• RovC - a novel type of hexameric transcriptional activator promoting type VI secretion gene expression.

      Knittel, Vanessa; Sadana, Pooja; Seekircher, Stephanie; Stolle, Anne-Sophie; Körner, Britta; Volk, Marcel; Jeffries, Cy M; Svergun, Dmitri I; Heroven, Ann Kathrin; Scrima, Andrea; et al. (PLOS, 2020-09-23)
      Type VI secretion systems (T6SSs) are complex macromolecular injection machines which are widespread in Gram-negative bacteria. They are involved in host-cell interactions and pathogenesis, required to eliminate competing bacteria, or are important for the adaptation to environmental stress conditions. Here we identified regulatory elements controlling the T6SS4 of Yersinia pseudotuberculosis and found a novel type of hexameric transcription factor, RovC. RovC directly interacts with the T6SS4 promoter region and activates T6SS4 transcription alone or in cooperation with the LysR-type regulator RovM. A higher complexity of regulation was achieved by the nutrient-responsive global regulator CsrA, which controls rovC expression on the transcriptional and post-transcriptional level. In summary, our work unveils a central mechanism in which RovC, a novel key activator, orchestrates the expression of the T6SS weapons together with a global regulator to deploy the system in response to the availability of nutrients in the species' native environment.
    • Cholesterol sensing by CD81 is important for hepatitis C virus entry.

      Palor, Machaela; Stejskal, Lenka; Mandal, Piya; Lenman, Annasara; Alberione, Maria Pia; Kirui, Jared; Moeller, Rebecca; Ebner, Stefan; Meissner, Felix; Gerold, Gisa; et al. (DeGruyter, 2020-09-08)
      CD81 plays a role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus. Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81-cholesterol association, but had disparate effects on HCV, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified an allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol unbound) or closed (cholesterol bound) conformation. The open mutant of CD81 exhibited reduced receptor activity whereas the closed mutant was enhanced. These data are consistent with cholesterol switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81-partner networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry and CD81's function as a molecular scaffold; these insights are relevant to CD81's varied roles in health and disease.
    • Strategic Anti-SARS-CoV-2 Serology Testing in a Low Prevalence Setting: The COVID-19 Contact (CoCo) Study in Healthcare Professionals.

      Behrens, Georg M N; Cossmann, Anne; Stankov, Metodi V; Schulte, Bianca; Streeck, Hendrik; Förster, Reinhold; Bosnjak, Berislav; Willenzon, Stefanie; Boeck, Anna-Lena; Thu Tran, Anh; et al. (Springer Healthcare, 2020-09-04)
      Background: Serology testing is explored for epidemiological research and to inform individuals after suspected infection. During the coronavirus disease 2019 (COVID-19) pandemic, frontline healthcare professionals (HCP) may be at particular risk for infection. No longitudinal data on functional seroconversion in HCP in regions with low COVID-19 prevalence and low pre-test probability exist. Methods: In a large German university hospital, we performed weekly questionnaire assessments and anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G (IgG) measurements with various commercial tests, a novel surrogate virus neutralisation test, and a neutralisation assay using live SARS-CoV-2. Results: From baseline to week 6, 1080 screening measurements for anti-SARS CoV-2 (S1) IgG from 217 frontline HCP (65% female) were performed. Overall, 75.6% of HCP reported at least one symptom of respiratory infection. Self-perceived infection probability declined over time (from mean 20.1% at baseline to 12.4% in week 6, p < 0.001). In sera of convalescent patients with PCR-confirmed COVID-19, we measured high anti-SARS-CoV-2 IgG levels, obtained highly concordant results from enzyme-linked immunosorbent assays (ELISA) using e.g. the spike 1 (S1) protein domain and the nucleocapsid protein (NCP) as targets, and confirmed antiviral neutralisation. However, in HCP the cumulative incidence for anti-SARS-CoV-2 (S1) IgG was 1.86% for positive and 0.93% for equivocal positive results over the study period of 6 weeks. Except for one HCP, none of the eight initial positive results were confirmed by alternative serology tests or showed in vitro neutralisation against live SARS-CoV-2. The only true seroconversion occurred without symptoms and mounted strong functional humoral immunity. Thus, the confirmed cumulative incidence for neutralizing anti-SARS-CoV-2 IgG was 0.47%. Conclusion: When assessing anti-SARS-CoV-2 immune status in individuals with low pre-test probability, we suggest confirming positive results from single measurements by alternative serology tests or functional assays. Our data highlight the need for a methodical serology screening approach in regions with low SARS-CoV-2 infection rates.
    • Impact of Von Willebrand Factor on Bacterial Pathogenesis.

      Steinert, Michael; Ramming, Isabell; Bergmann, Simone; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2020-09-03)
      Von Willebrand factor (VWF) is a mechano-sensitive protein with crucial functions in normal hemostasis, which are strongly dependant on the shear-stress mediated defolding and multimerization of VWF in the blood stream. Apart from bleeding disorders, higher plasma levels of VWF are often associated with a higher risk of cardiovascular diseases. Herein, the disease symptoms are attributed to the inflammatory response of the activated endothelium and share high similarities to the reaction of the host vasculature to systemic infections caused by pathogenic bacteria such as Staphylococcus aureus and Streptococcus pneumoniae. The bacteria recruit circulating VWF, and by binding to immobilized VWF on activated endothelial cells in blood flow, they interfere with the physiological functions of VWF, including platelet recruitment and coagulation. Several bacterial VWF binding proteins have been identified and further characterized by biochemical analyses. Moreover, the development of a combination of sophisticated cell culture systems simulating shear stress levels of the blood flow with microscopic visualization also provided valuable insights into the interaction mechanism between bacteria and VWF-strings. In vivo studies using mouse models of bacterial infection and zebrafish larvae provided evidence that the interaction between bacteria and VWF promotes bacterial attachment, coagulation, and thrombus formation, and thereby contributes to the pathophysiology of severe infectious diseases such as infective endocarditis and bacterial sepsis. This mini-review summarizes the current knowledge of the interaction between bacteria and the mechano-responsive VWF, and corresponding pathophysiological disease symptoms.
    • Recombinant protein production-associated metabolic burden reflects anabolic constraints and reveals similarities to a carbon overfeeding response.

      Li, Zhaopeng; Rinas, Ursula; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2020-09-03)
      A comparison of the metabolic response of Escherichia coli BL21 (DE3) towards the production of human basic fibroblast growth factor (hFGF-2) or towards carbon overfeeding revealed similarities which point to constraints in anabolic pathways. Contrary to expectations, neither energy generation (e.g., ATP) nor provision of precursor molecules for nucleotides (e.g., uracil) and amino acids (e.g., pyruvate, glutamate) limit host cell and plasmid-encoded functions. Growth inhibition is assumed to occur when hampered anabolic capacities do not match with the ongoing and overwhelming carbon catabolism. Excessive carbon uptake leads to by-product secretion, for example, pyruvate, acetate, glutamate, and energy spillage, for example, accumulation and degradation of adenine nucleotides with concomitant accumulation of extracellular hypoxanthine. The cellular response towards compromised anabolic capacities involves downregulation of cAMP formation, presumably responsible for subsequently better-controlled glucose uptake and resultant accumulation of glucose in the culture medium. Growth inhibition is neglectable under conditions of reduced carbon availability when hampered anabolic capacities also match with catabolic carbon processing. The growth inhibitory effect with accompanying energy spillage, respectively, hypoxanthine secretion and cessation of cAMP formation is not unique to the production of hFGF-2 but observed during the production of other proteins and also during overexpression of genes without transcript translation.
    • Epigenome-wide association study of DNA methylation and adult asthma in the Agricultural Lung Health Study.

      Hoang, Thanh T; Sikdar, Sinjini; Xu, Cheng-Jian; Lee, Mi Kyeong; Cardwell, Jonathan; Forno, Erick; Imboden, Medea; Jeong, Ayoung; Madore, Anne-Marie; Qi, Cancan; et al. (European Respiratory Society (ERS), 2020-09-03)
      Epigenome-wide studies of methylation in children support a role for epigenetic mechanisms in asthma; however, studies in adults are rare and few have examined non-atopic asthma. We conducted the largest epigenome-wide association study (EWAS) of blood DNA methylation in adults in relation to non-atopic and atopic asthma.We measured DNA methylation in blood using the Illumina MethylationEPIC array among 2286 participants in a case-control study of current adult asthma nested within a United States agricultural cohort. Atopy was defined by serum specific immunoglobulin E (IgE). Participants were categorised as atopy without asthma (n=185), non-atopic asthma (n=673), atopic asthma (n=271), or a reference group of neither atopy nor asthma (n=1157). Analyses were conducted using logistic regression.No associations were observed with atopy without asthma. Numerous cytosine-phosphate-guanine (CpG) sites were differentially methylated in non-atopic asthma (eight at family-wise error rate (FWER) p<9×10-8, 524 at false discovery rate (FDR) less than 0.05) and implicated 382 novel genes. More CpG sites were identified in atopic asthma (181 at FWER, 1086 at FDR) and implicated 569 novel genes. 104 FDR CpG sites overlapped. 35% of CpG sites in non-atopic asthma and 91% in atopic asthma replicated in studies of whole blood, eosinophils, airway epithelium, or nasal epithelium. Implicated genes were enriched in pathways related to the nervous system or inflammation.We identified numerous, distinct differentially methylated CpG sites in non-atopic and atopic asthma. Many CpG sites from blood replicated in asthma-relevant tissues. These circulating biomarkers reflect risk and sequelae of disease, as well as implicate novel genes associated with non-atopic and atopic asthma.
    • Staphylococcus aureus Alpha-Toxin Limits Type 1 While Fostering Type 3 Immune Responses.

      Bonifacius, Agnes; Goldmann, Oliver; Floess, Stefan; Holtfreter, Silva; Robert, Philippe A; Nordengrün, Maria; Kruse, Friederike; Lochner, Matthias; Falk, Christine S; Schmitz, Ingo; et al. (Frontiers, 2020-08-07)
      Staphylococcus aureus can cause life-threatening diseases, and hospital- as well as community-associated antibiotic-resistant strains are an emerging global public health problem. Therefore, prophylactic vaccines or immune-based therapies are considered as alternative treatment opportunities. To develop such novel treatment approaches, a better understanding of the bacterial virulence and immune evasion mechanisms and their potential effects on immune-based therapies is essential. One important staphylococcal virulence factor is alpha-toxin, which is able to disrupt the epithelial barrier in order to establish infection. In addition, alpha-toxin has been reported to modulate other cell types including immune cells. Since CD4+ T cell-mediated immunity is required for protection against S. aureus infection, we were interested in the ability of alpha-toxin to directly modulate CD4+ T cells. To address this, murine naïve CD4+ T cells were differentiated in vitro into effector T cell subsets in the presence of alpha-toxin. Interestingly, alpha-toxin induced death of Th1-polarized cells, while cells polarized under Th17 conditions showed a high resistance toward increasing concentrations of this toxin. These effects could neither be explained by differential expression of the cellular alpha-toxin receptor ADAM10 nor by differential activation of caspases, but might result from an increased susceptibility of Th1 cells toward Ca2+-mediated activation-induced cell death. In accordance with the in vitro findings, an alpha-toxin-dependent decrease of Th1 and concomitant increase of Th17 cells was observed in vivo during S. aureus bacteremia. Interestingly, corresponding subsets of innate lymphoid cells and γδ T cells were similarly affected, suggesting a more general effect of alpha-toxin on the modulation of type 1 and type 3 immune responses. In conclusion, we have identified a novel alpha-toxin-dependent immunomodulatory strategy of S. aureus, which can directly act on CD4+ T cells and might be exploited for the development of novel immune-based therapeutic approaches to treat infections with antibiotic-resistant S. aureus strains.
    • Purification of the human fibroblast growth factor 2 using novel animal-component free materials

      Bolten, Svenja Nicolin; Knoll, Anne-Sophie; Li, Zhaopeng; Gellermann, Pia; Pepelanova, Iliyana; Rinas, Ursula; Scheper, Thomas; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier BV, 2020-08)
      This paper analyzes the use of animal-component free chromatographic materials for the efficient purifi- cation of the human fibroblast growth factor 2 (hFGF-2). hFGF-2 is produced in Escherichia coli and pu- rified via three different chromatography steps, which include a strong cation exchange chromatography as a capture step, followed by heparin affinity chromatography and an anion exchange chromatography as a polishing step. The affinity chromatography step is based on the animal-derived material heparin. Chemically produced ligands provide a viable alternative to animal-derived components in production processes, since they are characterized by a defined structure which leads to reproducible results and a broad range of applications. The alternative ligands can be assigned to adsorber of the mixed-mode chromatography (MMC) and pseudo-affinity chromatography. Eight different animal-component free materials used as adsorbers in MMC or pseudo-affinity chromatog- raphy were tested as a substitute for heparin. The MMCs were cation exchangers characterized with fur- ther functional residues. The ligands of the pseudo-affinity chromatography were heparin-like ligands which are based on heparin’s molecular structure. The alternative methods were tested as a capture step and in combination with another chromatographic step in the purification procedure of hFGF-2. In each downstream step purity, recovery and yield were analysed and compared to the conventional downstream process. Two types of MMC –the column Foresight TM Nuvia TM cPrime TM from Bio-Rad Laboratories and the col- umn HiTrap TM Capto TM MMC from GE Healthcare Life Sciences - can be regarded as effective animal- component free alternatives to the heparin - based adsorber.
    • 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. (Elsevier, 2020-07-30)
      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.
    • 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.
    • 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.
    • Varying the sustained release of BMP-2 from chitosan nanogel-functionalized polycaprolactone fiber mats by different polycaprolactone surface modifications.

      Sundermann, Julius; Oehmichen, Sarah; Sydow, Steffen; Burmeister, Laura; Quaas, Bastian; Hänsch, Robert; Rinas, Ursula; Hoffmann, Andrea; Menzel, Henning; Bunjes, Heike; et al. (Wiley and sons, 2020-06-30)
      Polycaprolactone (PCL) fiber mats with different surface modifications were functionalized with a chitosan nanogel coating to attach the growth factor human bone morphogenetic protein 2 (BMP-2). Three different hydrophilic surface modifications were compared with regard to the binding and in vitro release of BMP-2. The type of surface modification and the specific surface area derived from the fiber thickness had an important influence on the degree of protein loading. Coating the PCL fibers with polydopamine resulted in the binding of the largest BMP-2 quantity per surface area. However, most of the binding was irreversible over the investigated period of time, causing a low release in vitro. PCL fiber mats with a chitosan-graft-PCL coating and an additional alginate layer, as well as PCL fiber mats with an air plasma surface modification boundless BMP-2, but the immobilized protein could almost completely be released. With polydopamine and plasma modifications as well as with unmodified PCL, high amounts of BMP-2 could also be attached directly to the surface. Integration of BMP-2 into the chitosan nanogel functionalization considerably increased binding on all hydrophilized surfaces and resulted in a sustained release with an initial burst release of BMP-2 without detectable loss of bioactivity in vitro.
    • Deconvolution of bulk blood eQTL effects into immune cell subpopulations.

      Aguirre-Gamboa, Raúl; de Klein, Niek; di Tommaso, Jennifer; Claringbould, Annique; van der Wijst, Monique Gp; de Vries, Dylan; Brugge, Harm; Oelen, Roy; Võsa, Urmo; Zorro, Maria M; et al. (BMC, 2020-06-12)
      A novel planctomycetal strain, designated Pla85_3_4T, was isolated from the surface of wood incubated at the discharge of a wastewater treatment plant in the Warnow river near Rostock, Germany. Cells of the novel strain have a cell envelope architecture resembling that of Gram-negative bacteria, are round to pear-shaped (length: 2.2 ± 0.4 µm, width: 1.2 ± 0.3 µm), form aggregates and divide by polar budding. Colonies have a cream colour. Strain Pla85_3_4T grows at ranges of 10-30 °C (optimum 26 °C) and at pH 6.5-10.0 (optimum 7.5), and has a doubling time of 26 h. Phylogenetically, strain Pla85_3_4T (DSM 103796T = LMG 29741T) is concluded to represent a novel species of a novel genus within the family Pirellulaceae, for which we propose the name Lignipirellula cremea gen. nov., sp. nov.
    • Relevance of inducible nitric oxide synthase for immune control of Mycobacterium avium subspecies paratuberculosis infection in mice.

      Abdissa, Ketema; Ruangkiattikul, Nanthapon; Ahrend, Wiebke; Nerlich, Andreas; Beineke, Andreas; Laarmann, Kristin; Janze, Nina; Lobermeyer, Ulrike; Suwandi, Abdulhadi; Falk, Christine; et al. (Taylor & Francis, 2020-05-14)
      Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease (JD), an incurable chronic intestinal bowel disease in ruminants. JD occurs worldwide and causes enormous economic burden in dairy industry. Research on JD pathobiology is hampered by its complexity which cannot completely be mimicked by small animal models. As a model the mouse allows dissecting some pathogenicity features of MAP. However, for unknown reasons MAP exhibits reduced growth in granulomas of infected mice compared to other Mycobacterium avium subspecies. Here, we characterized immune reactions of MAP-infected C57BL/6 mice. After infection, mice appeared fully immunocompetent. A strong antigen-specific T cell response was elicited indicated by IFNγ production of splenic T cells re-stimulated with MAP antigens. Function of splenic dendritic cells and proliferation of adoptively transferred antigen-specific CD4+ T cells was unaltered. Isolated splenic myeloid cells from infected mice revealed that MAP resides in CD11b+ macrophages. Importantly, sorted CD11b+CD11c- cells expressed high level of type 2 nitric oxide synthase (NOS2) but only low levels of pro- and anti-inflammatory cytokines. Correspondingly, MAP-infected MAC2 expressing myeloid cells in spleen and liver granuloma displayed strong expression of NOS2. In livers of infected Nos2-/-mice higher bacterial loads, more granuloma and larger areas of tissue damage were observed 5 weeks post infection compared to wild type mice. In vitro, MAP was sensitive to NO released by a NO-donor. Thus, a strong T cell response and concomitant NOS2/NO activity appears to control MAP infection, but allows development of chronicity and pathogen persistence. A similar mechanism might explain persistence of MAP in ruminants.
    • 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
    • Pathological mechanism and antisense oligonucleotide-mediated rescue of a non-coding variant suppressing factor 9 RNA biogenesis leading to hemophilia B.

      Krooss, Simon; Werwitzke, Sonja; Kopp, Johannes; Rovai, Alice; Varnholt, Dirk; Wachs, Amelie S; Goyenvalle, Aurelie; Aarstma-Rus, Annemieke; Ott, Michael; Tiede, Andreas; et al. (PLOS, 2020-04-08)
      Loss-of-function mutations in the human coagulation factor 9 (F9) gene lead to hemophilia B. Here, we dissected the consequences and the pathomechanism of a non-coding mutation (c.2545A>G) in the F9 3' untranslated region. Using wild type and mutant factor IX (FIX) minigenes we revealed that the mutation leads to reduced F9 mRNA and FIX protein levels and to lower coagulation activity of cell culture supernatants. The phenotype could not be compensated by increased transcription. The pathomechanism comprises the de novo creation of a binding site for the spliceosomal component U1snRNP, which is able to suppress the nearby F9 poly(A) site. This second, splicing-independent function of U1snRNP was discovered previously and blockade of U1snRNP restored mutant F9 mRNA expression. In addition, we explored the vice versa approach and masked the mutation by antisense oligonucleotides resulting in significantly increased F9 mRNA expression and coagulation activity. This treatment may transform the moderate/severe hemophilia B into a mild or subclinical form in the patients. This antisense based strategy is applicable to other mutations in untranslated regions creating deleterious binding sites for cellular proteins.
    • Recombinant protein production associated growth inhibition results mainly from transcription and not from translation.

      Li, Zhaopeng; Rinas, Ursula; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (BMC (part of Springer), 2020-04-06)
      Background: Recombinant protein production can be stressful to the host organism. The extent of stress is determined by the specific properties of the recombinant transcript and protein, by the rates of transcription and translation, and by the environmental conditions encountered during the production process. Results: The impact of the transcription of the T7-promoter controlled genes encoding human basic fibroblast growth factor (hFGF-2) and green fluorescent protein (GFP) as well as the translation into the recombinant protein on the growth properties of the production host E. coli BL21(DE3) were investigated. This was done by using expression vectors where the promoter region or the ribosome binding site(s) or both were removed. It is shown that already transcription without protein translation imposes a metabolic burden on the host cell. Translation of the transcript into large amounts of a properly folded protein does not show any effect on cell growth in the best case, e.g. high-level production of GFP in Luria-Bertani medium. However, translation appears to contribute to the metabolic burden if it is connected to protein folding associated problems, e.g. inclusion body formation. Conclusion: The so-called metabolic burden of recombinant protein production is mainly attributed to transcription but can be enhanced through translation and those processes following translation (e.g. protein folding and degradation, heat-shock responses).
    • TLR4 abrogates the Th1 immune response through IRF1 and IFN-β to prevent immunopathology during L. infantum infection.

      Sacramento, Laís Amorim; Benevides, Luciana; Maruyama, Sandra Regina; Tavares, Lucas; Fukutani, Kiyoshi Ferreira; Francozo, Marcela; Sparwasser, Tim; Cunha, Fernando Queiroz; Almeida, Roque Pacheco; da Silva, João Santana; et al. (PLOS, 2020-03-25)
      A striking feature of human visceral leishmaniasis (VL) is chronic inflammation in the spleen and liver, and VL patients present increased production levels of multiple inflammatory mediators, which contribute to tissue damage and disease severity. Here, we combined an experimental model with the transcriptional profile of human VL to demonstrate that the TLR4-IFN-β pathway regulates the chronic inflammatory process and is associated with the asymptomatic form of the disease. Tlr4-deficient mice harbored fewer parasites in their spleen and liver than wild-type mice. TLR4 deficiency enhanced the Th1 immune response against the parasite, which was correlated with an increased activation of dendritic cells (DCs). Gene expression analyses demonstrated that IRF1 and IFN-β were expressed downstream of TLR4 after infection. Accordingly, IRF1- and IFNAR-deficient mice harbored fewer parasites in the target organs than wild-type mice due to having an increased Th1 immune response. However, the absence of TLR4 or IFNAR increased the serum transaminase levels in infected mice, indicating the presence of liver damage in these animals. In addition, IFN-β limits IFN-γ production by acting directly on Th1 cells. Using RNA sequencing analysis of human samples, we demonstrated that the transcriptional signature for the TLR4 and type I IFN (IFN-I) pathways was positively modulated in asymptomatic subjects compared with VL patients and thus provide direct evidence demonstrating that the TLR4-IFN-I pathway is related to the nondevelopment of the disease. In conclusion, our results demonstrate that the TLR4-IRF1 pathway culminates in IFN-β production as a mechanism for dampening the chronic inflammatory process and preventing immunopathology development.
    • Glutathione Restricts Serine Metabolism to Preserve Regulatory T Cell Function.

      Kurniawan, Henry; Franchina, Davide G; Guerra, Luana; Bonetti, Lynn; -Baguet, Leticia Soriano; Grusdat, Melanie; Schlicker, Lisa; Hunewald, Oliver; Dostert, Catherine; Merz, Myriam P; et al. (Elsevier (Cell Press), 2020-03-25)
      Regulatory T cells (Tregs) maintain immune homeostasis and prevent autoimmunity. Serine stimulates glutathione (GSH) synthesis and feeds into the one-carbon metabolic network (1CMet) essential for effector T cell (Teff) responses. However, serine's functions, linkage to GSH, and role in stress responses in Tregs are unknown. Here, we show, using mice with Treg-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), that GSH loss in Tregs alters serine import and synthesis and that the integrity of this feedback loop is critical for Treg suppressive capacity. Although Gclc ablation does not impair Treg differentiation, mutant mice exhibit severe autoimmunity and enhanced anti-tumor responses. Gclc-deficient Tregs show increased serine metabolism, mTOR activation, and proliferation but downregulated FoxP3. Limitation of cellular serine in vitro and in vivo restores FoxP3 expression and suppressive capacity of Gclc-deficient Tregs. Our work reveals an unexpected role for GSH in restricting serine availability to preserve Treg functionality.
    • Variations in microbiota composition of laboratory mice influence Citrobacter rodentium infection via variable short-chain fatty acid production.

      Osbelt, Lisa; Thiemann, Sophie; Smit, Nathiana; Lesker, Till Robin; Schröter, Madita; Gálvez, Eric J C; Schmidt-Hohagen, Kerstin; Pils, Marina C; Mühlen, Sabrina; Dersch, Petra; et al. (PLOS, 2020-03-24)
      The composition of the intestinal microbiota influences the outcome of enteric infections in human and mice. However, the role of specific members and their metabolites contributing to disease severity is largely unknown. Using isogenic mouse lines harboring distinct microbiota communities, we observed highly variable disease kinetics of enteric Citrobacter rodentium colonization after infection. Transfer of communities from susceptible and resistant mice into germ-free mice verified that the varying susceptibilities are determined by microbiota composition. The strongest differences in colonization were observed in the cecum and could be maintained in vitro by coculturing cecal bacteria with C. rodentium. Cohousing of animals as well as the transfer of cultivable bacteria from resistant to susceptible mice led to variable outcomes in the recipient mice. Microbiome analysis revealed that a higher abundance of butyrate-producing bacteria was associated with the resistant phenotype. Quantification of short-chain fatty acid (SCFA) levels before and after infection revealed increased concentrations of acetate, butyrate and propionate in mice with delayed colonization. Addition of physiological concentrations of butyrate, but not of acetate and/or propionate strongly impaired growth of C. rodentium in vitro. In vivo supplementation of susceptible, antibiotic-treated and germ-free mice with butyrate led to the same level of protection, notably only when cecal butyrate concentration reached a concentration higher than 50 nmol/mg indicating a critical threshold for protection. In the recent years, commensal-derived primary and secondary bacterial metabolites emerged as potent modulators of hosts susceptibility to infection. Our results provide evidence that variations in SCFA production in mice fed fibre-rich chow-based diets modulate susceptibility to colonization with Enterobacteriaceae not only in antibiotic-disturbed ecosystems but even in undisturbed microbial communities. These findings emphasise the need for microbiota normalization across laboratory mouse lines for infection experiments with the model-pathogen C. rodentium independent of investigations of diet and antibiotic usage.