• Murine toll-like receptor 2 activation induces type I interferon responses from endolysosomal compartments.

      Dietrich, Nicole; Lienenklaus, Stefan; Weiss, Siegfried; Gekara, Nelson O; Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2010)
      BACKGROUND: Toll-like receptors (TLRs) are among the first-line sentinels for immune detection and responsiveness to pathogens. The TLR2 subfamily of TLRs (TLR1, TLR2, TLR6) form heterodimers with each other and are thus able to recognize a broad range of components from several microbes such as yeast, Gram-positive bacteria and protozoa. Until now, TLR2 activation by bacterial ligands has long been associated with pro-inflammatory cytokines but not type I interferon responses. METHODOLOGY/PRINCIPAL FINDINGS: Using a variety of transgenic mice, here we provide in vivo and in vitro data showing that TLR2 activation does in fact induce interferon-beta and that this occurs via MyD88-IRF1 and -IRF7 pathways. Interestingly, by microscopy we demonstrate that although a cell surface receptor, TLR2 dependent induction of type I interferons occurs in endolysosomal compartments where it is translocated to upon ligand engagement. Furthermore, we could show that blocking receptor internalization or endolysosomal acidification inhibits the ability of TLR2 to trigger the induction type I interferon but not pro-inflammatory responses. CONCLUSION/SIGNIFICANCE: The results indicate that TLR2 activation induces pro-inflammatory and type I interferon responses from distinct subcellular sites: the plasma membrane and endolysosomal compartments respectively. Apart from identifying and characterizing a novel pathway for induction of type I interferons, the present study offers new insights into how TLR signaling discriminates and regulates the nature of responses to be elicited against extracellular and endocytosed microbes. These findings may also have clinical implication. Excessive production of pro-inflammatory cytokines and type I IFNs following activation of TLRs is a central pathologic event in several hyper-inflammatory conditions. The discovery that the induction of pro-inflammatory and type I IFN responses can be uncoupled through pharmacological manipulation of endolysosomal acidification suggests new avenues for potential therapeutic intervention against inflammations and sepsis.
    • The mycobacterial thioredoxin peroxidase can act as a one-cysteine peroxiredoxin.

      Trujillo, Madia; Mauri, PierLuigi; Benazzi, Louise; Comini, Marcelo; De Palma, Antonella; Flohé, Leopold; Radi, Rafael; Stehr, Matthias; Singh, Mahavir; Ursini, Fulvio; et al. (2006-07-21)
      Thioredoxin peroxidase (TPx) has been reported to dominate the defense against H(2)O(2), other hydroperoxides, and peroxynitrite at the expense of thioredoxin (Trx) B and C in Mycobacterium tuberculosis (Mt). By homology, the enzyme has been classified as an atypical 2-C-peroxiredoxin (Prx), with Cys(60) as the "peroxidatic" cysteine (C(P)) forming a complex catalytic center with Cys(93) as the "resolving" cysteine (C(R)). Site-directed mutagenesis confirms Cys(60) to be C(P) and Cys(80) to be catalytically irrelevant. Replacing Cys(93) with serine leads to fast inactivation as seen by conventional activity determination, which is associated with oxidation of Cys(60) to a sulfinic acid derivative. However, in comparative stopped-flow analysis, WT-MtTPx and MtTPx C93S reduce peroxynitrite and react with TrxB and -C similarly fast. Reduction of pre-oxidized WT-MtTPx and MtTPx C93S by MtTrxB is demonstrated by monitoring the redox-dependent tryptophan fluorescence of MtTrxB. Furthermore, MtTPx C93S remains stable for 10 min at a morpholinosydnonimine hydrochloride-generated low flux of peroxynitrite and excess MtTrxB in a dihydrorhodamine oxidation model. Liquid chromatography-tandem mass spectrometry analysis revealed disulfide bridges between Cys(60) and Cys(93) and between Cys(60) and Cys(80) in oxidized WT-MtTPx. Reaction of pre-oxidized WT-MtTPx and MtTPx C93S with MtTrxB C34S or MtTrxC C40S yielded dead-end intermediates in which the Trx mutants are preferentially linked via disulfide bonds to Cys(60) and never to Cys(93) of the TPx. It is concluded that neither Cys(80) nor Cys(93) is required for the catalytic cycle of the peroxidase. Instead, MtTPx can react as a 1-C-Prx with Cys(60) being the site of attack for both the oxidizing and the reducing substrate. The role of Cys(93) is likely to conserve the oxidation equivalents of the sulfenic acid state of C(P) as a disulfide bond to prevent overoxidation of Cys(60) under a restricted supply of reducing substrate.
    • The Mycobacterium avium ssp. paratuberculosis specific mptD gene is required for maintenance of the metabolic homeostasis necessary for full virulence in mouse infections.

      Meißner, Thorsten; Eckelt, Elke; Basler, Tina; Meens, Jochen; Heinzmann, Julia; Suwandi, Abdulhadi; Oelemann, Walter M R; Trenkamp, Sandra; Holst, Otto; Weiss, Siegfried; et al. (2014)
      Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease, a chronic granulomatous enteritis in ruminants. Furthermore, infections of humans with MAP have been reported and a possible association with Crohn's disease and diabetes type I is currently discussed. MAP owns large sequence polymorphisms (LSPs) that were exclusively found in this mycobacteria species. The relevance of these LSPs in the pathobiology of MAP is still unclear. The mptD gene (MAP3733c) of MAP belongs to a small group of functionally uncharacterized genes, which are not present in any other sequenced mycobacteria species. mptD is part of a predicted operon (mptABCDEF), encoding a putative ATP binding cassette-transporter, located on the MAP-specific LSP14. In the present study, we generated an mptD knockout strain (MAPΔmptD) by specialized transduction. In order to investigate the potential role of mptD in the host, we performed infection experiments with macrophages. By this, we observed a significantly reduced cell number of MAPΔmptD early after infection, indicating that the mutant was hampered with respect to adaptation to the early macrophage environment. This important role of mptD was supported in mouse infection experiments where MAPΔmptD was significantly attenuated after peritoneal challenge. Metabolic profiling was performed to determine the cause for the reduced virulence and identified profound metabolic disorders especially in the lipid metabolism of MAPΔmptD. Overall our data revealed the mptD gene to be an important factor for the metabolic adaptation of MAP required for persistence in the host.
    • Mycobacterium tuberculosis Is a Natural Ornithine Aminotransferase (rocD) Mutant and Depends on Rv2323c for Growth on Arginine.

      Hampel, Annegret; Huber, Claudia; Geffers, Robert; Spona-Friedl, Marina; Eisenreich, Wolfgang; Bange, Franz-Christoph; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2015)
      Mycobacterium tuberculosis (Mtb) possesses a genetic repertoire for metabolic pathways, which are specific and fit to its intracellular life style. Under in vitro conditions, Mtb is known to use arginine as a nitrogen source, but the metabolic pathways for arginine utilization have not been identified. Here we show that, in the presence of arginine, Mtb upregulates a gene cluster which includes an ornithine aminotransferase (rocD) and Rv2323c, a gene of unknown function. Isotopologue analysis by using 13C- or 15N-arginine revealed that in Mtb arginine is not only used as nitrogen source but also as carbon source for the formation of amino acids, in particular of proline. Surprisingly, rocD, which is widespread in other bacteria and is part of the classical arginase pathway turned out to be naturally deleted in Mtb, but not in non-tuberculous mycobacteria. Mtb lacking Rv2323c showed a growth defect on arginine, did not produce proline from arginine, and incorporated less nitrogen derived from arginine in its core nitrogen metabolism. We conclude that the highly induced pathway for arginine utilization in Mtb differs from that of other bacteria including non-tuberculous mycobacteria, probably reflecting a specific metabolic feature of intracellular Mtb.
    • Mycobacterium tuberculosis isolates from Rio de Janeiro reveal unusually low correlation between pyrazinamide resistance and mutations in the pncA gene.

      Bhuju, Sabin; Fonseca, Leila de Souza; Marsico, Anna Grazia; de Oliveira Vieira, Gisele Betzler; Sobral, Luciana Fonseca; Stehr, Matthias; Singh, Mahavir; Saad, Maria Helena Féres; Department of Genome Analytics, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2013-06-14)
      It has been widely accepted, that pyrazinamide (PZA) resistance in Mycobacterium tuberculosis is correlated with mutations in the pncA gene. But since years researchers have been puzzled by the fact that up to 30% of PZA resistant strains do not show any correlation between PZA resistance and mutations in the pncA gene, and thus may vary with geographic area. The objective of the study was to investigate the correlation between PZA susceptibility and mutations in pncA gene in M. tuberculosis isolates from individuals living in a highly endemic area. Therefore we analyzed drug resistant and multidrug resistant (MDR) isolates from patients in Rio de Janeiro, Brazil. From a total of 97 clinical isolates of M. tuberculosis 35 were identified as PZA resistant, 24/35 strains did not show PZase activity and 15/24 (62.5%) strains possess mutation in the pncA gene. This is a low correlation between PZA resistance and PZase activity (68.6%) and even lower correlation between PZA resistance and the presence of mutation in pncA gene (45.7%). Most of the mutations found were conserved near the active site or metal binding site of PZase. The 146A>C mutation was found both in PZA resistant and susceptible isolates, suggesting that this mutation may not fully associated with PZA resistance. Of the mutations found, three have not been previously described. The insertions 192-193 TCCTCGTC and 388-389 AGGTCGATG, although found before, here was found to be a short tandem repeat and in one strain, insertion of the IS6110 was observed 55nt upstream of the gene. All PZA resistant isolates had no mutation in the gene coding ribosomal protein S1 (rpsA), which has recently been proposed as alternate target for pyrazinoic acid (POA). The results show a low association of PZA resistance and pncA gene mutations in a selected patient group from an highly endemic area. Our findings point out that the phenotypic susceptibility testing remains important for the detection of PZA-resistant M. tuberculosis.
    • The mycolyltransferase 85A, a putative drug target of Mycobacterium tuberculosis: development of a novel assay and quantification of glycolipid-status of the mycobacterial cell wall.

      Elamin, Ayssar A; Stehr, Matthias; Oehlmann, Wulf; Singh, Mahavir; Department of Genome Analysis, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany. (2009-12)
      The enzymes of the antigen 85 complex (Ag85A, B, and C) possess mycolyltransferase activity and catalyze the synthesis of the most abundant glycolipid of the mycobacterial cell wall, the cord factor. The cord factor (trehalose 6,6'-dimycolate, TDM) is essential for the integrity of the mycobacterial cell wall and pathogenesis of the bacillus. Thus, TDM biosynthesis is regarded as a potential drug target for control of Mycobacterium tuberculosis infections. Trehalose 6,6'-dimycolate (TDM) is synthesized from two molecules of trehalose-6'-monomycolate (TMM) by antigen 85A. We report here a novel enzyme assay using the natural substrate TMM. The novel colorimetric assay is based on the quantification of glucose from the degradation of trehalose, which is the product from catalytic activity of antigen 85A. Using the new assay, K(m) and K(cat) were determined with values of 129.6+/-8.1 microM and 65.4+/-4.1 min(-1), respectively. This novel assay is also suitable for robust high-throughput screening (HTS) for compound library screening against mycolyltransferase (antigen 85A). The assay is significantly faster and more convenient to use than all assays currently in use. The assay has a very low coefficient of variance (0.04) in 96-well plates and shows a Z' factor of 0.67-0.73, indicating the robustness of the assay. In addition, this new assay is highly suitable for the quantification of total TMM of the mycobacterial cell envelope.
    • Natural Compound Library Screening Identifies New Molecules for the Treatment of Cardiac Fibrosis and Diastolic Dysfunction.

      Schimmel, Katharina; Jung, Mira; Foinquinos, Ariana; José, Gorka San; Beaumont, Javier; Bock, Katharina; Grote-Levi, Lea; Xiao, Ke; Bär, Christian; Pfanne, Angelika; et al. (Lippincott, Williams & Wilkins, 2020-01-17)
      High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds.
    • Neoplastic MiR-17~92 deregulation at a DNA fragility motif (SIDD).

      Schneider, Björn; Nagel, Stefan; Ehrentraut, Stefan; Kaufmann, Maren; Meyer, Corinna; Geffers, Robert; Drexler, Hans G; MacLeod, Roderick A F; Department of Human and Animal Cell Cultures, DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7b, 38124 Braunschweig, Germany. bjoern.schneider@med.uni-rostock.de (2012-03)
      Chromosomal or mutational activation of BCL6 (at 3q27) typifies diffuse large B-cell lymphoma (DLBCL) which in the germinal center subtype may be accompanied by focal amplification of chromosome band 13q31 effecting upregulation of miR-17~92. Using long distance inverse-polymerase chain reaction, we mapped and sequenced six breakpoints of a complex BCL6 rearrangement t(3;13)(q27;q31)t(12;13)(p11;q31) in DLBCL cells, which places miR-17~92 antisense within the resulting ITPR2-BCL6 chimeric fusion gene rearrangement. MiR-17~92 members were upregulated ~15-fold over controls in a copy number independent manner consistent with structural deregulation. MIR17HG and ITPR2-BCL6 were, despite their close configuration, independently expressed, discounting antisense regulation. MIR17HG in t(3;13)t(12;13) cells proved highly responsive to treatment with histone deacetylase inhibitors implicating epigenetic deregulation, consistent with which increased histone-H3 acetylation was detected by chromatin immunoprecipitation near the upstream MIR17HG breakpoint. Remarkably, 5/6 DNA breaks in the t(3;13)t(12;13) precisely cut at stress-induced DNA duplex destabilization (SIDD) peaks reminiscent of chromosomal fragile sites, while the sixth lay 150 bp distant. Extended SIDD profiling showed that additional oncomiRs also map to SIDD peaks. Fluorescence in situ hybridization analysis showed that 11 of 52 (21%) leukemia-lymphoma (L-L) cell lines with 13q31 involvement bore structural rearrangements at/near MIR17HG associated with upregulation. As well as fueling genome instability, SIDD peaks mark regulatory nuclear-scaffold matrix attachment regions open to nucleosomal acetylation. Collectively, our data indict a specific DNA instability motif (SIDD) in chromosome rearrangement, specifically alterations activating miR-17~92 epigenetically via promoter hyperacetylation, and supply a model for the clustering of oncomiRs near cancer breakpoints.
    • Neutrophils responsive to endogenous IFN-beta regulate tumor angiogenesis and growth in a mouse tumor model.

      Jablonska, Jadwiga; Leschner, Sara; Westphal, Kathrin; Lienenklaus, Stefan; Weiss, Siegfried; Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany. jja@gbf.de (2010-04)
      Angiogenesis is a hallmark of malignant neoplasias, as the formation of new blood vessels is required for tumors to acquire oxygen and nutrients essential for their continued growth and metastasis. However, the signaling pathways leading to tumor vascularization are not fully understood. Here, using a transplantable mouse tumor model, we have demonstrated that endogenous IFN-beta inhibits tumor angiogenesis through repression of genes encoding proangiogenic and homing factors in tumor-infiltrating neutrophils. We determined that IFN-beta-deficient mice injected with B16F10 melanoma or MCA205 fibrosarcoma cells developed faster-growing tumors with better-developed blood vessels than did syngeneic control mice. These tumors displayed enhanced infiltration by CD11b+Gr1+ neutrophils expressing elevated levels of the genes encoding the proangiogenic factors VEGF and MMP9 and the homing receptor CXCR4. They also expressed higher levels of the transcription factors c-myc and STAT3, known regulators of VEGF, MMP9, and CXCR4. In vitro, treatment of these tumor-infiltrating neutrophils with low levels of IFN-beta restored expression of proangiogenic factors to control levels. Moreover, depletion of these neutrophils inhibited tumor growth in both control and IFN-beta-deficient mice. We therefore suggest that constitutively produced endogenous IFN-beta is an important mediator of innate tumor surveillance. Further, we believe our data help to explain the therapeutic effect of IFN treatment during the early stages of cancer development.
    • Neutrophils-related host factors associated with severe disease and fatality in patients with influenza infection.

      Tang, Benjamin M; Shojaei, Maryam; Teoh, Sally; Meyers, Adrienne; Ho, John; Ball, T Blake; Keynan, Yoav; Pisipati, Amarnath; Kumar, Aseem; Eisen, Damon P; et al. (Springer-Nature, 2019-07-31)
      Severe influenza infection has no effective treatment available. One of the key barriers to developing host-directed therapy is a lack of reliable prognostic factors needed to guide such therapy. Here, we use a network analysis approach to identify host factors associated with severe influenza and fatal outcome. In influenza patients with moderate-to-severe diseases, we uncover a complex landscape of immunological pathways, with the main changes occurring in pathways related to circulating neutrophils. Patients with severe disease display excessive neutrophil extracellular traps formation, neutrophil-inflammation and delayed apoptosis, all of which have been associated with fatal outcome in animal models. Excessive neutrophil activation correlates with worsening oxygenation impairment and predicted fatal outcome (AUROC 0.817-0.898). These findings provide new evidence that neutrophil-dominated host response is associated with poor outcomes. Measuring neutrophil-related changes may improve risk stratification and patient selection, a critical first step in developing host-directed immune therapy.
    • The NF-κB-dependent and -independent transcriptome and chromatin landscapes of human coronavirus 229E-infected cells.

      Poppe, Michael; Wittig, Sascha; Jurida, Liane; Bartkuhn, Marek; Wilhelm, Jochen; Müller, Helmut; Beuerlein, Knut; Karl, Nadja; Bhuju, Sabin; Ziebuhr, John; et al. (2017-03)
      Coronavirus replication takes place in the host cell cytoplasm and triggers inflammatory gene expression by poorly characterized mechanisms. To obtain more insight into the signals and molecular events that coordinate global host responses in the nucleus of coronavirus-infected cells, first, transcriptome dynamics was studied in human coronavirus 229E (HCoV-229E)-infected A549 and HuH7 cells, respectively, revealing a core signature of upregulated genes in these cells. Compared to treatment with the prototypical inflammatory cytokine interleukin(IL)-1, HCoV-229E replication was found to attenuate the inducible activity of the transcription factor (TF) NF-κB and to restrict the nuclear concentration of NF-κB subunits by (i) an unusual mechanism involving partial degradation of IKKβ, NEMO and IκBα and (ii) upregulation of TNFAIP3 (A20), although constitutive IKK activity and basal TNFAIP3 expression levels were shown to be required for efficient virus replication. Second, we characterized actively transcribed genomic regions and enhancers in HCoV-229E-infected cells and systematically correlated the genome-wide gene expression changes with the recruitment of Ser5-phosphorylated RNA polymerase II and prototypical histone modifications (H3K9ac, H3K36ac, H4K5ac, H3K27ac, H3K4me1). The data revealed that, in HCoV-infected (but not IL-1-treated) cells, an extensive set of genes was activated without inducible p65 NF-κB being recruited. Furthermore, both HCoV-229E replication and IL-1 were shown to upregulate a small set of genes encoding immunomodulatory factors that bind p65 at promoters and require IKKβ activity and p65 for expression. Also, HCoV-229E and IL-1 activated a common set of 440 p65-bound enhancers that differed from another 992 HCoV-229E-specific enhancer regions by distinct TF-binding motif combinations. Taken together, the study shows that cytoplasmic RNA viruses fine-tune NF-κB signaling at multiple levels and profoundly reprogram the host cellular chromatin landscape, thereby orchestrating the timely coordinated expression of genes involved in multiple signaling, immunoregulatory and metabolic processes.
    • NK cell activation in visceral leishmaniasis requires TLR9, myeloid DCs, and IL-12, but is independent of plasmacytoid DCs.

      Schleicher, Ulrike; Liese, Jan; Knippertz, Ilka; Kurzmann, Claudia; Hesse, Andrea; Heit, Antje; Fischer, Jens A A; Weiss, Siegfried; Kalinke, Ulrich; Kunz, Stefanie; et al. (2007-04-16)
      Natural killer (NK) cells are sentinel components of the innate response to pathogens, but the cell types, pathogen recognition receptors, and cytokines required for their activation in vivo are poorly defined. Here, we investigated the role of plasmacytoid dendritic cells (pDCs), myeloid DCs (mDCs), Toll-like receptors (TLRs), and of NK cell stimulatory cytokines for the induction of an NK cell response to the protozoan parasite Leishmania infantum. In vitro, pDCs did not endocytose Leishmania promastigotes but nevertheless released interferon (IFN)-alpha/beta and interleukin (IL)-12 in a TLR9-dependent manner. mDCs rapidly internalized Leishmania and, in the presence of TLR9, produced IL-12, but not IFN-alpha/beta. Depletion of pDCs did not impair the activation of NK cells in L. infantum-infected mice. In contrast, L. infantum-induced NK cell cytotoxicity and IFN-gamma production were abolished in mDC-depleted mice. The same phenotype was observed in TLR9(-/-) mice, which lacked IL-12 expression by mDCs, and in IL-12(-/-) mice, whereas IFN-alpha/beta receptor(-/-) mice showed only a minor reduction of NK cell IFN-gamma expression. This study provides the first direct evidence that mDCs are essential for eliciting NK cell cytotoxicity and IFN-gamma release in vivo and demonstrates that TLR9, mDCs, and IL-12 are functionally linked to the activation of NK cells in visceral leishmaniasis.
    • Non-invasive, ratiometric determination of intracellular pH in Pseudomonas species using a novel genetically encoded indicator.

      Arce-Rodríguez, Alejandro; Volke, Daniel C; Bense, Sarina; Häussler, Susanne; Nikel, Pablo I; HZI, Helmholtz -Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (Wiley Open, 2019-07-01)
      The ability of Pseudomonas species to thrive in all major natural environments (i.e. terrestrial, freshwater and marine) is based on its exceptional capability to adapt to physicochemical changes. Thus, environmental bacteria have to tightly control the maintenance of numerous physiological traits across different conditions. The intracellular pH (pHi ) homoeostasis is a particularly important feature, since the pHi influences a large portion of the biochemical processes in the cell. Despite its importance, relatively few reliable, easy-to-implement tools have been designed for quantifying in vivo pHi changes in Gram-negative bacteria with minimal manipulations. Here we describe a convenient, non-invasive protocol for the quantification of the pHi in bacteria, which is based on the ratiometric fluorescent indicator protein PHP (pH indicator for Pseudomonas). The DNA sequence encoding PHP was thoroughly adapted to guarantee optimal transcription and translation of the indicator in Pseudomonas species. Our PHP-based quantification method demonstrated that pHi is tightly regulated over a narrow range of pH values not only in Pseudomonas, but also in other Gram-negative bacterial species such as Escherichia coli. The maintenance of the cytoplasmic pH homoeostasis in vivo could also be observed upon internal (e.g. redirection of glucose consumption pathways in P. putida) and external (e.g. antibiotic exposure in P. aeruginosa) perturbations, and the PHP indicator was also used to follow dynamic changes in the pHi upon external pH shifts. In summary, our work describes a reliable method for measuring pHi in Pseudomonas, allowing for the detailed investigation of bacterial pHi homoeostasis and its regulation.
    • A Novel Mechanism of Host-Pathogen Interaction through sRNA in Bacterial Outer Membrane Vesicles.

      Koeppen, Katja; Hampton, Thomas H; Jarek, Michael; Scharfe, Maren; Gerber, Scott A; Mielcarz, Daniel W; Demers, Elora G; Dolben, Emily L; Hammond, John H; Hogan, Deborah A; et al. (2016-06)
      Bacterial outer membrane vesicle (OMV)-mediated delivery of proteins to host cells is an important mechanism of host-pathogen communication. Emerging evidence suggests that OMVs contain differentially packaged short RNAs (sRNAs) with the potential to target host mRNA function and/or stability. In this study, we used RNA-Seq to characterize differentially packaged sRNAs in Pseudomonas aeruginosa OMVs, and to show transfer of OMV sRNAs to human airway cells. We selected one sRNA for further study based on its stable secondary structure and predicted mRNA targets. Our candidate sRNA (sRNA52320), a fragment of a P. aeruginosa methionine tRNA, was abundant in OMVs and reduced LPS-induced as well as OMV-induced IL-8 secretion by cultured primary human airway epithelial cells. We also showed that sRNA52320 attenuated OMV-induced KC cytokine secretion and neutrophil infiltration in mouse lung. Collectively, these findings are consistent with the hypothesis that sRNA52320 in OMVs is a novel mechanism of host-pathogen interaction whereby P. aeruginosa reduces the host immune response.
    • Optimizing Salmonella enterica serovar Typhimurium for bacteria-mediated tumor therapy.

      Felgner, Sebastian; Kocijancic, Dino; Frahm, Michael; Curtiss, Roy; Erhardt, Marc; Weiss, Siegfried; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
      Bacteria-mediated tumor therapy using Salmonella enterica serovar Typhimurium is a therapeutic option with great potential. Numerous studies explored the potential of Salmonella Typhimurium for therapeutic applications, however reconciling safety with vectorial efficacy remains a major issue. Recently we have described a conditionally attenuated Salmonella vector that is based on genetic lipopolysaccharide modification. This vector combines strong attenuation with appropriate anti-tumor properties by targeting various cancerous tissues in vivo. Therefore, it was promoted as an anti-tumor agent. In this addendum, we summarize these findings and demonstrate additional optimization steps that may further improve the therapeutic efficacy of our vector strain.
    • An oral multispecies biofilm model for high content screening applications.

      Kommerein, Nadine; Stumpp, Sascha N; Müsken, Mathias; Ehlert, Nina; Winkel, Andreas; Häussler, Susanne; Behrens, Peter; Buettner, Falk F R; Stiesch, Meike; Helmholtz Centre for infection research, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017)
      Peri-implantitis caused by multispecies biofilms is a major complication in dental implant treatment. The bacterial infection surrounding dental implants can lead to bone loss and, in turn, to implant failure. A promising strategy to prevent these common complications is the development of implant surfaces that inhibit biofilm development. A reproducible and easy-to-use biofilm model as a test system for large scale screening of new implant surfaces with putative antibacterial potency is therefore of major importance. In the present study, we developed a highly reproducible in vitro four-species biofilm model consisting of the highly relevant oral bacterial species Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis. The application of live/dead staining, quantitative real time PCR (qRT-PCR), scanning electron microscopy (SEM) and urea-NaCl fluorescence in situ hybridization (urea-NaCl-FISH) revealed that the four-species biofilm community is robust in terms of biovolume, live/dead distribution and individual species distribution over time. The biofilm community is dominated by S. oralis, followed by V. dispar, A. naeslundii and P. gingivalis. The percentage distribution in this model closely reflects the situation in early native plaques and is therefore well suited as an in vitro model test system. Furthermore, despite its nearly native composition, the multispecies model does not depend on nutrient additives, such as native human saliva or serum, and is an inexpensive, easy to handle and highly reproducible alternative to the available model systems. The 96-well plate format enables high content screening for optimized implant surfaces impeding biofilm formation or the testing of multiple antimicrobial treatment strategies to fight multispecies biofilm infections, both exemplary proven in the manuscript.
    • Packaging of Dinoroseobacter shibae DNA into Gene Transfer Agent Particles Is Not Random.

      Tomasch, Jürgen; Wang, Hui; Hall, April T K; Patzelt, Diana; Preusse, Matthias; Petersen, Jörn; Brinkmann, Henner; Bunk, Boyke; Bhuju, Sabin; Jarek, Michael; et al. (2018-01-01)
      Gene transfer agents (GTAs) are phage-like particles which contain a fragment of genomic DNA of the bacterial or archaeal producer and deliver this to a recipient cell. GTA gene clusters are present in the genomes of almost all marine Rhodobacteraceae (Roseobacters) and might be important contributors to horizontal gene transfer in the world's oceans. For all organisms studied so far, no obvious evidence of sequence specificity or other nonrandom process responsible for packaging genomic DNA into GTAs has been found. Here, we show that knock-out of an autoinducer synthase gene of Dinoroseobacter shibae resulted in overproduction and release of functional GTA particles (DsGTA). Next-generation sequencing of the 4.2-kb DNA fragments isolated from DsGTAs revealed that packaging was not random. DNA from low-GC conjugative plasmids but not from high-GC chromids was excluded from packaging. Seven chromosomal regions were strongly overrepresented in DNA isolated from DsGTA. These packaging peaks lacked identifiable conserved sequence motifs that might represent recognition sites for the GTA terminase complex. Low-GC regions of the chromosome, including the origin and terminus of replication, were underrepresented in DNA isolated from DsGTAs. DNA methylation reduced packaging frequency while the level of gene expression had no influence. Chromosomal regions found to be over- and underrepresented in DsGTA-DNA were regularly spaced. We propose that a "headful" type of packaging is initiated at the sites of coverage peaks and, after linearization of the chromosomal DNA, proceeds in both directions from the initiation site. GC-content, DNA-modifications, and chromatin structure might influence at which sides GTA packaging can be initiated.
    • Parallel evolutionary paths to produce more than one biofilm phenotype.

      Thöming, Janne G; Tomasch, Jürgen; Preusse, Matthias; Koska, Michal; Grahl, Nora; Pohl, Sarah; Willger, Sven D; Kaever, Volkhard; Müsken, Mathias; Häussler, Susanne; et al. (Nature publishing group, 2020-01-01)
      Studying parallel evolution of similar traits in independent within-species lineages provides an opportunity to address evolutionary predictability of molecular changes underlying adaptation. In this study, we monitored biofilm forming capabilities, motility, and virulence phenotypes of a plethora of phylogenetically diverse clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa. We also recorded biofilm-specific and planktonic transcriptional responses. We found that P. aeruginosa isolates could be stratified based on the production of distinct organismal traits. Three major biofilm phenotypes, which shared motility and virulence phenotypes, were produced repeatedly in several isolates, indicating that the phenotypes evolved via parallel or convergent evolution. Of note, while we found a restricted general response to the biofilm environment, the individual groups of biofilm phenotypes reproduced biofilm transcriptional profiles that included the expression of well-known biofilm features, such as surface adhesive structures and extracellular matrix components. Our results provide insights into distinct ways to make a biofilm and indicate that genetic adaptations can modulate multiple pathways for biofilm development that are followed by several independent clinical isolates. Uncovering core regulatory pathways that drive biofilm-associated growth and tolerance towards environmental stressors promises to give clues to host and environmental interactions and could provide useful targets for new clinical interventions.
    • The peptide chain release factor methyltransferase PrmC is essential for pathogenicity and environmental adaptation of Pseudomonas aeruginosa PA14.

      Pustelny, Christian; Brouwer, Stephan; Müsken, Mathias; Bielecka, Agata; Dötsch, Andreas; Nimtz, Manfred; Häussler, Susanne; Department of Molecular Bacteriology, Helmholtz Center for Infection Research, Braunschweig, Germany. Christian.pustelny@helmholtz-hzi.de (2013-02)
      Pseudomonas aeruginosa pathogenicity and its capability to adapt to multiple environments are dependent on the production of diverse virulence factors, controlled by the sophisticated quorum sensing (QS) network of P. aeruginosa. To better understand the molecular mechanisms that underlie this adaptation we searched for novel key regulators of virulence factor production by screening a PA14 transposon mutant library for potential candidates acting downstream of the unique 2-alkyl-4-quinolone (AQ) QS system of P. aeruginosa. We focused the work on a protein named HemK with high homology to PrmC of Escherichia coli displaying a similar enzymatic activity (therefore also referred to as PrmC). In this study, we demonstrate that PrmC is an S-adenosyl-l-methionine (AdoMet)-dependent methyltransferase of peptide chain release factors (RFs) essential for the expression of several virulence factors, such as pyocyanin, rhamnolipids and the type III-secreted toxin ExoT. Furthermore, the PA14_prmC mutant strain is unable to grow under anoxic conditions and has a significantly reduced pathogenicity in the infection model Galleria mellonella. Along with transcriptomic and proteomic analyses, the presented data indicate that the methylation of RFs in P. aeruginosa seems to have a global effect on cellular processes related to the virulence of this nosocomial pathogen.
    • Peripheral T-cell lymphoma cell line T8ML-1 highlights conspicuous targeting of PVRL2 by t(14;19)(q11.2;q13.3).

      Ehrentraut, Stefan; Nagel, Stefan; Pommerenke, Claudia; Dirks, Wilhelm G; Quentmeier, Hilmar; Kaufmann, Maren; Meyer, Corinna; Zaborski, Margarete; Geffers, Robert; Fujiwara, Hiroshi; et al. (2017-01-01)
      Focal amplifications and chromosome translocations involving the long arm of chromosome 19 (19q13.3) are recurrent in T-cell lymphoma, where neighboring BCL3 and PVRL2 are competing target genes. Here we present the oncogenomic characterization of a peripheral T-cell lymphoma (PTCL) cell line T8ML-1 to reveal t(14;19)(q11.2;q13.3) juxtaposing TRA@ and PVRL2. Parallel mRNA and protein expression data for the 19q13.3 region of interest pinpointed PVRL2 as the sole conspicuous target therein. Collectively, our findings endorse T8ML-1 as the first proven cell line model for t(14;19)/PTCL.