• Dissecting Herpes Simplex Virus 1-Induced Host Shutoff at the RNA Level.

      Friedel, Caroline C; Whisnant, Adam W; Djakovic, Lara; Rutkowski, Andrzej J; Friedl, Marie-Sophie; Kluge, Michael; Williamson, James C; Sai, Somesh; Vidal, Ramon Oliveira; Sauer, Sascha; et al. (American Society for Microbilogy (ASM), 2020-11-04)
      Herpes simplex virus 1 (HSV-1) induces a profound host shut-off during lytic infection. The virion host shut-off (vhs) protein plays a key role in this process by efficiently cleaving host and viral mRNAs. Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in host transcriptional activity. To dissect relative contributions of both mechanisms and elucidate gene-specific host transcriptional responses throughout the first 8h of lytic HSV-1 infection, we employed RNA-seq of total, newly transcribed (4sU-labelled) and chromatin-associated RNA in wild-type (WT) and Δvhs infection of primary human fibroblasts. Following virus entry, vhs activity rapidly plateaued at an elimination rate of around 30% of cellular mRNAs per hour until 8h p.i. In parallel, host transcriptional activity dropped to 10-20%. While the combined effects of both phenomena dominated infection-induced changes in total RNA, extensive gene-specific transcriptional regulation was observable in chromatin-associated RNA and was surprisingly concordant between WT and Δvhs infection. Both induced strong transcriptional up-regulation of a small subset of genes that were poorly expressed prior to infection but already primed by H3K4me3 histone marks at their promoters. Most interestingly, analysis of chromatin-associated RNA revealed vhs-nuclease-activity-dependent transcriptional down-regulation of at least 150 cellular genes, in particular of many integrin adhesome and extracellular matrix components. This was accompanied by a vhs-dependent reduction in protein levels by 8h p.i. for many of these genes. In summary, our study provides a comprehensive picture of the molecular mechanisms that govern cellular RNA metabolism during the first 8h of lytic HSV-1 infection.IMPORTANCE The HSV-1 virion host shut-off (vhs) protein efficiently cleaves both host and viral mRNAs in a translation-dependent manner. In this study, we model and quantify changes in vhs activity as well as virus-induced global loss of host transcriptional activity during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA levels were dominated by these two global effects. In contrast, chromatin-associated RNA depicted gene-specific transcriptional changes. This revealed highly concordant transcriptional changes in WT and Δvhs infection, confirmed DUX4 as a key transcriptional regulator in HSV-1 infection and depicted vhs-dependent, transcriptional down-regulation of the integrin adhesome and extracellular matrix components. The latter explained seemingly gene-specific effects previously attributed to vhs-mediated mRNA degradation and resulted in a concordant loss in protein levels by 8h p.i. for many of the respective genes.
    • Drug-Induced Dynamics of Bile Colloids.

      Hanio, Simon; Schlauersbach, Jonas; Lenz, Bettina; Spiegel, Franziska; Böckmann, Rainer A; Schweins, Ralf; Nischang, Ivo; Schubert, Ulrich S; Endres, Sebastian; Pöppler, Ann-Christin; et al. (American Society for Chemistry (ACS), 2021-02-15)
      Bile colloids containing taurocholate and lecithin are essential for the solubilization of hydrophobic molecules including poorly water-soluble drugs such as Perphenazine. We detail the impact of Perphenazine concentrations on taurocholate/lecithin colloids using analytical ultracentrifugation, dynamic light scattering, small-angle neutron scattering, nuclear magnetic resonance spectroscopy, coarse-grained molecular dynamics simulations, and isothermal titration calorimetry. Perphenazine impacted colloidal molecular arrangement, structure, and binding thermodynamics in a concentration-dependent manner. At low concentration, Perphenazine was integrated into stable and large taurocholate/lecithin colloids and close to lecithin. Integration of Perphenazine into these colloids was exothermic. At higher Perphenazine concentration, the taurocholate/lecithin colloids had an approximately 5-fold reduction in apparent hydrodynamic size, heat release was less exothermic upon drug integration into the colloids, and Perphenazine interacted with both lecithin and taurocholate. In addition, Perphenazine induced a morphological transition from vesicles to wormlike micelles as indicated by neutron scattering. Despite these surprising colloidal dynamics, these natural colloids successfully ensured stable relative amounts of free Perphenazine throughout the entire drug concentration range tested here. Future studies are required to further detail these findings both on a molecular structural basis and in terms of in vivo relevance.
    • Dual RNA-seq of Orientia tsutsugamushi informs on host-pathogen interactions for this neglected intracellular human pathogen.

      Mika-Gospodorz, Bozena; Giengkam, Suparat; Westermann, Alexander J; Wongsantichon, Jantana; Kion-Crosby, Willow; Chuenklin, Suthida; Wang, Loo Chien; Sunyakumthorn, Piyanate; Sobota, Radoslaw M; Subbian, Selvakumar; et al. (Nature Publishing Group, 2020-07-03)
      Studying emerging or neglected pathogens is often challenging due to insufficient information and absence of genetic tools. Dual RNA-seq provides insights into host-pathogen interactions, and is particularly informative for intracellular organisms. Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), an obligate intracellular bacterium that causes the vector-borne human disease scrub typhus. Half the Ot genome is composed of repetitive DNA, and there is minimal collinearity in gene order between strains. Integrating RNA-seq, comparative genomics, proteomics, and machine learning to study the transcriptional architecture of Ot, we find evidence for wide-spread post-transcriptional antisense regulation. Comparing the host response to two clinical isolates, we identify distinct immune response networks for each strain, leading to predictions of relative virulence that are validated in a mouse infection model. Thus, dual RNA-seq can provide insight into the biology and host-pathogen interactions of a poorly characterized and genetically intractable organism such as Ot.
    • Einzelzell-RNA-Sequenzierung beleuchtet den Infektionsprozess

      Saliba, Antoine-Emmanuel; Westermann, Alexander J.; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Schneider-Straße 2, 97080 Würzburg. Germany. (2017-10-11)
    • Frugal Innovation for Point-of-Care Diagnostics Controlling Outbreaks and Epidemics.

      Miesler, Tobias; Wimschneider, Christine; Brem, Alexander; Meinel, Lorenz; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (American Chemical Society (ACS), 2020-04-13)
      Today epidemics of infectious diseases occur more often and spread both faster and further due to globalization and changes in our lifestyle. One way to meet these biological threats are so-called "Frugal Innovations", which focus on the development of affordable, rapid, and easy-to-use diagnostics with widespread use. In this context, point-of-care-tests (POCTs), performed at the patient's bedside, reduce extensive waiting times and unnecessary treatments and enable effective containment measures. This Perspective covers advances in POCT diagnostics on the basis of frugal innovation characteristics that will enable a faster, less expensive, and more convenient reaction to upcoming epidemics. Established POCT systems on the health care market, as well as currently evolving technological advancements in that sector are discussed. Progress in POCT technology and insights on how to most effectively use them allows the handling of more patients in a shorter time frame and consequently improves clinical outcomes at lower cost.
    • Genome organization and DNA accessibility control antigenic variation in trypanosomes.

      Müller, Laura S M; Cosentino, Raúl O; Förstner, Konrad U; Guizetti, Julien; Wedel, Carolin; Kaplan, Noam; Janzen, Christian J; Arampatzi, Panagiota; Vogel, Jörg; Steinbiss, Sascha; et al. (2018-01-01)
      Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host1. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility2,3. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding4. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses—Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing—that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation.
    • A genome-wide transcriptomic analysis of embryos fathered by obese males in a murine model of diet-induced obesity.

      Bernhardt, Laura; Dittrich, Marcus; El-Merahbi, Rabih; Saliba, Antoine-Emmanuel; Müller, Tobias; Sumara, Grzegorz; Vogel, Jörg; Nichols-Burns, Stefanie; Mitchell, Megan; Haaf, Thomas; et al. (Nature Pulishing Group, 2021-01-21)
      Paternal obesity is known to have a negative impact on the male's reproductive health as well as the health of his offspring. Although epigenetic mechanisms have been implicated in the non-genetic transmission of acquired traits, the effect of paternal obesity on gene expression in the preimplantation embryo has not been fully studied. To this end, we investigated whether paternal obesity is associated with gene expression changes in eight-cell stage embryos fathered by males on a high-fat diet. We used single embryo RNA-seq to compare the gene expression profile of embryos generated by males on a high fat (HFD) versus control (CD) diet. This analysis revealed significant upregulation of the Samd4b and Gata6 gene in embryos in response to a paternal HFD. Furthermore, we could show a significant increase in expression of both Gata6 and Samd4b during differentiation of stromal vascular cells into mature adipocytes. These findings suggest that paternal obesity may induce changes in the male germ cells which are associated with the gene expression changes in the resulting preimplantation embryos.
    • A global data-driven census of Salmonella small proteins and their potential functions in bacterial virulence

      Venturini, Elisa; Svensson, Sarah L; Maaß, Sandra; Gelhausen, Rick; Eggenhofer, Florian; Li, Lei; Cain, Amy K; Parkhill, Julian; Becher, Dörte; Backofen, Rolf; et al. (Oxford University Press (OUP), 2020-10-17)
      Small proteins are an emerging class of gene products with diverse roles in bacterial physiology. However, a full understanding of their importance has been hampered by insufficient genome annotations and a lack of comprehensive characterization in microbes other than Escherichia coli. We have taken an integrative approach to accelerate the discovery of small proteins and their putative virulence-associated functions in Salmonella Typhimurium. We merged the annotated small proteome of Salmonella with new small proteins predicted with in silico and experimental approaches. We then exploited existing and newly generated global datasets that provide information on small open reading frame expression during infection of epithelial cells (dual RNA-seq), contribution to bacterial fitness inside macrophages (Transposon-directed insertion sequencing), and potential engagement in molecular interactions (Grad-seq). This integrative approach suggested a new role for the small protein MgrB beyond its known function in regulating PhoQ. We demonstrate a virulence and motility defect of a Salmonella ΔmgrB mutant and reveal an effect of MgrB in regulating the Salmonella transcriptome and proteome under infection-relevant conditions. Our study highlights the power of interpreting available ‘omics’ datasets with a focus on small proteins, and may serve as a blueprint for a data integration-based survey of small proteins in diverse bacteria.
    • Global discovery of bacterial RNA-binding proteins by RNase-sensitive gradient profiles reports a new FinO domain protein.

      Gerovac, Milan; El Mouali, Youssef; Kuper, Jochen; Kisker, Caroline; Barquist, Lars; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Cold Spring Habor Laboratory Press and RNA Society, 2020-07-09)
      RNA-binding proteins (RBPs) play important roles in bacterial gene expression and physiology but their true number and functional scope remain little understood even in model microbes. To advance global RBP discovery in bacteria, we here establish glycerol gradient sedimentation with RNase treatment and mass spectrometry (GradR). Applied to Salmonella enterica, GradR confirms many known RBPs such as CsrA, Hfq, and ProQ by their RNase-sensitive sedimentation profiles, and discovers the FopA protein as a new member of the emerging family of FinO/ProQ-like RBPs. FopA, encoded on resistance plasmid pCol1B9, primarily targets a small RNA associated with plasmid replication. The target suite of FopA dramatically differs from the related global RBP ProQ, revealing context-dependent selective RNA recognition by FinO-domain RBPs. Numerous other unexpected RNase-induced changes in gradient profiles suggest that cellular RNA helps to organize macromolecular complexes in bacteria. By enabling poly(A)-independent generic RBP discovery, GradR provides an important element in the quest to build a comprehensive catalog of microbial RBPs.
    • Global Maps of ProQ Binding In Vivo Reveal Target Recognition via RNA Structure and Stability Control at mRNA 3' Ends.

      Holmqvist, Erik; Li, Lei; Bischler, Thorsten; Barquist, Lars; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Elsevier, 2018-06-07)
      The conserved RNA-binding protein ProQ has emerged as the centerpiece of a previously unknown third large network of post-transcriptional control in enterobacteria. Here, we have used in vivo UV crosslinking and RNA sequencing (CLIP-seq) to map hundreds of ProQ binding sites in Salmonella enterica and Escherichia coli. Our analysis of these binding sites, many of which are conserved, suggests that ProQ recognizes its cellular targets through RNA structural motifs found in small RNAs (sRNAs) and at the 3′ end of mRNAs. Using the cspE mRNA as a model for 3′ end targeting, we reveal a function for ProQ in protecting mRNA against exoribonucleolytic activity. Taken together, our results underpin the notion that ProQ governs a post-transcriptional network distinct from those of the well-characterized sRNA-binding proteins, CsrA and Hfq, and suggest a previously unrecognized, sRNA-independent role of ProQ in stabilizing mRNAs.
    • Global RNA profiles show target selectivity and physiological effects of peptide-delivered antisense antibiotics.

      Popella, Linda; Jung, Jakob; Popova, Kristina; Ðurica-Mitić, Svetlana; Barquist, Lars; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
      Antisense peptide nucleic acids (PNAs) inhibiting mRNAs of essential genes provide a straight-forward way to repurpose our knowledge of bacterial regulatory RNAs for development of programmable species-specific antibiotics. While there is ample proof of PNA efficacy, their target selectivity and impact on bacterial physiology are poorly understood. Moreover, while antibacterial PNAs are typically designed to block mRNA translation, effects on target mRNA levels are not well-investigated. Here, we pioneer the use of global RNA-seq analysis to decipher PNA activity in a transcriptome-wide manner. We find that PNA-based antisense oligomer conjugates robustly decrease mRNA levels of the widely-used target gene, acpP, in Salmonella enterica, with limited off-target effects. Systematic analysis of several different PNA-carrier peptides attached not only shows different bactericidal efficiency, but also activation of stress pathways. In particular, KFF-, RXR- and Tat-PNA conjugates especially induce the PhoP/Q response, whereas the latter two additionally trigger several distinct pathways. We show that constitutive activation of the PhoP/Q response can lead to Tat-PNA resistance, illustrating the utility of RNA-seq for understanding PNA antibacterial activity. In sum, our study establishes an experimental framework for the design and assessment of PNA antimicrobials in the long-term quest to use these for precision editing of microbiota.
    • Global snapshots of bacterial RNA networks.

      Hör, Jens; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung,Josef-Schneider-Straße 2, 97080 Würzburg, Germany. (2017-02-01)
    • Grad-seq in a Gram-positive bacterium reveals exonucleolytic sRNA activation in competence control.

      Hör, Jens; Garriss, Geneviève; Di Giorgio, Silvia; Hack, Lisa-Marie; Vanselow, Jens T; Förstner, Konrad U; Schlosser, Andreas; Henriques-Normark, Birgitta; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (EMBO Press, 2020-03-30)
      RNA-protein interactions are the crucial basis for many steps of bacterial gene expression, including post-transcriptional control by small regulatory RNAs (sRNAs). In stark contrast to recent progress in the analysis of Gram-negative bacteria, knowledge about RNA-protein complexes in Gram-positive species remains scarce. Here, we used the Grad-seq approach to draft a comprehensive landscape of such complexes in Streptococcus pneumoniae, in total determining the sedimentation profiles of ~ 88% of the transcripts and ~ 62% of the proteins of this important human pathogen. Analysis of in-gradient distributions and subsequent tag-based protein capture identified interactions of the exoribonuclease Cbf1/YhaM with sRNAs that control bacterial competence for DNA uptake. Unexpectedly, the nucleolytic activity of Cbf1 stabilizes these sRNAs, thereby promoting their function as repressors of competence. Overall, these results provide the first RNA/protein complexome resource of a Gram-positive species and illustrate how this can be utilized to identify new molecular factors with functions in RNA-based regulation of virulence-relevant pathways.
    • Grad-seq shines light on unrecognized RNA and protein complexes in the model bacterium Escherichia coli.

      Hör, Jens; Di Giorgio, Silvia; Gerovac, Milan; Venturini, Elisa; Förstner, Konrad U; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Oxford University Press, 2020-08-19)
      Stable protein complexes, including those formed with RNA, are major building blocks of every living cell. Escherichia coli has been the leading bacterial organism with respect to global protein-protein networks. Yet, there has been no global census of RNA/protein complexes in this model species of microbiology. Here, we performed Grad-seq to establish an RNA/protein complexome, reconstructing sedimentation profiles in a glycerol gradient for ∼85% of all E. coli transcripts and ∼49% of the proteins. These include the majority of small noncoding RNAs (sRNAs) detectable in this bacterium as well as the general sRNA-binding proteins, CsrA, Hfq and ProQ. In presenting use cases for utilization of these RNA and protein maps, we show that a stable association of RyeG with 30S ribosomes gives this seemingly noncoding RNA of prophage origin away as an mRNA of a toxic small protein. Similarly, we show that the broadly conserved uncharacterized protein YggL is a 50S subunit factor in assembled 70S ribosomes. Overall, this study crucially extends our knowledge about the cellular interactome of the primary model bacterium E. coli through providing global RNA/protein complexome information and should facilitate functional discovery in this and related species.
    • A Grad-seq View of RNA and Protein Complexes in Pseudomonas aeruginosa under Standard and Bacteriophage Predation Conditions.

      Gerovac, Milan; Wicke, Laura; Chihara, Kotaro; Schneider, Cornelius; Lavigne, Rob; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (American Society for Microbiology (ASM), 2021-02-09)
      The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and posttranscriptional regulation through the RNA-binding proteins Hfq, RsmA, and RsmN have been studied in detail, global information about stable RNA-protein complexes in this human pathogen is currently lacking. Here, we implement gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73% of all transcripts and ∼40% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa, including 6S RNA with RNA polymerase and associated product RNAs (pRNAs). We observe specific clusters of noncoding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain-containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased cosedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded noncoding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell.IMPORTANCE Stable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation and physiology in any bacterium of interest. It is therefore crucial to globally determine these complexes in order to identify and characterize new molecular players and regulation mechanisms. Pseudomonads harbor some of the largest genomes known in bacteria, encoding ∼5,500 different proteins. Here, we provide a first glimpse on which proteins and cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa We additionally performed this analysis with bacteria subjected to the important and frequently encountered biological stress of a bacteriophage infection. We identified several molecules with established roles in a variety of cellular pathways, which were affected by the phage and can now be explored for their role during phage infection. Most importantly, we observed strong colocalization of phage transcripts and host ribosomes, indicating the existence of specialized translation mechanisms during phage infection. All data are publicly available in an interactive and easy to use browser.
    • Herpes simplex virus blocks host transcription termination via the bimodal activities of ICP27.

      Wang, Xiuye; Hennig, Thomas; Whisnant, Adam W; Erhard, Florian; Prusty, Bhupesh K; Friedel, Caroline C; Forouzmand, Elmira; Hu, William; Erber, Luke; Chen, Yue; et al. (Nature publishing group, 2020-01-15)
      Infection by viruses, including herpes simplex virus-1 (HSV-1), and cellular stresses causewidespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) inhost genes. However, the underlying mechanisms remain unclear. Here, we demonstrate thatthe HSV-1 immediate early protein ICP27 induces DoTT by directly binding to the essentialmRNA 3’processing factor CPSF. It thereby induces the assembly of a dead-end 3’processing complex, blocking mRNA 3’cleavage. Remarkably, ICP27 also acts as a sequence-dependent activator of mRNA 3’processing for viral and a subset of host transcripts.Our results unravel a bimodal activity of ICP27 that plays a key role in HSV-1-induced hostshutoff and identify CPSF as an important factor that mediates regulation of transcriptiontermination. Thesefindings have broad implications for understanding the regulation oftranscription termination by other viruses, cellular stress and cancer.
    • HHV-6 encoded small non-coding RNAs define an intermediate and early stage in viral reactivation.

      Prusty, Bhupesh K; Gulve, Nitish; Chowdhury, Suvagata Roy; Schuster, Michael; Strempel, Sebastian; Descamps, Vincent; Rudel, Thomas; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (2018-01-01)
      Human herpesvirus 6A and 6B frequently acquires latency. HHV-6 activation has been associated with various human diseases. Germ line inheritance of chromosomally integrated HHV-6 makes viral DNA-based analysis difficult for determination of early stages of viral activation. We characterized early stages of HHV-6 activation using high throughput transcriptomics studies and applied the results to understand virus activation under clinical conditions. Using a latent HHV-6A cell culture model in U2OS cells, we identified an early stage of viral reactivation, which we define as transactivation that is marked by transcription of several viral small non-coding RNAs (sncRNAs) in the absence of detectable increase in viral replication and proteome. Using deep sequencing approaches, we detected previously known as well as a new viral sncRNAs that characterized viral transactivation and differentiated it from latency. Here we show changes in human transcriptome upon viral transactivation that reflect multiple alterations in mitochondria-associated pathways, which was supported by observation of increased mitochondrial fragmentation in virus reactivated cells. Furthermore, we present here a unique clinical case of DIHS/DRESS associated death where HHV-6 sncRNA-U14 was abundantly detected throughout the body of the patient in the presence of low viral DNA. In this study, we have identified a unique and early stage of viral activation that is characterized by abundant transcription of viral sncRNAs, which can serve as an ideal biomarker under clinical conditions.
    • How Insertion of a Single Tryptophan in the N-Terminus of a Cecropin A-Melittin Hybrid Peptide Changes Its Antimicrobial and Biophysical Profile.

      Ferreira, Ana Rita; Teixeira, Cátia; Sousa, Carla F; Bessa, Lucinda J; Gomes, Paula; Gameiro, Paula; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (MDPI, 2021-01-12)
      n the era of antibiotic resistance, there is an urgent need for efficient antibiotic therapies to fight bacterial infections. Cationic antimicrobial peptides (CAMP) are promising lead compounds given their membrane-targeted mechanism of action, and high affinity towards the anionic composition of bacterial membranes. We present a new CAMP, W-BP100, derived from the highly active BP100, holding an additional tryptophan at the N-terminus. W-BP100 showed a broader antibacterial activity, demonstrating a potent activity against Gram-positive strains. Revealing a high partition constant towards anionic over zwitterionic large unilamellar vesicles and inducing membrane saturation at a high peptide/lipid ratio, W-BP100 has a preferential location for hydrophobic environments. Contrary to BP100, almost no aggregation of anionic vesicles is observed around saturation conditions and at higher concentrations no aggregation is observed. With these results, it is possible to state that with the incorporation of a single tryptophan to the N-terminus, a highly active peptide was obtained due to the π-electron system of tryptophan, resulting in negatively charged clouds, that participate in cation-π interactions with lysine residues. Furthermore, we propose that W-BP100 action can be achieved by electrostatic interactions followed by peptide translocation.
    • Humanized mice for modeling human infectious disease: challenges, progress, and outlook.

      Legrand, Nicolas; Ploss, Alexander; Balling, Rudi; Becker, Pablo D; Borsotti, Chiara; Brezillon, Nicolas; Debarry, Jennifer; de Jong, Ype; Deng, Hongkui; Di Santo, James P; et al. (2009-07-23)
      Over 800 million people worldwide are infected with hepatitis viruses, human immunodeficiency virus (HIV), and malaria, resulting in more than 5 million deaths annually. Here we discuss the potential and challenges of humanized mouse models for developing effective and affordable therapies and vaccines, which are desperately needed to combat these diseases.
    • Identification of a Novel LysR-Type Transcriptional Regulator in Staphylococcus aureus That Is Crucial for Secondary Tissue Colonization during Metastatic Bloodstream Infection.

      Groma, Michaela; Horst, Sarah A; Das, Sudip; Huettel, Bruno; Klepsch, Maximilian; Rudel, Thomas; Medina, Eva; Fraunholz, Martin; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (ASM, 2020-08-25)
      Staphylococcus aureus is a common cause of bacteremia that can lead to severe complications once the bacteria exit the bloodstream and establish infection in secondary organs. Despite its clinical relevance, little is known about the bacterial factors facilitating the development of these metastatic infections. Here, we used an S. aureus transposon mutant library coupled to transposon insertion sequencing (Tn-Seq) to identify genes that are critical for efficient bacterial colonization of secondary organs in a murine model of metastatic bloodstream infection. Our transposon screen identified a LysR-type transcriptional regulator (LTTR), which was required for efficient colonization of secondary organs such as the kidneys in infected mice. The critical role of LTTR in secondary organ colonization was confirmed using an isogenic mutant deficient in the expression of LTTR. To identify the set of genes controlled by LTTR, we used an S. aureus strain carrying the LTTR gene in an inducible expression plasmid. Gene expression analysis upon induction of LTTR showed increased transcription of genes involved in branched-chain amino acid biosynthesis, a methionine sulfoxide reductase, and a copper transporter as well as decreased transcription of genes coding for urease and components of pyrimidine nucleotides. Furthermore, we show that transcription of LTTR is repressed by glucose, is induced under microaerobic conditions, and required trace amounts of copper ions. Our data thus pinpoints LTTR as an important element that enables a rapid adaptation of S. aureus to the changing host microenvironment.IMPORTANCEStaphylococcus aureus is an important pathogen that can disseminate via the bloodstream and establish metastatic infections in distant organs. To achieve a better understanding of the bacterial factors facilitating the development of these metastatic infections, we used in this study a Staphylococcus aureus transposon mutant library in a murine model of intravenous infection, where bacteria first colonize the liver as the primary infection site and subsequently progress to secondary sites such as the kidney and bones. We identified a novel LysR-type transcriptional regulator (LTTR), which was specifically required by S. aureus for efficient colonization of secondary organs. We also determined the transcriptional activation as well as the regulon of LTTR, which suggests that this regulator is involved in the metabolic adaptation of S. aureus to the host microenvironment found in secondary infection sites.