• 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.
    • No impact of a short-term climatic "El Niño" fluctuation on gut microbial diversity in populations of the Galápagos marine iguana (Amblyrhynchus cristatus).

      Ibáñez, Alejandro; Bletz, Molly C; Quezada, Galo; Geffers, Robert; Jarek, Michael; Vences, Miguel; Steinfartz, Sebastian; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Springer, 2021-02-02)
      Gut microorganisms are crucial for many biological functions playing a pivotal role in the host's well-being. We studied gut bacterial community structure of marine iguana populations across the Galápagos archipelago. Marine iguanas depend heavily on their specialized gut microbiome for the digestion of dietary algae, a resource whose growth was strongly reduced by severe "El Niño"-related climatic fluctuations in 2015/2016. As a consequence, marine iguana populations showed signs of starvation as expressed by a poor body condition. Body condition indices (BCI) varied between island populations indicating that food resources (i.e., algae) are affected differently across the archipelago during 'El Niño' events. Though this event impacted food availability for marine iguanas, we found that reductions in body condition due to "El Niño"-related starvation did not result in differences in bacterial gut community structure. Species richness of gut microorganisms was instead correlated with levels of neutral genetic diversity in the distinct host populations. Our data suggest that marine iguana populations with a higher level of gene diversity and allelic richness may harbor a more diverse gut microbiome than those populations with lower genetic diversity. Since low values of these diversity parameters usually correlate with small census and effective population sizes, we use our results to propose a novel hypothesis according to which small and genetically less diverse host populations might be characterized by less diverse microbiomes. Whether such genetically depauperate populations may experience additional threats from reduced dietary flexibility due to a limited intestinal microbiome is currently unclear and calls for further investigation.
    • 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.
    • Opuntisines, 14-membered cyclopeptide alkaloids from fruits of Opuntia stricta var. dillenii isolated by high-performance countercurrent chromatography.

      Surup, Frank; Minh Thi Tran, Thu; Pfütze, Sebastian; Budde, Jarmo; Moussa-Ayoub, Tamer E; Rohn, Sascha; Jerz, Gerold; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2020-07-13)
      Extracts of Opuntia stricta var. dillenii fruits were fractionated by semi-preparative high-performance countercurrent chromatography (HPCCC) to study the secondary metabolite formation, whereby HPCCC showed a superior separation capacity to fractionate minor metabolites compared to HPLC. A family of new peptides was detected in semi-polar fractions when monitoring the HPCCC separation by off-line injections of fractions to ESI-MS/MS. Planar structures of the major compounds, two 14-ring-membered cyclopeptide alkaloids, which were named opuntisines A and B, were elucidated by 1D- and 2D-NMR spectroscopy and HR-ESI-MS/MS spectrometry, while a combination of chemical derivatisation and degradation revealed the stereo-configurations. Specifically, the methods of Marfey and Mosher indicated l-Glu, l-Ile, l-Phe and 1S-configurations, respectively; ROESY correlations revealed 8S, 9S. The novel opuntisine A showed moderate activity against the Gram-negative bacterium Escherichia coli, but no further antibacterial, antifungal nor cytotoxic effects. This bioactive natural product class is reported for the first time in the plant family Cactaceae.
    • 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.
    • Organism-specific depletion of highly abundant RNA species from bacterial total RNA.

      Engelhardt, Florian; Tomasch, Jürgen; Häussler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Microbiology Society, 2020-09-09)
      High-throughput sequencing has become a standard tool for transcriptome analysis. The depletion of overrepresented RNA species from sequencing libraries plays a key role in establishing potent and cost-efficient RNA-seq routines. Commercially available kits are known to obtain good results for the reduction of ribosomal RNA (rRNA). However, we found that the transfer-messenger RNA (tmRNA) was frequently highly abundant in rRNA-depleted samples of Pseudomonas aeruginosa , consuming up to 25 % of the obtained reads. The tmRNA fraction was particularly high in samples taken from stationary cultures. This suggests that overrepresentation of this RNA species reduces the mRNA fraction when cells are grown under challenging conditions. Here, we present an RNase-H-based depletion protocol that targets the tmRNA in addition to ribosomal RNAs. We were able to increase the mRNA fraction to 93-99% and therefore outperform not only the commercially Ribo-off kit (Vazyme) operating by the same principle but also the formerly widely used Ribo-Zero kit (Illumina). Maximizing the read share of scientifically interesting RNA species enhances the discriminatory potential of next-generation RNA-seq experiments and, therefore, can contribute to a better understanding of the transcriptomic landscape of bacterial pathogens and their used mechanisms in host infection.
    • p53-Independent Induction of p21 Fails to Control Regeneration and Hepatocarcinogenesis in a Murine Liver Injury Model.

      Buitrago-Molina, Laura Elisa; Marhenke, Silke; Becker, Diana; Geffers, Robert; Itzel, Timo; Teufel, Andreas; Jaeschke, Hartmut; Lechel, André; Unger, Kristian; Markovic, Jovana; et al. (Elsevier, 2021-01-21)
      Background & aims: A coordinated stress and regenerative response is important after hepatocyte damage. Here, we investigate the phenotypes that result from genetic abrogation of individual components of the checkpoint kinase 2/transformation-related protein 53 (p53)/cyclin-dependent kinase inhibitor 1A (p21) pathway in a murine model of metabolic liver injury. Methods: Nitisinone was reduced or withdrawn in Fah-/- mice lacking Chk2, p53, or p21, and survival, tumor development, liver injury, and regeneration were analyzed. Partial hepatectomies were performed and mice were challenged with the Fas antibody Jo2. Results: In a model of metabolic liver injury, loss of p53, but not Chk2, impairs the oxidative stress response and aggravates liver damage, indicative of a direct p53-dependent protective effect on hepatocytes. Cell-cycle control during chronic liver injury critically depends on the presence of both p53 and its downstream effector p21. In p53-deficient hepatocytes, unchecked proliferation occurs despite a strong induction of p21, showing a complex interdependency between p21 and p53. The increased regenerative potential in the absence of p53 cannot fully compensate the surplus injury and is not sufficient to promote survival. Despite the distinct phenotypes associated with the loss of individual components of the DNA damage response, gene expression patterns are dominated by the severity of liver injury, but reflect distinct effects of p53 on proliferation and the anti-oxidative stress response. Conclusions: Characteristic phenotypes result from the genetic abrogation of individual components of the DNA damage-response cascade in a liver injury model. The extent to which loss of gene function can be compensated, or affects injury and proliferation, is related to the level at which the cascade is interrupted. Accession numbers of repository for expression microarray data: GSE156983, GSE156263, GSE156852, and GSE156252.
    • 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 Influences the Pseudomonas aeruginosa PA14 Endo- and Exometabolome.

      Depke, Tobias; Häussler, Susanne; Brönstrup, Mark; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-10-18)
      Pseudomonas aeruginosa is one of the most important nosocomial pathogens and understanding its virulence is the key to effective control of P. aeruginosa infections. The regulatory network governing virulence factor production in P. aeruginosa is exceptionally complex. Previous studies have shown that the peptide chain release factor methyltransferase PrmC plays an important role in bacterial pathogenicity. Yet, the underlying molecular mechanism is incompletely understood. In this study, we used untargeted liquid and gas chromatography coupled to mass spectrometry to characterise the metabolome of a prmC defective P. aeruginosa PA14 strain in comparison with the corresponding strain complemented with prmC in trans. The comprehensive metabolomics data provided new insight into the influence of prmC on virulence and metabolism. prmC deficiency had broad effects on the endo- and exometabolome of P. aeruginosa PA14, with a marked decrease of the levels of aromatic compounds accompanied by reduced precursor supply from the shikimate pathway. Furthermore, a pronounced decrease of phenazine production was observed as well as lower abundance of alkylquinolones. Unexpectedly, the metabolomics data showed no prmC-dependent effect on rhamnolipid production and an increase in pyochelin levels. A putative virulence biomarker identified in a previous study was significantly less abundant in the prmC deficient strain.
    • 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.
    • pH-degradable imidazoquinoline-ligated nanogels for lymph node-focused immune activation.

      Nuhn, Lutz; Vanparijs, Nane; De Beuckelaer, Ans; Lybaert, Lien; Verstraete, Glenn; Deswarte, Kim; Lienenklaus, Stefan; Shukla, Nikunj M; Salyer, Alex C D; Lambrecht, Bart N; et al. (2016-07-19)
      Agonists of Toll-like receptors (TLRs) are potent activators of the innate immune system and hold promise as vaccine adjuvant and for anticancer immunotherapy. Unfortunately, in soluble form they readily enter systemic circulation and cause systemic inflammatory toxicity. Here we demonstrate that by covalent ligation of a small-molecule imidazoquinoline-based TLR7/8 agonist to 50-nm-sized degradable polymeric nanogels the potency of the agonist to activate TLR7/8 in in vitro cultured dendritic cells is largely retained. Importantly, imidazoquinoline-ligated nanogels focused the in vivo immune activation on the draining lymph nodes while dramatically reducing systemic inflammation. Mechanistic studies revealed a prevalent passive diffusion of the nanogels to the draining lymph node. Moreover, immunization studies in mice have shown that relative to soluble TLR7/8 agonist, imidazoquinoline-ligated nanogels induce superior antibody and T-cell responses against a tuberculosis antigen. This approach opens possibilities to enhance the therapeutic benefit of small-molecule TLR agonist for a variety of applications.