• A Listeria monocytogenes ST2 clone lacking chitinase ChiB from an outbreak of non-invasive gastroenteritis.

      Halbedel, Sven; Prager, Rita; Banerji, Sangeeta; Kleta, Sylvia; Trost, Eva; Nishanth, Gopala; Alles, Georg; Hölzel, Christina; Schlesiger, Friederike; Pietzka, Ariane; et al. (Springer Nature, 2019-01-01)
      An outbreak with a remarkable Listeria monocytogenes clone causing 163 cases of non-invasive listeriosis occurred in Germany in 2015. Core genome multi locus sequence typing grouped non-invasive outbreak isolates and isolates obtained from related food samples into a single cluster, but clearly separated genetically close isolates obtained from invasive listeriosis cases. A comparative genomic approach identified a premature stop codon in the chiB gene, encoding one of the two L. monocytogenes chitinases, which clustered with disease outcome. Correction of this premature stop codon in one representative gastroenteritis outbreak isolate restored chitinase production, but effects in infection experiments were not found. While the exact role of chitinases in virulence of L. monocytogenes is still not fully understood, our results now clearly show that ChiB-derived activity is not required to establish L. monocytogenes gastroenteritis in humans. This limits a possible role of ChiB in human listeriosis to later steps of the infection.
    • Macrophage entrapped silica coated superparamagnetic iron oxide particles for controlled drug release in a 3D cancer model.

      Ullah, Sami; Seidel, Katja; Türkkan, Sibel; Warwas, Dawid Peter; Dubich, Tatyana; Rohde, Manfred; Hauser, Hansjörg; Behrens, Peter; Kirschning, Andreas; Köster, Mario; et al. (2018-12-23)
      Targeted delivery of drugs is a major challenge in treatment of diverse diseases. Systemically administered drugs demand high doses and are accompanied by poor selectivity and side effects on non-target cells. Here, we introduce a new principle for targeted drug delivery. It is based on macrophages as transporters for nanoparticle-coupled drugs as well as controlled release of drugs by hyperthermia mediated disruption of the cargo cells and simultaneous deliberation of nanoparticle-linked drugs. Hyperthermia is induced by an alternating electromagnetic field (AMF) that induces heat from silica-coated superparamagnetic iron oxide nanoparticles (SPIONs). We show proof-of-principle of controlled release by the simultaneous disruption of the cargo cells and the controlled, AMF induced release of a toxin, which was covalently linked to silica-coated SPIONs via a thermo-sensitive linker. Cells that had not been loaded with SPIONs remain unaffected. Moreover, in a 3D co-culture model we demonstrate specific killing of associated tumour cells when employing a ratio as low as 1:40 (SPION-loaded macrophage: tumour cells). Overall, our results demonstrate that AMF induced drug release from macrophage-entrapped nanoparticles is tightly controlled and may be an attractive novel strategy for targeted drug release.
    • MAIT cells are enriched and highly functional in ascites of patients with decompensated liver cirrhosis.

      Niehaus, Christian E; Strunz, Benedikt; Cornillet, Martin; Falk, Christine S; Schnieders, Ansgar; Maasoumy, Benjamin; Hardtke, Svenja; Manns, Michael P; Rm Kraft, Anke; Björkström, Niklas K; et al. (Wiley Online Open, 2020-02-03)
      Patients with advanced liver cirrhosis have an increased susceptibility to infections. As part of the cirrhosis-associated immune dysfunction, mucosal associated invariant T (MAIT) cells, that have the capacity to respond towards bacteria, are severely diminished in circulation and liver tissue. However, MAIT cell presence and function in the peritoneal cavity, a common anatomical site for infections in cirrhosis, remain elusive. To study this, matched peripheral blood and ascites fluid were collected from 35 patients with decompensated cirrhosis, with or without spontaneous bacterial peritonitis (SBP). MAIT cell phenotype and function were analyzed using high-dimensional flow cytometry and obtained data was compared to blood samples of healthy controls (n=24) and patients with compensated cirrhosis (n=11). We found circulating MAIT cells to be severely decreased in cirrhotic patients as compared to controls. In contrast, in ascites fluid, MAIT cells were significantly increased together with CD14+ CD16+ monocytes, ILCs, and NK cells. This was paralleled by elevated levels of several pro-inflammatory cytokines and chemokines in ascites fluid as compared to plasma. Peritoneal MAIT cells displayed an activated tissue-resident phenotype and this was corroborated by increased functional responses following stimulation with E. coli or lL-12 + IL-18 as compared to circulating MAIT cells. During SBP, peritoneal MAIT cell frequencies increased most among all major immune cell subsets, suggestive of active homing of MAIT cells to the site of infection. CONCLUSIONS: Despite severely diminished MAIT cell numbers and impaired phenotype in circulation, peritoneal MAIT cells remain abundant, activated, and highly functional in decompensated cirrhosis and are further enriched in SBP. This suggests that peritoneal MAIT cells could be of interest for immune intervention strategies in patients with decompensated liver cirrhosis and SBP.
    • Metagenome Survey of Biofilms in Drinking-Water Networks

      Schmeisser, C.; Stöckigt, C.; Raasch, C.; Wingender, J.; Timmis, K. N.; Wenderoth, D. F.; Flemming, H.-C.; Liesegang, H.; Schmitz, R. A.; Jaeger, K.-E.; et al. (American Society for Microbiology, 2003-12)
    • Microbial β-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail

      Del Pozo, Mercedes V; Fernández-Arrojo, Lucía; Gil-Martínez, Jorge; Montesinos, Alejandro; Chernikova, Tatyana N; Nechitaylo, Taras Y; Waliszek, Agnes; Tortajada, Marta; Rojas, Antonia; Huws, Sharon A; et al. (2012-09-21)
      Abstract Background A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product. Results In the present work, we have identified and then successfully cloned, expressed, purified and characterised 4 highly active beta-glucosidases from fibre-adherent microbial community from the cow rumen. The enzymes were most active at temperatures 45–55°C and pH 4.0-7.0 and exhibited high affinity and activity towards synthetic substrates such as p-nitrophenyl-beta-D-glucopyranoside (pNPbetaG) and pNP-beta-cellobiose, as well as to natural cello-oligosaccharides ranging from cellobiose to cellopentaose. The apparent capability of the most active beta-glucosidase, herein named LAB25g2, was tested for its ability to improve, at low dosage (31.25 units g-1 dry biomass, using pNPbetaG as substrate), the hydrolysis of pre-treated corn stover (dry matter content of 20%; 350 g glucan kg-1 dry biomass) in combination with a beta-glucosidase-deficient commercial Trichoderma reseei cellulase cocktail (5 units g-1 dry biomass in the basis of pNPbetaG). LAB25g2 increased the final hydrolysis yield by a factor of 20% (44.5 ± 1.7% vs. 34.5 ± 1.5% in control conditions) after 96–120 h as compared to control reactions in its absence or in the presence of other commercial beta-glucosidase preparations. The high stability (half-life higher than 5 days at 50°C and pH 5.2) and 2–38000 fold higher (as compared with reported beta-glucosidases) activity towards cello-oligosaccharides may account for its performance in supplementation assays. Conclusions The results suggest that beta-glucosidases from yet uncultured bacteria from animal digestomes may be of a potential interest for biotechnological processes related to the effective bio-ethanol production in combination with low dosage of commercial cellulases.
    • Microbial β-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail.

      Del Pozo, Mercedes V; Fernández-Arrojo, Lucía; Gil-Martínez, Jorge; Montesinos, Alejandro; Chernikova, Tatyana N; Nechitaylo, Taras Y; Waliszek, Agnes; Tortajada, Marta; Rojas, Antonia; Huws, Sharon A; et al. (2012)
      A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product.
    • MicroRNA-125b-5p Regulates Hepatocyte Proliferation During the Termination Phase of Liver Regeneration.

      Yang, Dakai; Dai, Zhen; Yang, Taihua; Balakrishnan, Asha; Yuan, Qinggong; Vondran, Florian W R; Manns, Michael P; Ott, Michael; Cantz, Tobias; Sharma, Amar Deep; et al. (Wiley, 2020-09-15)
      The ability of the liver to regenerate and restore mass limits the increasing mortality rate due to life-threatening liver diseases. Successful liver regeneration is accomplished in multiple stages, of which the priming and proliferation phases are well studied. However, the regulatory pathways, specifically microRNA (miRNA)-mediated posttranscriptional regulation, which prevent uncontrolled proliferation and mediate the termination of liver regeneration, are not well understood. We identified differentially regulated miRNAs during the termination phase after 2/3 partial hepatectomy (PH) in mice, which is a well-established mouse model of liver regeneration. We further evaluated the function of differentially regulated miRNAs in primary mouse hepatocytes by using mimics and inhibitors and in vivo by using adeno-associated virus (AAV) serotype 8. A candidate miRNA target was identified by messenger RNA array in silico analyses and validated in primary mouse and human hepatocytes. Using miRNA profiling, we discovered miR-125b-5p as a novel regulator of hepatocyte proliferation in the late phase of liver regeneration. AAV-mediated miR-125b-5p delivery in mice enhanced the endogenous regenerative capacity and resulted in improved restoration of liver mass after 2/3 PH. Further, we found that ankyrin repeat and BTB/POZ domain containing protein 1 (Abtb1) is a direct target of miR-125b-5p in primary mouse and human hepatocytes and contributes to the pro-proliferative activity of miR-125b-5p by forkhead box G1 (FOXG1) and the cyclin-dependent kinase inhibitor 1A (p21) pathway. Conclusion: miR-125b-5p has an important role in regulating hepatocyte proliferation in the termination phase of liver regeneration and may serve as a potential therapeutic target in various liver diseases that often exhibit deregulated hepatocyte proliferation.
    • MicroRNA-221: A Fine Tuner and Potential Biomarker of Chronic Liver Injury.

      Markovic, Jovana; Sharma, Amar Deep; Balakrishnan, Asha; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-07-23)
      The last decade has witnessed significant advancements in our understanding of how small noncoding RNAs, such as microRNAs (miRNAs), regulate disease progression. One such miRNA, miR-221, has been shown to play a key role in the progression of liver fibrosis, a common feature of most liver diseases. Many reports have demonstrated the upregulation of miR-221 in liver fibrosis caused by multiple etiologies such as viral infections and nonalcoholic steatohepatitis. Inhibition of miR-221 via different strategies has shown promising results in terms of the suppression of fibrogenic gene signatures in vitro, as well as in vivo, in independent mouse models of liver fibrosis. In addition, miR-221 has also been suggested as a noninvasive serum biomarker for liver fibrosis and cirrhosis. In this review, we discuss the biology of miR-221, its significance and use as a biomarker during progression of liver fibrosis, and finally, potential and robust approaches that can be utilized to suppress liver fibrosis via inhibition of miR-221.
    • MicroRNA-342-3p is a potent tumour suppressor in hepatocellular carcinoma

      Komoll, Ronja Melinda; Hu, Qingluan; Olarewaju, Olaniyi; von Döhlen, Lena; Yuan, Qinggong; Xie, Yu; Tsay, Hsin Chieh; Daon, Joel; Qin, Renyi; Manns, Michael P; et al. (2021-01-01)
      Background & aims: Hepatocellular carcinoma (HCC) is a cancer with multiple aetiologies and widespread prevalence. Largely refractory to current treatments, HCC is the fourth leading cause of cancer-related deaths worldwide. MicroRNAs (miRNAs) are important regulators in HCCs. We aimed to identify tumour suppressor miRNAs during tumour regression in a conditional c-MYC-driven mouse model (LT2/MYC) of HCC, and to evaluate their therapeutic potential for HCC treatment. Methods: We performed miRNA expression profiling of developed and regressing LT2/MYC tumours and in-depth in vitro gain- and loss-of-function analyses. The effect of adeno-associated virus (AAV) vector-mediated miR-342-3p treatment was evaluated in 3 HCC mouse models. Results: We identified miR-342-3p as a tumour suppressor miRNA in HCC, with increased expression in regressing tumours. Forced miR-342-3p expression in hepatoma cells showed significantly decreased cell proliferation, migration, and colony formation. In vivo administration of AAV-miR-342-3p led to significant attenuation of tumour development and increased overall survival. We identified monocarboxylic acid transporter 1 (MCT1) as a bona fide target of miR-342-3p in HCC. We show that the tumour suppressor role of miR-342-3p is executed partly by modulating the lactate transport function of MCT1. Importantly, we find miR-342-3p downregulated in tumours from patients with HCC compared with matched non-tumour tissues, inversely correlating with MCT1 expression. We observed similar findings in TCGA-LIHC data. Conclusions: In our study, we identified and validated miR-342-3p as a tumour suppressor miRNA in HCC. We demonstrated its therapeutic efficacy in significantly attenuating tumour development, and prolonging survival, in different HCC mouse models. Identification of miR-342-3p as an effective tumour suppressor opens a therapeutic avenue for miRNA-mediated attenuation of HCC development. Lay summary: Hepatocellular carcinoma (HCC), the most common type of liver cancer, affects diverse populations and has a global impact, being the fourth leading cause of cancer deaths worldwide. There are currently no systemic therapies for HCC that can significantly prolong long-term survival. Thus, novel effective treatment options are urgently required. To understand the molecular basis of tumour regression, we compared tumours and regressing liver tumours in mice. We show that a small non-coding miRNA, miR-342-3p, is a tumour suppressor in HCC. Expression of miR-342-3p is low in tumours and high in regressing tumours. When miR-342-3p is delivered to mouse livers with HCC, it can significantly slow down liver tumour development and improve survival. Our study highlights the promising therapeutic potential of miR-342-3p intervention in HCC.
    • miR-181a/b-1 controls thymic selection of Treg cells and tunes their suppressive capacity.

      Łyszkiewicz, Marcin; Winter, Samantha J; Witzlau, Katrin; Föhse, Lisa; Brownlie, Rebecca; Puchałka, Jacek; Verheyden, Nikita A; Kunze-Schumacher, Heike; Imelmann, Esther; Blume, Jonas; et al. (PLOS, 2019-03-01)
      The interdependence of selective cues during development of regulatory T cells (Treg cells) in the thymus and their suppressive function remains incompletely understood. Here, we analyzed this interdependence by taking advantage of highly dynamic changes in expression of microRNA 181 family members miR-181a-1 and miR-181b-1 (miR-181a/b-1) during late T-cell development with very high levels of expression during thymocyte selection, followed by massive down-regulation in the periphery. Loss of miR-181a/b-1 resulted in inefficient de novo generation of Treg cells in the thymus but simultaneously permitted homeostatic expansion in the periphery in the absence of competition. Modulation of T-cell receptor (TCR) signal strength in vivo indicated that miR-181a/b-1 controlled Treg-cell formation via establishing adequate signaling thresholds. Unexpectedly, miR-181a/b-1-deficient Treg cells displayed elevated suppressive capacity in vivo, in line with elevated levels of cytotoxic T-lymphocyte-associated 4 (CTLA-4) protein, but not mRNA, in thymic and peripheral Treg cells. Therefore, we propose that intrathymic miR-181a/b-1 controls development of Treg cells and imposes a developmental legacy on their peripheral function.
    • Misinterpretation of the odds ratios.

      Fernández, Nathalie; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2020-05-11)
      No abstract available
    • More than just a metabolic regulator--elucidation and validation of new targets of PdhR in Escherichia coli.

      Göhler, Anna-Katharina; Kökpinar, Öznur; Schmidt-Heck, Wolfgang; Geffers, Robert; Guthke, Reinhard; Rinas, Ursula; Schuster, Stefan; Jahreis, Knut; Kaleta, Christoph; Department of Genetics, University of Osnabrück, Osnabrück, Germany. (2011)
      The pyruvate dehydrogenase regulator protein (PdhR) of Escherichia coli acts as a transcriptional regulator in a pyruvate dependent manner to control central metabolic fluxes. However, the complete PdhR regulon has not yet been uncovered. To achieve an extended understanding of its gene regulatory network, we combined large-scale network inference and experimental verification of results obtained by a systems biology approach.
    • Multi-omics examination of Q fever fatigue syndrome identifies similarities with chronic fatigue syndrome.

      Raijmakers, Ruud P H; Roerink, Megan E; Jansen, Anne F M; Keijmel, Stephan P; Gacesa, Ranko; Li, Yang; Joosten, Leo A B; van der Meer, Jos W M; Netea, Mihai G; Bleeker-Rovers, Chantal P; et al. (BMC, 2020-11-26)
      Inflammatory markers, including 4E-BP1 (P = 9.60-16 and 1.41-7) and MMP-1 (P = 7.09-9 and 3.51-9), are significantly more expressed in both QFS and CFS patients compared to HC. Blood metabolite profiles show significant differences when comparing QFS (319 metabolites) and CFS (441 metabolites) patients to HC, and are significantly enriched in pathways like sphingolipid (P = 0.0256 and 0.0033) metabolism. When comparing QFS to CFS patients, almost no significant differences in metabolome were found. Comparison of microbiome taxonomy of QFS and CFS patients with that of HC, shows both in- and decreases in abundancies in Bacteroidetes (with emphasis on Bacteroides and Alistiples spp.), and Firmicutes and Actinobacteria (with emphasis on Ruminococcus and Bifidobacterium spp.). When we compare QFS patients to CFS patients, there is a striking resemblance and hardly any significant differences in microbiome taxonomy are found.
    • Mutation in a "tesB-like" hydroxyacyl-coenzyme A-specific thioesterase gene causes hyperproduction of extracellular polyhydroxyalkanoates by Alcanivorax borkumensis SK2.

      Sabirova, Julia S; Ferrer, Manuel; Lünsdorf, Heinrich; Wray, Victor; Kalscheuer, Rainer; Steinbüchel, Alexander; Timmis, Kenneth N; Golyshin, Peter N; Department of Environmental Microbiology, HZI-Helmholtz Center fro Infection Research, Braunschweig, Germany. jsa05@helmholtz-hzi.de (2006-12)
      A novel mutant of the marine oil-degrading bacterium Alcanivorax borkumensis SK2, containing a mini-Tn5 transposon disrupting a "tesB-like" acyl-coenzyme A (CoA) thioesterase gene, was found to hyperproduce polyhydroxyalkanoates (PHA), resulting in the extracellular deposition of this biotechnologically important polymer when grown on alkanes. The tesB-like gene encodes a distinct novel enzyme activity, which acts exclusively on hydroxylated acyl-CoAs and thus represents a hydroxyacyl-CoA-specific thioesterase. Inactivation of this enzyme results in the rechanneling of CoA-activated hydroxylated fatty acids, the cellular intermediates of alkane degradation, towards PHA production. These findings may open up new avenues for the development of simplified biotechnological processes for the production of PHA as a raw material for the production of bioplastics.
    • Nasal DNA methylation profiling of asthma and rhinitis.

      Qi, Cancan; Jiang, Yale; Yang, Ivana V; Forno, Erick; Wang, Ting; Vonk, Judith M; Gehring, Ulrike; Smit, Henriëtte A; Milanzi, Edith B; Carpaij, Orestes A; et al. (2020-01-14)
    • NeutrobodyPlex-monitoring SARS-CoV-2 neutralizing immune responses using nanobodies.

      Wagner, Teresa R; Ostertag, Elena; Kaiser, Philipp D; Gramlich, Marius; Ruetalo, Natalia; Junker, Daniel; Haering, Julia; Traenkle, Bjoern; Becker, Matthias; Dulovic, Alex; et al. (EMBO Press, 2021-04-27)
      In light of the COVID-19 pandemic, there is an ongoing need for diagnostic tools to monitor the immune status of large patient cohorts and the effectiveness of vaccination campaigns. Here, we present 11 unique nanobodies (Nbs) specific for the SARS-CoV-2 spike receptor-binding domain (RBD), of which 8 Nbs potently inhibit the interaction of RBD with angiotensin-converting enzyme 2 (ACE2) as the major viral docking site. Following detailed epitope mapping and structural analysis, we select two inhibitory Nbs, one of which binds an epitope inside and one of which binds an epitope outside the RBD:ACE2 interface. Based on these, we generate a biparatopic nanobody (bipNb) with viral neutralization efficacy in the picomolar range. Using bipNb as a surrogate, we establish a competitive multiplex binding assay ("NeutrobodyPlex") for detailed analysis of the presence and performance of neutralizing RBD-binding antibodies in serum of convalescent or vaccinated patients. We demonstrate that NeutrobodyPlex enables high-throughput screening and detailed analysis of neutralizing immune responses in infected or vaccinated individuals, to monitor immune status or to guide vaccine design.
    • New lineage of filamentous, spore-forming, gram-positive bacteria from soil.

      Cavaletti, Linda; Monciardini, Paolo; Bamonte, Ruggiero; Schumann, Peter; Rohde, Manfred; Sosio, Margherita; Donadio, Stefano; Vicuron Pharmaceuticals, 21040 Gerenzano, Italy. (2006-06)
      A novel bacterial strain that was isolated from an Italian soil and was designated SOSP1-21T forms branched mycelia in solid and liquid media and has a filamentous morphology similar to that of some genera belonging to the Actinobacteria. Electron microscopy showed that this organism has a grape-like appearance, resulting from interlacing of spores originating from sporophoric hyphae. Ten strains that are morphologically related to SOSP1-21T were recovered from soil. Phylogenetic analyses of 16S rRNA gene segments confirmed the relatedness of these strains to SOSP1-21T and indicated that the newly isolated strains form separate clades in a deeply branching lineage. The closest matches for the 16S rRNA sequences of all the strains (around 79% identity) were matches with representatives of the Chloroflexi, although the affiliation with this division was not supported by high bootstrap values. The strains are mesophilic aerobic heterotrophs and are also capable of growing under microaerophilic conditions. They all stain gram positive. Strain SOSP1-21T contains ornithine, alanine, glutamic acid, serine, and glycine as the peptidoglycan amino acids. In addition, an unusual level of C16:1 2OH (30%) was found in the cellular fatty acids. The G+C content of SOSP1-21T genomic DNA is 53.9%, and MK-9(H2) was the only menaquinone detected. All these data suggest that SOSP1-21T and the related strains may constitute a new division of filamentous, spore-forming, gram-positive bacteria. We propose the name Ktedobacter racemifer gen. nov., sp. nov. for strain SOSP1-21T.
    • Non-Targeted Mass Isotopolome Analysis Using Stable Isotope Patterns to Identify Metabolic Changes.

      Dudek, Christian-Alexander; Schlicker, Lisa; Hiller, Karsten; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
      Gas chromatography coupled with mass spectrometry can provide an extensive overview of the metabolic state of a biological system. Analysis of raw mass spectrometry data requires powerful data processing software to generate interpretable results. Here we describe a data processing workflow to generate metabolite levels, mass isotopomer distribution, similarity and variability analysis of metabolites in a nontargeted manner, using stable isotope labeling. Using our data analysis software, no bioinformatic or programming background is needed to generate results from raw mass spectrometry data.
    • Notch and TLR signaling coordinate monocyte cell fate and inflammation.

      Gamrekelashvili, Jaba; Kapanadze, Tamar; Sablotny, Stefan; Ratiu, Corina; Dastagir, Khaled; Lochner, Matthias; Karbach, Susanne; Wenzel, Philip; Sitnow, Andre; Fleig, Susanne; et al. (elife Sciences, 2020-07-29)
      Conventional Ly6Chi monocytes have developmental plasticity for a spectrum of differentiated phagocytes. Here we show, using conditional deletion strategies in a mouse model of Toll-like receptor (TLR) 7-induced inflammation, that the spectrum of developmental cell fates of Ly6Chi monocytes, and the resultant inflammation, is coordinately regulated by TLR and Notch signaling. Cell-intrinsic Notch2 and TLR7-Myd88 pathways independently and synergistically promote Ly6Clo patrolling monocyte development from Ly6Chi monocytes under inflammatory conditions, while impairment in either signaling axis impairs Ly6Clo monocyte development. At the same time, TLR7 stimulation in the absence of functional Notch2 signaling promotes resident tissue macrophage gene expression signatures in monocytes in the blood and ectopic differentiation of Ly6Chi monocytes into macrophages and dendritic cells, which infiltrate the spleen and major blood vessels and are accompanied by aberrant systemic inflammation. Thus, Notch2 is a master regulator of Ly6Chi monocyte cell fate and inflammation in response to TLR signaling.
    • Novel Cycloheximide Derivatives Targeting the Moonlighting Protein Mip Exhibit Specific Antimicrobial Activity Against Legionella pneumophila.

      Rasch, Janine; Theuerkorn, Martin; Ünal, Can; Heinsohn, Natascha; Tran, Stefan; Fischer, Gunter; Weiwad, Matthias; Steinert, Michael; Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany. (2015)
      Macrophage infectivity potentiator (Mip) and Mip-like proteins are virulence factors in a wide range of pathogens including Legionella pneumophila. These proteins belong to the FK506 binding protein (FKBP) family of peptidyl-prolyl-cis/trans-isomerases (PPIases). In L. pneumophila, the PPIase activity of Mip is required for invasion of macrophages, transmigration through an in vitro lung-epithelial barrier, and full virulence in the guinea pig infection model. Additionally, Mip is a moonlighting protein that binds to collagen IV in the extracellular matrix. Here, we describe the development and synthesis of cycloheximide derivatives with adamantyl moieties as novel FKBP ligands, and analyze their effect on the viability of L. pneumophila and other bacteria. All compounds efficiently inhibited PPIase activity of the prototypic human FKBP12 as well as Mip with IC50-values as low as 180 nM and 1.7 μM, respectively. Five of these derivatives inhibited the growth of L. pneumophila at concentrations of 30-40 μM, but exhibited no effect on other tested bacterial species indicating a specific spectrum of antibacterial activity. The derivatives carrying a 3,5-dimethyladamantan-1-[yl]acetamide substitution (MT_30.32), and a 3-ethyladamantan-1-[yl]acetamide substitution (MT_30.51) had the strongest effects in PPIase- and liquid growth assays. MT_30.32 and MT_30.51 were also inhibitory in macrophage infection studies without being cytotoxic. Accordingly, by applying a combinatorial approach, we were able to generate novel, hybrid inhibitors consisting of cycloheximide and adamantane, two known FKBP inhibitors that interact with different parts of the PPIase domain, respectively. Interestingly, despite the proven Mip-inhibitory activity, the viability of a Mip-deficient strain was affected to the same degree as its wild type. Hence, we also propose that cycloheximide derivatives with adamantyl moieties are potent PPIase inhibitors with multiple targets in L. pneumophila.