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-151856 VL - 11 IS - 6 ER - TY - JOUR A1 - Blättner, Sebastian A1 - Das, Sudip A1 - Paprotka, Kerstin A1 - Eilers, Ursula A1 - Krischke, Markus A1 - Kretschmer, Dorothee A1 - Remmele, Christian W. A1 - Dittrich, Marcus A1 - Müller, Tobias A1 - Schuelein-Voelk, Christina A1 - Hertlein, Tobias A1 - Mueller, Martin J. A1 - Huettel, Bruno A1 - Reinhardt, Richard A1 - Ohlsen, Knut A1 - Rudel, Thomas A1 - Fraunholz, Martin J. T1 - Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes JF - PLoS Pathogens N2 - Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection. KW - cell death KW - cytotoxicity KW - Staphylococcus aureus KW - host cells KW - neutrophils KW - macrophages KW - transposable elements KW - epithelial cells Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-180380 VL - 12 IS - 9 ER - TY - JOUR A1 - Selle, Martina A1 - Hertlein, Tobias A1 - Oesterreich, Babett A1 - Klemm, Theresa A1 - Kloppot, Peggy A1 - Müller, Elke A1 - Ehricht, Ralf A1 - Stentzel, Sebastian A1 - Bröker, Barbara M. A1 - Engelmann, Susanne A1 - Ohlsen, Knut T1 - Global antibody response to Staphylococcus aureus live-cell vaccination JF - Scientific Reports N2 - The pathogen Staphylococcus aureus causes a broad range of severe diseases and is feared for its ability to rapidly develop resistance to antibiotic substances. The increasing number of highly resistant S. aureus infections has accelerated the search for alternative treatment options to close the widening gap in anti-S. aureus therapy. This study analyses the humoral immune response to vaccination of Balb/c mice with sublethal doses of live S. aureus. The elicited antibody pattern in the sera of intravenously and intramuscularly vaccinated mice was determined using of a recently developed protein array. We observed a specific antibody response against a broad set of S. aureus antigens which was stronger following i.v. than i.m. vaccination. Intravenous but not intramuscular vaccination protected mice against an intramuscular challenge infection with a high bacterial dose. Vaccine protection was correlated with the strength of the anti-S. aureus antibody response. This study identified novel vaccine candidates by using protein microarrays as an effective tool and showed that successful vaccination against S. aureus relies on the optimal route of administration. KW - pathogens KW - bacterial infection KW - cell vaccines KW - Staphylococcus aureus Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-181245 VL - 6 ER - TY - JOUR A1 - Ashraf, Kerolos A1 - Yasrebi, Kaveh A1 - Hertlein, Tobias A1 - Ohlsen, Knut A1 - Lalk, Michael A1 - Hilgeroth, Andreas T1 - Novel effective small-molecule antibacterials against \(Enterococcus\) strains JF - Molecules N2 - \(Enterococcus\) species cause increasing numbers of infections in hospitals. They contribute to the increasing mortality rates, mostly in patients with comorbidities, who suffer from severe diseases. \(Enterococcus\) resistances against most antibiotics have been described, including novel antibiotics. Therefore, there is an ongoing demand for novel types of antibiotics that may overcome bacterial resistances. We discovered a novel class of antibiotics resulting from a simple one-pot reaction of indole and \(o\)-phthaldialdehyde. Differently substituted indolyl benzocarbazoles were yielded. Both the indole substitution and the positioning at the molecular scaffold influence the antibacterial activity towards the various strains of \(Enterococcus\) species with the highest relevance to nosocomial infections. Structure-activity relationships are discussed, and the first lead compounds were identified as also being effective in the case of a vancomycin resistance. KW - medicine KW - antibacterial activity KW - synthesis KW - derivatives KW - structure-activity KW - lead structure Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-172628 VL - 22 IS - 12 ER - TY - JOUR A1 - García-Betancur, Juan-Carlos A1 - Goñi-Moreno, Angel A1 - Horger, Thomas A1 - Schott, Melanie A1 - Sharan, Malvika A1 - Eikmeier, Julian A1 - Wohlmuth, Barbara A1 - Zernecke, Alma A1 - Ohlsen, Knut A1 - Kuttler, Christina A1 - Lopez, Daniel T1 - Cell differentiation defines acute and chronic infection cell types in Staphylococcus aureus JF - eLife N2 - A central question to biology is how pathogenic bacteria initiate acute or chronic infections. Here we describe a genetic program for cell-fate decision in the opportunistic human pathogen Staphylococcus aureus, which generates the phenotypic bifurcation of the cells into two genetically identical but different cell types during the course of an infection. Whereas one cell type promotes the formation of biofilms that contribute to chronic infections, the second type is planktonic and produces the toxins that contribute to acute bacteremia. We identified a bimodal switch in the agr quorum sensing system that antagonistically regulates the differentiation of these two physiologically distinct cell types. We found that extracellular signals affect the behavior of the agr bimodal switch and modify the size of the specialized subpopulations in specific colonization niches. For instance, magnesium-enriched colonization niches causes magnesium binding to S. aureusteichoic acids and increases bacterial cell wall rigidity. This signal triggers a genetic program that ultimately downregulates the agr bimodal switch. Colonization niches with different magnesium concentrations influence the bimodal system activity, which defines a distinct ratio between these subpopulations; this in turn leads to distinct infection outcomes in vitro and in an in vivo murine infection model. Cell differentiation generates physiological heterogeneity in clonal bacterial infections and helps to determine the distinct infection types. KW - Staphylococcus aureus KW - infection KW - cell differentiation KW - pathogenic bacteria Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170346 VL - 6 IS - e28023 ER - TY - JOUR A1 - Mielich-Süss, Benjamin A1 - Wagner, Rabea M. A1 - Mietrach, Nicole A1 - Hertlein, Tobias A1 - Marincola, Gabriella A1 - Ohlsen, Knut A1 - Geibel, Sebastian A1 - Lopez, Daniel T1 - Flotillin scaffold activity contributes to type VII secretion system assembly in Staphylococcus aureus JF - PLoS Pathogens N2 - Scaffold proteins are ubiquitous chaperones that promote efficient interactions between partners of multi-enzymatic protein complexes; although they are well studied in eukaryotes, their role in prokaryotic systems is poorly understood. Bacterial membranes have functional membrane microdomains (FMM), a structure homologous to eukaryotic lipid rafts. Similar to their eukaryotic counterparts, bacterial FMM harbor a scaffold protein termed flotillin that is thought to promote interactions between proteins spatially confined to the FMM. Here we used biochemical approaches to define the scaffold activity of the flotillin homolog FloA of the human pathogen Staphylococcus aureus, using assembly of interacting protein partners of the type VII secretion system (T7SS) as a case study. Staphylococcus aureus cells that lacked FloA showed reduced T7SS function, and thus reduced secretion of T7SS-related effectors, probably due to the supporting scaffold activity of flotillin. We found that the presence of flotillin mediates intermolecular interactions of T7SS proteins. We tested several small molecules that interfere with flotillin scaffold activity, which perturbed T7SS activity in vitro and in vivo. Our results suggest that flotillin assists in the assembly of S. aureus membrane components that participate in infection and influences the infective potential of this pathogen. KW - flotillin KW - scaffold protein KW - Staphylococcus aureus KW - type VII secretion system Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170035 VL - 13 IS - 11 ER - TY - JOUR A1 - Jarick, Marcel A1 - Bertsche, Ute A1 - Stahl, Mark A1 - Schultz, Daniel A1 - Methling, Karen A1 - Lalk, Michael A1 - Stigloher, Christian A1 - Steger, Mirco A1 - Schlosser, Andreas A1 - Ohlsen, Knut T1 - The serine/threonine kinase Stk and the phosphatase Stp regulate cell wall synthesis in Staphylococcus aureus JF - Scientific Reports N2 - The cell wall synthesis pathway producing peptidoglycan is a highly coordinated and tightly regulated process. Although the major components of bacterial cell walls have been known for decades, the complex regulatory network controlling peptidoglycan synthesis and many details of the cell division machinery are not well understood. The eukaryotic-like serine/threonine kinase Stk and the cognate phosphatase Stp play an important role in cell wall biosynthesis and drug resistance in S. aureus. We show that stp deletion has a pronounced impact on cell wall synthesis. Deletion of stp leads to a thicker cell wall and decreases susceptibility to lysostaphin. Stationary phase Δstp cells accumulate peptidoglycan precursors and incorporate higher amounts of incomplete muropeptides with non-glycine, monoglycine and monoalanine interpeptide bridges into the cell wall. In line with this cell wall phenotype, we demonstrate that the lipid II:glycine glycyltransferase FemX can be phosphorylated by the Ser/Thr kinase Stk in vitro. Mass spectrometric analyses identify Thr32, Thr36 and Ser415 as phosphoacceptors. The cognate phosphatase Stp dephosphorylates these phosphorylation sites. Moreover, Stk interacts with FemA and FemB, but is unable to phosphorylate them. Our data indicate that Stk and Stp modulate cell wall synthesis and cell division at several levels. KW - bacterial transcription KW - pathogens KW - cell wall synthesis Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177333 VL - 8 IS - 13693 ER - TY - JOUR A1 - Bruchhagen, Christin A1 - Jarick, Marcel A1 - Mewis, Carolin A1 - Hertlein, Tobias A1 - Niemann, Silke A1 - Ohlsen, Knut A1 - Peters, Georg A1 - Planz, Oliver A1 - Ludwig, Stephan A1 - Ehrhardt, Christina T1 - Metabolic conversion of CI-1040 turns a cellular MEK-inhibitor into an antibacterial compound JF - Scientific Reports N2 - Influenza virus (IV) infections cause severe respiratory illnesses that can be complicated by bacterial super-infections. Previously, we identified the cellular Raf-MEK-ERK cascade as a promising antiviral target. Inhibitors of MEK, such as CI-1040, showed potent antiviral activity. However, it remained unclear if this inhibitor and its active form, ATR-002, might sensitize host cells to either IV or secondary bacterial infections. To address these questions, we studied the anti-pathogen activity of ATR-002 in comparison to CI-1040, particularly, its impact on Staphylococcus aureus (S. aureus), which is a major cause of IV super-infections. We analysed IV and S. aureus titres in vitro during super-infection in the presence and absence of the drugs and characterized the direct impact of ATR-002 on bacterial growth and phenotypic changes. Importantly, neither CI-1040 nor ATR-002 treatment led to increased bacterial titres during super-infection, indicating that the drug does not sensitize cells for bacterial infection. In contrast, we rather observed reduced bacterial titres in presence of ATR-002. Surprisingly, ATR-002 also led to reduced bacterial growth in suspension cultures, reduced stress- and antibiotic tolerance without resistance induction. Our data identified for the first time that a particular MEK-inhibitor metabolite exhibits direct antibacterial activity, which is likely due to interference with the bacterial PknB kinase/Stp phosphatase signalling system. KW - antimicrobials KW - pathogens Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-221648 VL - 8 ER - TY - JOUR A1 - Seethaler, Marius A1 - Hertlein, Tobias A1 - Wecklein, Björn A1 - Ymeraj, Alba A1 - Ohlsen, Knut A1 - Lalk, Michael A1 - Hilgeroth, Andreas T1 - Novel small-molecule antibacterials against Gram-positive pathogens of Staphylococcus and Enterococcus species JF - Antibiotics N2 - Defeat of the antibiotic resistance of pathogenic bacteria is one great challenge today and for the future. In the last century many classes of effective antibacterials have been developed, so that upcoming resistances could be met with novel drugs of various compound classes. Meanwhile, there is a certain lack of research of the pharmaceutical companies, and thus there are missing developments of novel antibiotics. Gram-positive bacteria are the most important cause of clinical infections. The number of novel antibacterials in clinical trials is strongly restricted. There is an urgent need to find novel antibacterials. We used synthetic chemistry to build completely novel hybrid molecules of substituted indoles and benzothiophene. In a simple one-pot reaction, two novel types of thienocarbazoles were yielded. Both indole substituted compound classes have been evaluated as completely novel antibacterials against the Staphylococcus and Enterococcus species. The evaluated partly promising activities depend on the indole substituent type. First lead compounds have been evaluated within in vivo studies. They confirmed the in vitro results for the new classes of small-molecule antibacterials. KW - antibacterial activity KW - synthesis KW - substituent KW - structure-activity KW - inhibition Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193130 SN - 2079-6382 VL - 8 IS - 4 ER - TY - JOUR A1 - Gomes, Sara F. Martins A1 - Westermann, Alexander J. A1 - Sauerwein, Till A1 - Hertlein, Tobias A1 - Förstner, Konrad U. A1 - Ohlsen, Knut A1 - Metzger, Marco A1 - Shusta, Eric V. A1 - Kim, Brandon J. A1 - Appelt-Menzel, Antje A1 - Schubert-Unkmeir, Alexandra T1 - Induced pluripotent stem cell-derived brain endothelial cells as a cellular model to study Neisseria meningitidis infection JF - Frontiers in Microbiology N2 - Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs. KW - Neisseria meningitidis KW - meningococcus KW - bacteria KW - stem cells KW - blood-cerebrospinal fluid barrier KW - blood-brain barrier KW - brain endothelial cells Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201562 VL - 10 IS - 1181 ER - TY - JOUR A1 - Mühlberg, Eric A1 - Umstätter, Florian A1 - Domhan, Cornelius A1 - Hertlein, Tobias A1 - Ohlsen, Knut A1 - Krause, Andreas A1 - Kleist, Christian A1 - Beijer, Barbro A1 - Zimmermann, Stefan A1 - Haberkorn, Uwe A1 - Mier, Walter A1 - Uhl, Philipp T1 - Vancomycin-lipopeptide conjugates with high antimicrobial activity on vancomycin-resistant enterococci JF - Pharmaceuticals N2 - Multidrug-resistant bacteria represent one of the most important health care problems worldwide. While there are numerous drugs available for standard therapy, there are only a few compounds capable of serving as a last resort for severe infections. Therefore, approaches to control multidrug-resistant bacteria must be implemented. Here, a strategy of reactivating the established glycopeptide antibiotic vancomycin by structural modification with polycationic peptides and subsequent fatty acid conjugation to overcome the resistance of multidrug-resistant bacteria was followed. This study especially focuses on the structure–activity relationship, depending on the modification site and fatty acid chain length. The synthesized conjugates showed high antimicrobial potential on vancomycin-resistant enterococci. We were able to demonstrate that the antimicrobial activity of the vancomycin-lipopeptide conjugates depends on the chain length of the attached fatty acid. All conjugates showed good cytocompatibility in vitro and in vivo. Radiolabeling enabled the in vivo determination of pharmacokinetics in Wistar rats by molecular imaging and biodistribution studies. An improved biodistribution profile in comparison to unmodified vancomycin was observed. While vancomycin is rapidly excreted by the kidneys, the most potent conjugate shows a hepatobiliary excretion profile. In conclusion, these results demonstrate the potential of the structural modification of already established antibiotics to provide highly active compounds for tackling multidrug-resistant bacteria. KW - antibiotics KW - multidrug-resistant bacteria KW - enterococci KW - vancomycin KW - structural modification KW - fatty acids KW - polycationic peptides Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-205879 SN - 1424-8247 VL - 13 IS - 6 ER - TY - JOUR A1 - Umstätter, Florian A1 - Domhan, Cornelius A1 - Hertlein, Tobias A1 - Ohlsen, Knut A1 - Mühlberg, Eric A1 - Kleist, Christian A1 - Zimmermann, Stefan A1 - Beijer, Barbro A1 - Klika, Karel D. A1 - Haberkorn, Uwe A1 - Mier, Walter A1 - Uhl, Philipp T1 - Vancomycin Resistance Is Overcome by Conjugation of Polycationic Peptides JF - Angewandte Chemie International Edition N2 - Multidrug‐resistant bacteria represent one of the biggest challenges facing modern medicine. The increasing prevalence of glycopeptide resistance compromises the efficacy of vancomycin, for a long time considered as the last resort for the treatment of resistant bacteria. To reestablish its activity, polycationic peptides were conjugated to vancomycin. By site‐specific conjugation, derivatives that bear the peptide moiety at four different sites of the antibiotic were synthesized. The most potent compounds exhibited an approximately 1000‐fold increased antimicrobial activity and were able to overcome the most important types of vancomycin resistance. Additional blocking experiments using d‐Ala‐d‐Ala revealed a mode of action beyond inhibition of cell‐wall formation. The antimicrobial potential of the lead candidate FU002 for bacterial infection treatments could be demonstrated in an in vivo study. Molecular imaging and biodistribution studies revealed that conjugation engenders superior pharmacokinetics. KW - antibiotics KW - bacterial resistance KW - glycopeptide antibiotics KW - peptide conjugates KW - vancomycin Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-215550 VL - 59 IS - 23 SP - 8823 EP - 8827 ER - TY - JOUR A1 - Liang, Chunguang A1 - Rios-Miguel, Ana B. A1 - Jarick, Marcel A1 - Neurgaonkar, Priya A1 - Girard, Myriam A1 - François, Patrice A1 - Schrenzel, Jacques A1 - Ibrahim, Eslam S. A1 - Ohlsen, Knut A1 - Dandekar, Thomas T1 - Staphylococcus aureus transcriptome data and metabolic modelling investigate the interplay of Ser/Thr kinase PknB, its phosphatase Stp, the glmR/yvcK regulon and the cdaA operon for metabolic adaptation JF - Microorganisms N2 - Serine/threonine kinase PknB and its corresponding phosphatase Stp are important regulators of many cell functions in the pathogen S. aureus. Genome-scale gene expression data of S. aureus strain NewHG (sigB\(^+\)) elucidated their effect on physiological functions. Moreover, metabolic modelling from these data inferred metabolic adaptations. We compared wild-type to deletion strains lacking pknB, stp or both. Ser/Thr phosphorylation of target proteins by PknB switched amino acid catabolism off and gluconeogenesis on to provide the cell with sufficient components. We revealed a significant impact of PknB and Stp on peptidoglycan, nucleotide and aromatic amino acid synthesis, as well as catabolism involving aspartate transaminase. Moreover, pyrimidine synthesis was dramatically impaired by stp deletion but only slightly by functional loss of PknB. In double knockouts, higher activity concerned genes involved in peptidoglycan, purine and aromatic amino acid synthesis from glucose but lower activity of pyrimidine synthesis from glucose compared to the wild type. A second transcriptome dataset from S. aureus NCTC 8325 (sigB\(^−\)) validated the predictions. For this metabolic adaptation, PknB was found to interact with CdaA and the yvcK/glmR regulon. The involved GlmR structure and the GlmS riboswitch were modelled. Furthermore, PknB phosphorylation lowered the expression of many virulence factors, and the study shed light on S. aureus infection processes. KW - metabolism KW - flux balance analysis KW - phosphorylation KW - regulation KW - riboswitch KW - PknB KW - Stp KW - yvcK/glmR operon Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-248459 SN - 2076-2607 VL - 9 IS - 10 ER - TY - JOUR A1 - Masota, Nelson E. A1 - Vogg, Gerd A1 - Ohlsen, Knut A1 - Holzgrabe, Ulrike T1 - Reproducibility challenges in the search for antibacterial compounds from nature JF - PLoS One N2 - Background Reproducibility of reported antibacterial activities of plant extracts has long remained questionable. Although plant-related factors should be well considered in serious pharmacognostic research, they are often not addressed in many research papers. Here we highlight the challenges in reproducing antibacterial activities of plant extracts. Methods Plants with reported antibacterial activities of interest were obtained from a literature review. Antibacterial activities against Escherichia coli and Klebsiella pneumoniae were tested using extracts’ solutions in 10% DMSO and acetone. Compositions of working solutions from both solvents were established using LC-MS analysis. Moreover, the availability of details likely to affect reproducibility was evaluated in articles which reported antibacterial activities of studied plants. Results Inhibition of bacterial growth at MIC of 256–1024 μg/mL was observed in only 15.4% of identical plant species. These values were 4–16-fold higher than those reported earlier. Further, 18.2% of related plant species had MICs of 128–256 μg/mL. Besides, 29.2% and 95.8% of the extracts were soluble to sparingly soluble in 10% DMSO and acetone, respectively. Extracts’ solutions in both solvents showed similar qualitative compositions, with differing quantities of corresponding phytochemicals. Details regarding seasons and growth state at collection were missing in 65% and 95% of evaluated articles, respectively. Likewise, solvents used to dissolve the extracts were lacking in 30% of the articles, whereas 40% of them used unidentified bacterial isolates. Conclusion Reproducibility of previously reported activities from plants’ extracts is a multi-factorial aspect. Thus, collective approaches are necessary in addressing the highlighted challenges. KW - acetones KW - antibacterials KW - leaves KW - phytochemicals KW - solubility KW - plants KW - liquid chromatography-mass spectrometry KW - ethanol Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-260239 VL - 16 IS - 7 ER - TY - JOUR A1 - Gehrmann, Robin A1 - Hertlein, Tobias A1 - Hopke, Elisa A1 - Ohlsen, Knut A1 - Lalk, Michael A1 - Hilgeroth, Andreas T1 - Novel small-molecule hybrid-antibacterial agents against S. aureus and MRSA strains JF - Molecules N2 - Ongoing resistance developments against antibiotics that also affect last-resort antibiotics require novel antibacterial compounds. Strategies to discover such novel structures have been dimerization or hybridization of known antibacterial agents. We found novel antibacterial agents by dimerization of indols and hybridization with carbazoles. They were obtained in a simple one-pot reaction as bisindole tetrahydrocarbazoles. Further oxidation led to bisindole carbazoles with varied substitutions of both the indole and the carbazole scaffold. Both the tetrahydrocarbazoles and the carbazoles have been evaluated in various S. aureus strains, including MRSA strains. Those 5-cyano substituted derivatives showed best activities as determined by MIC values. The tetrahydrocarbazoles partly exceed the activity of the carbazole compounds and thus the activity of the used standard antibiotics. Thus, promising lead compounds could be identified for further studies. KW - antibacterial activity KW - synthesis KW - substituent KW - structure–activity KW - inhibition Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-252371 SN - 1420-3049 VL - 27 IS - 1 ER - TY - JOUR A1 - Stelzner, Kathrin A1 - Boyny, Aziza A1 - Hertlein, Tobias A1 - Sroka, Aneta A1 - Moldovan, Adriana A1 - Paprotka, Kerstin A1 - Kessie, David A1 - Mehling, Helene A1 - Potempa, Jan A1 - Ohlsen, Knut A1 - Fraunholz, Martin J. A1 - Rudel, Thomas T1 - Intracellular Staphylococcus aureus employs the cysteine protease staphopain A to induce host cell death in epithelial cells JF - PLoS Pathogens N2 - Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Uptake by host cells is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death after translocation of intracellular S. aureus into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication of S. aureus in epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. In phagocytic cells, where intracellular S. aureus is exclusively localized in the phagosome, staphopain A did not contribute to cytotoxicity. Our study suggests that staphopain A is utilized by S. aureus to exit the epithelial host cell and thus contributes to tissue destruction and dissemination of infection. Author summary Staphylococcus aureus is an antibiotic-resistant pathogen that emerges in hospital and community settings and can cause a variety of diseases ranging from skin abscesses to lung inflammation and blood poisoning. The bacterium can asymptomatically colonize the upper respiratory tract and skin of humans and take advantage of opportune conditions, like immunodeficiency or breached barriers, to cause infection. Although S. aureus was not regarded as intracellular bacterium, it can be internalized by human cells and subsequently exit the host cells by induction of cell death, which is considered to cause tissue destruction and spread of infection. The bacterial virulence factors and underlying molecular mechanisms involved in the intracellular lifestyle of S. aureus remain largely unknown. We identified a bacterial cysteine protease to contribute to host cell death of epithelial cells mediated by intracellular S. aureus. Staphopain A induced killing of the host cell after translocation of the pathogen into the cell cytosol, while bacterial proliferation was not required. Further, the protease enhanced survival of the pathogen during lung infection. These findings reveal a novel, intracellular role for the bacterial protease staphopain A. KW - Staphylococcus aureus KW - Staphylococcal infection KW - host cells KW - HeLa cells KW - cytotoxicity KW - intracellular pathogens KW - apoptosis KW - epithelial cells Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-263908 VL - 17 IS - 9 ER - TY - JOUR A1 - Umstätter, Florian A1 - Werner, Julia A1 - Zerlin, Leah A1 - Mühlberg, Eric A1 - Kleist, Christian A1 - Klika, Karel D. A1 - Hertlein, Tobias A1 - Beijer, Barbro A1 - Domhan, Cornelius A1 - Zimmermann, Stefan A1 - Ohlsen, Knut A1 - Haberkorn, Uwe A1 - Mier, Walter A1 - Uhl, Philipp T1 - Impact of linker modification and PEGylation of vancomycin conjugates on structure-activity relationships and pharmacokinetics JF - Pharmaceuticals N2 - As multidrug-resistant bacteria represent a concerning burden, experts insist on the need for a dramatic rethinking on antibiotic use and development in order to avoid a post-antibiotic era. New and rapidly developable strategies for antimicrobial substances, in particular substances highly potent against multidrug-resistant bacteria, are urgently required. Some of the treatment options currently available for multidrug-resistant bacteria are considerably limited by side effects and unfavorable pharmacokinetics. The glycopeptide vancomycin is considered an antibiotic of last resort. Its use is challenged by bacterial strains exhibiting various types of resistance. Therefore, in this study, highly active polycationic peptide-vancomycin conjugates with varying linker characteristics or the addition of PEG moieties were synthesized to optimize pharmacokinetics while retaining or even increasing antimicrobial activity in comparison to vancomycin. The antimicrobial activity of the novel conjugates was determined by microdilution assays on susceptible and vancomycin-resistant bacterial strains. VAN1 and VAN2, the most promising linker-modified derivatives, were further characterized in vivo with molecular imaging and biodistribution studies in rodents, showing that the linker moiety influences both antimicrobial activity and pharmacokinetics. Encouragingly, VAN2 was able to undercut the resistance breakpoint in microdilution assays on vanB and vanC vancomycin-resistant enterococci. Out of all PEGylated derivatives, VAN:PEG1 and VAN:PEG3 were able to overcome vanC resistance. Biodistribution studies of the novel derivatives revealed significant changes in pharmacokinetics when compared with vancomycin. In conclusion, linker modification of vancomycin-polycationic peptide conjugates represents a promising strategy for the modulation of pharmacokinetic behavior while providing potent antimicrobial activity. KW - glycopeptide antibiotics KW - antimicrobial resistance KW - vancomycin KW - polycationic peptides KW - linker influence KW - pharmacokinetics KW - PEGylation Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-255197 SN - 1424-8247 VL - 15 IS - 2 ER - TY - JOUR A1 - Ibrahim, Eslam S. A1 - Ohlsen, Knut T1 - The old yellow enzyme OfrA fosters Staphylococcus aureus survival via affecting thiol-dependent redox homeostasis JF - Frontiers in Microbiology N2 - Old yellow enzymes (OYEs) are widely found in the bacterial, fungal, and plant kingdoms but absent in humans and have been used as biocatalysts for decades. However, OYEs’ physiological function in bacterial stress response and infection situations remained enigmatic. As a pathogen, the Gram-positive bacterium Staphylococcus aureus adapts to numerous stress conditions during pathogenesis. Here, we show that in S. aureus genome, two paralogous genes (ofrA and ofrB) encode for two OYEs. We conducted a bioinformatic analysis and found that ofrA is conserved among all publicly available representative staphylococcal genomes and some Firmicutes. Expression of ofrA is induced by electrophilic, oxidative, and hypochlorite stress in S. aureus. Furthermore, ofrA contributes to S. aureus survival against reactive electrophilic, oxygen, and chlorine species (RES, ROS, and RCS) via thiol-dependent redox homeostasis. At the host–pathogen interface, S. aureusΔofrA has defective survival in macrophages and whole human blood and decreased staphyloxanthin production. Overall, our results shed the light onto a novel stress response strategy in the important human pathogen S. aureus. KW - MRSA KW - blood KW - phagocytes KW - quinone KW - ROS KW - stress response KW - electrophilic stress Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-274381 SN - 1664-302X VL - 13 ER - TY - JOUR A1 - Hung, Sophia A1 - Dreher, Liane A1 - Diessner, Joachim A1 - Schwarz, Stefan A1 - Ohlsen, Knut A1 - Hertlein, Tobias T1 - MRSA infection in the thigh muscle leads to systemic disease, strong inflammation, and loss of human monocytes in humanized mice JF - Frontiers in Immunology N2 - MRSA (Methicillin-resistant Staphylococcus aureus) is the second-leading cause of deaths by antibiotic-resistant bacteria globally, with more than 100,000 attributable deaths annually. Despite the high urgency to develop a vaccine to control this pathogen, all clinical trials with pre-clinically effective candidates failed so far. The recent development of “humanized” mice might help to edge the pre-clinical evaluation closer to the clinical situation and thus close this gap. We infected humanized NSG mice (huNSG: (NOD)-scid IL2R\(_γ\)\(^{null}\) mice engrafted with human CD34+ hematopoietic stem cells) locally with S. aureus USA300 LAC* lux into the thigh muscle in order to investigate the human immune response to acute and chronic infection. These mice proved not only to be more susceptible to MRSA infection than wild-type or “murinized” mice, but displayed furthermore inferior survival and signs of systemic infection in an otherwise localized infection model. The rate of humanization correlated directly with the severity of disease and survival of the mice. Human and murine cytokine levels in blood and at the primary site of infection were strongly elevated in huNSG mice compared to all control groups. And importantly, differences in human and murine immune cell lineages surfaced during the infection, with human monocyte and B cell numbers in blood and bone marrow being significantly reduced at the later time point of infection. Murine monocytes in contrast behaved conversely by increasing cell numbers. This study demonstrates significant differences in the in vivo behavior of human and murine cells towards S. aureus infection, which might help to sharpen the translational potential of pre-clinical models for future therapeutic approaches. KW - humanized mice KW - MRSA - methicillin-resistant Staphylococcus aureus KW - monocyte KW - bacterial infection model KW - inflammation KW - NSG KW - staphylocccal infection/epidemiology Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-278050 SN - 1664-3224 VL - 13 ER - TY - JOUR A1 - Masota, Nelson E. A1 - Ohlsen, Knut A1 - Schollmayer, Curd A1 - Meinel, Lorenz A1 - Holzgrabe, Ulrike T1 - Isolation and characterization of galloylglucoses effective against multidrug-resistant strains of Escherichia coli and Klebsiella pneumoniae JF - Molecules N2 - The search for new antibiotics against multidrug-resistant (MDR), Gram-negative bacteria is crucial with respect to filling the antibiotics development pipeline, which is subject to a critical shortage of novel molecules. Screening of natural products is a promising approach for identifying antimicrobial compounds hosting a higher degree of novelty. Here, we report the isolation and characterization of four galloylglucoses active against different MDR strains of Escherichia coli and Klebsiella pneumoniae. A crude acetone extract was prepared from Paeonia officinalis Linnaeus leaves, and bioautography-guided isolation of active compounds from the extract was performed by liquid–liquid extraction, as well as open column, flash, and preparative chromatographic methods. Isolated active compounds were characterized and elucidated by a combination of spectroscopic and spectrometric techniques. In vitro antimicrobial susceptibility testing was carried out on E. coli and K. pneumoniae using 2 reference strains and 13 strains hosting a wide range of MDR phenotypes. Furthermore, in vivo antibacterial activities were assessed using Galleria mellonella larvae, and compounds 1,2,3,4,6-penta-O-galloyl-β-d-glucose, 3-O-digalloyl-1,2,4,6-tetra-O-galloyl-β-d-glucose, 6-O-digalloyl-1,2,3,4-tetra-O-galloyl-β-d-glucose, and 3,6-bis-O-digalloyl-1,2,4-tri-O-galloyl-β-d-glucose were isolated and characterized. They showed minimum inhibitory concentration (MIC) values in the range of 2–256 µg/mL across tested bacterial strains. These findings have added to the number of known galloylglucoses from P. officinalis and highlight their potential against MDR Gram-negative bacteria. KW - antimicrobial resistance KW - Enterobacteriaceae KW - Paeonia KW - gallotannins KW - isolation KW - structural elucidation KW - Escherichia coli KW - Klebsiella pneumoniae Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-286179 SN - 1420-3049 VL - 27 IS - 15 ER - TY - JOUR A1 - Seethaler, Marius A1 - Hertlein, Tobias A1 - Hopke, Elisa A1 - Köhling, Paul A1 - Ohlsen, Knut A1 - Lalk, Michael A1 - Hilgeroth, Andreas T1 - Novel effective fluorinated benzothiophene-indole hybrid antibacterials against S. aureus and MRSA strains JF - Pharmaceuticals N2 - Increasing antibacterial drug resistance threatens global health, unfortunately, however, efforts to find novel antibacterial agents have been scaled back by the pharmaceutical industry due to concerns about a poor return on investment. Nevertheless, there is an urgent need to find novel antibacterial compounds to combat antibacterial drug resistance. The synthesis of novel drugs from natural sources is mostly cost-intensive due to those drugs’ complicated structures. Therefore, it is necessary to find novel antibacterials by simple synthesis to become more attractive for industrial production. We succeeded in the discovery of four antibacterial compound (sub)classes accessible in a simple one-pot reaction based on fluorinated benzothiophene-indole hybrids. They have been evaluated against various S. aureus and MRSA strains. Structure- and substituent-dependent activities have been found within the (sub)classes and promising lead compounds have been identified. In addition, bacterial pyruvate kinase was found to be the molecular target of the active compounds. In conclusion, simple one-pot synthesis of benzothiophene-indoles represents a promising strategy for the search of novel antimicrobial compounds. KW - antibacterial drug resistance KW - structure activity KW - synthesis KW - inhibition KW - substituent Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-288253 SN - 1424-8247 VL - 15 IS - 9 ER - TY - JOUR A1 - Hung, Sophia A1 - Kasperkowitz, Amelie A1 - Kurz, Florian A1 - Dreher, Liane A1 - Diessner, Joachim A1 - Ibrahim, Eslam S. A1 - Schwarz, Stefan A1 - Ohlsen, Knut A1 - Hertlein, Tobias T1 - Next-generation humanized NSG-SGM3 mice are highly susceptible to Staphylococcus aureus infection JF - Frontiers in Immunology N2 - Humanized hemato-lymphoid system mice, or humanized mice, emerged in recent years as a promising model to study the course of infection of human-adapted or human-specific pathogens. Though Staphylococcus aureus infects and colonizes a variety of species, it has nonetheless become one of the most successful human pathogens of our time with a wide armory of human-adapted virulence factors. Humanized mice showed increased vulnerability to S. aureus compared to wild type mice in a variety of clinically relevant disease models. Most of these studies employed humanized NSG (NOD-scid IL2Rgnull) mice which are widely used in the scientific community, but show poor human myeloid cell reconstitution. Since this immune cell compartment plays a decisive role in the defense of the human immune system against S. aureus, we asked whether next-generation humanized mice, like NSG-SGM3 (NOD-scid IL2Rgnull-3/GM/SF) with improved myeloid reconstitution, would prove to be more resistant to infection. To our surprise, we found the contrary when we infected humanized NSG-SGM3 (huSGM3) mice with S. aureus: although they had stronger human immune cell engraftment than humanized NSG mice, particularly in the myeloid compartment, they displayed even more pronounced vulnerability to S. aureus infection. HuSGM3 mice had overall higher numbers of human T cells, B cells, neutrophils and monocytes in the blood and the spleen. This was accompanied by elevated levels of pro-inflammatory human cytokines in the blood of huSGM3 mice. We further identified that the impaired survival of huSGM3 mice was not linked to higher bacterial burden nor to differences in the murine immune cell repertoire. Conversely, we could demonstrate a correlation of the rate of humanization and the severity of infection. Collectively, this study suggests a detrimental effect of the human immune system in humanized mice upon encounter with S. aureus which might help to guide future therapy approaches and analysis of virulence mechanisms. KW - humanized mice KW - Staphylococcus aureus KW - MRSA KW - NSG KW - NSG-SGM3 KW - staphylococcal abscess KW - Staphylococcus aureus immune response KW - humanized hemato-lymphoid mice Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-306966 VL - 14 ER -