@article{HungKasperkowitzKurzetal.2023, author = {Hung, Sophia and Kasperkowitz, Amelie and Kurz, Florian and Dreher, Liane and Diessner, Joachim and Ibrahim, Eslam S. and Schwarz, Stefan and Ohlsen, Knut and Hertlein, Tobias}, title = {Next-generation humanized NSG-SGM3 mice are highly susceptible to Staphylococcus aureus infection}, series = {Frontiers in Immunology}, volume = {14}, journal = {Frontiers in Immunology}, doi = {10.3389/fimmu.2023.1127709}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-306966}, year = {2023}, abstract = {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.}, language = {en} } @article{BruchhagenJarickMewisetal.2018, author = {Bruchhagen, Christin and Jarick, Marcel and Mewis, Carolin and Hertlein, Tobias and Niemann, Silke and Ohlsen, Knut and Peters, Georg and Planz, Oliver and Ludwig, Stephan and Ehrhardt, Christina}, title = {Metabolic conversion of CI-1040 turns a cellular MEK-inhibitor into an antibacterial compound}, series = {Scientific Reports}, volume = {8}, journal = {Scientific Reports}, doi = {10.1038/s41598-018-27445-7}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-221648}, year = {2018}, abstract = {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.}, language = {en} } @article{SeethalerHertleinHopkeetal.2022, author = {Seethaler, Marius and Hertlein, Tobias and Hopke, Elisa and K{\"o}hling, Paul and Ohlsen, Knut and Lalk, Michael and Hilgeroth, Andreas}, title = {Novel effective fluorinated benzothiophene-indole hybrid antibacterials against S. aureus and MRSA strains}, series = {Pharmaceuticals}, volume = {15}, journal = {Pharmaceuticals}, number = {9}, issn = {1424-8247}, doi = {10.3390/ph15091138}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-288253}, year = {2022}, abstract = {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.}, language = {en} } @article{MasotaOhlsenSchollmayeretal.2022, author = {Masota, Nelson E. and Ohlsen, Knut and Schollmayer, Curd and Meinel, Lorenz and Holzgrabe, Ulrike}, title = {Isolation and characterization of galloylglucoses effective against multidrug-resistant strains of Escherichia coli and Klebsiella pneumoniae}, series = {Molecules}, volume = {27}, journal = {Molecules}, number = {15}, issn = {1420-3049}, doi = {10.3390/molecules27155045}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-286179}, year = {2022}, abstract = {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.}, language = {en} } @article{UmstaetterWernerZerlinetal.2022, author = {Umst{\"a}tter, Florian and Werner, Julia and Zerlin, Leah and M{\"u}hlberg, Eric and Kleist, Christian and Klika, Karel D. and Hertlein, Tobias and Beijer, Barbro and Domhan, Cornelius and Zimmermann, Stefan and Ohlsen, Knut and Haberkorn, Uwe and Mier, Walter and Uhl, Philipp}, title = {Impact of linker modification and PEGylation of vancomycin conjugates on structure-activity relationships and pharmacokinetics}, series = {Pharmaceuticals}, volume = {15}, journal = {Pharmaceuticals}, number = {2}, issn = {1424-8247}, doi = {10.3390/ph15020159}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-255197}, year = {2022}, abstract = {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.}, language = {en} } @article{LiangRiosMiguelJaricketal.2021, author = {Liang, Chunguang and Rios-Miguel, Ana B. and Jarick, Marcel and Neurgaonkar, Priya and Girard, Myriam and Fran{\c{c}}ois, Patrice and Schrenzel, Jacques and Ibrahim, Eslam S. and Ohlsen, Knut and Dandekar, Thomas}, title = {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}, series = {Microorganisms}, volume = {9}, journal = {Microorganisms}, number = {10}, issn = {2076-2607}, doi = {10.3390/microorganisms9102148}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-248459}, year = {2021}, abstract = {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.}, language = {en} } @article{HungDreherDiessneretal.2022, author = {Hung, Sophia and Dreher, Liane and Diessner, Joachim and Schwarz, Stefan and Ohlsen, Knut and Hertlein, Tobias}, title = {MRSA infection in the thigh muscle leads to systemic disease, strong inflammation, and loss of human monocytes in humanized mice}, series = {Frontiers in Immunology}, volume = {13}, journal = {Frontiers in Immunology}, issn = {1664-3224}, doi = {10.3389/fimmu.2022.892053}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-278050}, year = {2022}, abstract = {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.}, language = {en} } @article{IbrahimOhlsen2022, author = {Ibrahim, Eslam S. and Ohlsen, Knut}, title = {The old yellow enzyme OfrA fosters Staphylococcus aureus survival via affecting thiol-dependent redox homeostasis}, series = {Frontiers in Microbiology}, volume = {13}, journal = {Frontiers in Microbiology}, issn = {1664-302X}, doi = {10.3389/fmicb.2022.888140}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-274381}, year = {2022}, abstract = {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.}, language = {en} } @article{GehrmannHertleinHopkeetal.2021, author = {Gehrmann, Robin and Hertlein, Tobias and Hopke, Elisa and Ohlsen, Knut and Lalk, Michael and Hilgeroth, Andreas}, title = {Novel small-molecule hybrid-antibacterial agents against S. aureus and MRSA strains}, series = {Molecules}, volume = {27}, journal = {Molecules}, number = {1}, issn = {1420-3049}, doi = {10.3390/molecules27010061}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-252371}, year = {2021}, abstract = {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.}, language = {en} } @article{MuehlbergUmstaetterDomhanetal.2020, author = {M{\"u}hlberg, Eric and Umst{\"a}tter, Florian and Domhan, Cornelius and Hertlein, Tobias and Ohlsen, Knut and Krause, Andreas and Kleist, Christian and Beijer, Barbro and Zimmermann, Stefan and Haberkorn, Uwe and Mier, Walter and Uhl, Philipp}, title = {Vancomycin-lipopeptide conjugates with high antimicrobial activity on vancomycin-resistant enterococci}, series = {Pharmaceuticals}, volume = {13}, journal = {Pharmaceuticals}, number = {6}, issn = {1424-8247}, doi = {10.3390/ph13060110}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-205879}, year = {2020}, abstract = {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.}, language = {en} }