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group leader: Prof. Dr. Vogel

Recent Submissions

  • Mechanism and consequences of herpes simplex virus 1-mediated regulation of host mRNA alternative polyadenylation.

    Wang, Xiuye; Liu, Liang; Whisnant, Adam W; Hennig, Thomas; Djakovic, Lara; Haque, Nabila; Bach, Cindy; Sandri-Goldin, Rozanne M; Erhard, Florian; Friedel, Caroline C; et al. (PLOS, 2021-03-08)
    Eukaryotic gene expression is extensively regulated by cellular stress and pathogen infections. We have previously shown that herpes simplex virus 1 (HSV-1) and several cellular stresses cause widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes and that the viral immediate early factor ICP27 plays an important role in HSV-1-induced DoTT. Here, we show that HSV-1 infection also leads to widespread changes in alternative polyadenylation (APA) of host mRNAs. In the majority of cases, polyadenylation shifts to upstream poly(A) sites (PAS), including many intronic PAS. Mechanistically, ICP27 contributes to HSV-1-mediated APA regulation. HSV-1- and ICP27-induced activation of intronic PAS is sequence-dependent and does not involve general inhibition of U1 snRNP. HSV1-induced intronic polyadenylation is accompanied by early termination of RNAPII. HSV-1-induced mRNAs polyadenylated at intronic PAS (IPA) are exported into the cytoplasm while APA isoforms with extended 3' UTRs are sequestered in the nuclei, both preventing the expression of the full-length gene products. Finally we provide evidence that HSV-induced IPA isoforms are translated. Together with other recent studies, our results suggest that viral infection and cellular stresses induce a multi-faceted host response that includes DoTT and changes in APA profiles.
  • Longitudinal Multi-omics Analyses Identify Responses of Megakaryocytes, Erythroid Cells, and Plasmablasts as Hallmarks of Severe COVID-19.

    Bernardes, Joana P; Mishra, Neha; Tran, Florian; Bahmer, Thomas; Best, Lena; Blase, Johanna I; Bordoni, Dora; Franzenburg, Jeanette; Geisen, Ulf; Josephs-Spaulding, Jonathan; et al. (Elsevier (Cell Press), 2020-11-26)
    Temporal resolution of cellular features associated with a severe COVID-19 disease trajectory is needed for understanding skewed immune responses and defining predictors of outcome. Here, we performed a longitudinal multi-omics study using a two-center cohort of 14 patients. We analyzed the bulk transcriptome, bulk DNA methylome, and single-cell transcriptome (>358,000 cells, including BCR profiles) of peripheral blood samples harvested from up to 5 time points. Validation was performed in two independent cohorts of COVID-19 patients. Severe COVID-19 was characterized by an increase of proliferating, metabolically hyperactive plasmablasts. Coinciding with critical illness, we also identified an expansion of interferon-activated circulating megakaryocytes and increased erythropoiesis with features of hypoxic signaling. Megakaryocyte- and erythroid-cell-derived co-expression modules were predictive of fatal disease outcome. The study demonstrates broad cellular effects of SARS-CoV-2 infection beyond adaptive immune cells and provides an entry point toward developing biomarkers and targeted treatments of patients with COVID-19.
  • The minimal meningococcal ProQ protein has an intrinsic capacity for structure-based global RNA recognition.

    Bauriedl, Saskia; Gerovac, Milan; Heidrich, Nadja; Bischler, Thorsten; Barquist, Lars; Vogel, Jörg; Schoen, Christoph; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Nature Research, 2020-06-04)
    FinO-domain proteins are a widespread family of bacterial RNA-binding proteins with regulatory functions. Their target spectrum ranges from a single RNA pair, in the case of plasmid-encoded FinO, to global RNA regulons, as with enterobacterial ProQ. To assess whether the FinO domain itself is intrinsically selective or promiscuous, we determine in vivo targets of Neisseria meningitidis, which consists of solely a FinO domain. UV-CLIP-seq identifies associations with 16 small non-coding sRNAs and 166 mRNAs. Meningococcal ProQ predominantly binds to highly structured regions and generally acts to stabilize its RNA targets. Loss of ProQ alters transcript levels of >250 genes, demonstrating that this minimal ProQ protein impacts gene expression globally. Phenotypic analyses indicate that ProQ promotes oxidative stress resistance and DNA damage repair. We conclude that FinO domain proteins recognize some abundant type of RNA shape and evolve RNA binding selectivity through acquisition of additional regions that constrain target recognition.
  • Integrative functional genomics decodes herpes simplex virus 1.

    Whisnant, Adam W; Jürges, Christopher S; Hennig, Thomas; Wyler, Emanuel; Prusty, Bhupesh; Rutkowski, Andrzej J; L'hernault, Anne; Djakovic, Lara; Göbel, Margarete; Döring, Kristina; et al. (NPG, 2020-04-27)
    The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including all known and 46 novel large ORFs. This includes a so far unknown ORF in the locus deleted in the FDA-approved oncolytic virus Imlygic. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of ORFs as well as N-terminal extensions (NTEs) and truncations. We show that NTEs with non-canonical start codons govern the subcellular protein localization and packaging of key viral regulators and structural proteins. We extend the current nomenclature to include all viral gene products and provide a genome browser that visualizes all the obtained data from whole genome to single-nucleotide resolution.
  • Biodistribution and serologic response in SARS-CoV-2 induced ARDS: A cohort study.

    Schlesinger, Tobias; Weißbrich, Benedikt; Wedekink, Florian; Notz, Quirin; Herrmann, Johannes; Krone, Manuel; Sitter, Magdalena; Schmid, Benedikt; Kredel, Markus; Stumpner, Jan; et al. (PLOS, 2020-11-24)
    Background: The viral load and tissue distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain important questions. The current study investigated SARS-CoV-2 viral load, biodistribution and anti-SARS-CoV-2 antibody formation in patients suffering from severe corona virus disease 2019 (COVID-19) induced acute respiratory distress syndrome (ARDS). Methods: This is a retrospective single-center study in 23 patients with COVID-19-induced ARDS. Data were collected within routine intensive care. SARS-CoV-2 viral load was assessed via reverse transcription quantitative polymerase chain reaction (RT-qPCR). Overall, 478 virology samples were taken. Anti-SARS-CoV-2-Spike-receptor binding domain (RBD) antibody detection of blood samples was performed with an enzyme-linked immunosorbent assay. Results: Most patients (91%) suffered from severe ARDS during ICU treatment with a 30-day mortality of 30%. None of the patients received antiviral treatment. Tracheal aspirates tested positive for SARS-CoV-2 in 100% of the cases, oropharyngeal swabs only in 77%. Blood samples were positive in 26% of the patients. No difference of viral load was found in tracheal or blood samples with regard to 30-day survival or disease severity. SARS-CoV-2 was never found in dialysate. Serologic testing revealed significantly lower concentrations of SARS-CoV-2 neutralizing IgM and IgA antibodies in survivors compared to non-survivors (p = 0.009). Conclusions: COVID-19 induced ARDS is accompanied by a high viral load of SARS-CoV-2 in tracheal aspirates, which remained detectable in the majority throughout intensive care treatment. Remarkably, SARS-CoV-2 RNA was never detected in dialysate even in patients with RNAemia. Viral load or the buildup of neutralizing antibodies was not associated with 30-day survival or disease severity.
  • Cross-species RNA-seq for deciphering host-microbe interactions.

    Westermann, Alexander J; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Nature research, 2021-02-17)
    The human body is constantly exposed to microorganisms, which entails manifold interactions between human cells and diverse commensal or pathogenic bacteria. The cellular states of the interacting cells are decisive for the outcome of these encounters such as whether bacterial virulence programmes and host defence or tolerance mechanisms are induced. This Review summarizes how next-generation RNA sequencing (RNA-seq) has become a primary technology to study host-microbe interactions with high resolution, improving our understanding of the physiological consequences and the mechanisms at play. We illustrate how the discriminatory power and sensitivity of RNA-seq helps to dissect increasingly complex cellular interactions in time and space down to the single-cell level. We also outline how future transcriptomics may answer currently open questions in host-microbe interactions and inform treatment schemes for microbial disorders.
  • 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.
  • MAPS integrates regulation of actin-targeting effector SteC into the virulence control network of Salmonella small RNA PinT.

    Correia Santos, Sara; Bischler, Thorsten; Westermann, Alexander J; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Elsevier (Cell Press), 2021-02-02)
    A full understanding of the contribution of small RNAs (sRNAs) to bacterial virulence demands knowledge of their target suites under infection-relevant conditions. Here, we take an integrative approach to capturing targets of the Hfq-associated sRNA PinT, a known post-transcriptional timer of the two major virulence programs of Salmonella enterica. Using MS2 affinity purification and RNA sequencing (MAPS), we identify PinT ligands in bacteria under in vitro conditions mimicking specific stages of the infection cycle and in bacteria growing inside macrophages. This reveals PinT-mediated translational inhibition of the secreted effector kinase SteC, which had gone unnoticed in previous target searches. Using genetic, biochemical, and microscopic assays, we provide evidence for PinT-mediated repression of steC mRNA, eventually delaying actin rearrangements in infected host cells. Our findings support the role of PinT as a central post-transcriptional regulator in Salmonella virulence and illustrate the need for complementary methods to reveal the full target suites of sRNAs.
  • 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.
  • 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.
  • Transcriptome profiling and protease inhibition experiments identify proteases that activate H3N2 influenza A and influenza B viruses in murine airways.

    Harbig, Anne; Mernberger, Marco; Bittel, Linda; Pleschka, Stephan; Schughart, Klaus; Steinmetzer, Torsten; Stiewe, Thorsten; Nist, Andrea; Böttcher-Friebertshäuser, Eva; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Elsevier, 2020-04-17)
    Cleavage of influenza virus hemagglutinin (HA) by host proteases is essential for virus infectivity. HA of most influenza A and B (IAV/IBV) viruses is cleaved at a monobasic motif by trypsin-like proteases. Previous studies have reported that transmembrane serine protease 2 (TMPRSS2) is essential for activation of H7N9 and H1N1pdm IAV in mice but that H3N2 IAV and IBV activation is independent of TMPRSS2 and carried out by as-yet-undetermined protease(s). Here, to identify additional H3 IAV- and IBV-activating proteases, we used RNA-Seq to investigate the protease repertoire of murine lower airway tissues, primary type II alveolar epithelial cells (AECIIs), and the mouse lung cell line MLE-15. Among 13 candidates identified, TMPRSS4, TMPRSS13, hepsin, and prostasin activated H3 and IBV HA in vitro IBV activation and replication was reduced in AECIIs from Tmprss2/Tmprss4-deficient mice compared with WT or Tmprss2-deficient mice, indicating that murine TMPRSS4 is involved in IBV activation. Multicycle replication of H3N2 IAV and IBV in AECIIs of Tmprss2/Tmprss4-deficient mice varied in sensitivity to protease inhibitors, indicating that different, but overlapping, sets of murine proteases facilitate H3 and IBV HA cleavages. Interestingly, human hepsin and prostasin orthologs did not activate H3, but they did activate IBV HA in vitro Our results indicate that TMPRSS4 is an IBV-activating protease in murine AECIIs and suggest that TMPRSS13, hepsin, and prostasin cleave H3 and IBV HA in mice. They further show that hepsin and prostasin orthologs might contribute to the differences observed in TMPRSS2-independent activation of H3 in murine and human airways.
  • Opposing Wnt signals regulate cervical squamocolumnar homeostasis and emergence of metaplasia.

    Chumduri, Cindrilla; Gurumurthy, Rajendra Kumar; Berger, Hilmar; Dietrich, Oliver; Kumar, Naveen; Koster, Stefanie; Brinkmann, Volker; Hoffmann, Kirstin; Drabkina, Marina; Arampatzi, Panagiota; et al. (Nature research, 2021-01-18)
    The transition zones of the squamous and columnar epithelia constitute hotspots for the emergence of cancer, often preceded by metaplasia, in which one epithelial type is replaced by another. It remains unclear how the epithelial spatial organization is maintained and how the transition zone niche is remodelled during metaplasia. Here we used single-cell RNA sequencing to characterize epithelial subpopulations and the underlying stromal compartment of endo- and ectocervix, encompassing the transition zone. Mouse lineage tracing, organoid culture and single-molecule RNA in situ hybridizations revealed that the two epithelia derive from separate cervix-resident lineage-specific stem cell populations regulated by opposing Wnt signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodelling and increases expression of the Wnt inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Our data indicate that homeostasis at the transition zone results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages.
  • 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.
  • 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.
  • Leveraging bile solubilization of poorly water-soluble drugs by rational polymer selection.

    Schlauersbach, Jonas; Hanio, Simon; Lenz, Bettina; Vemulapalli, Sahithya P B; Griesinger, Christian; Pöppler, Ann-Christin; Harlacher, Cornelius; Galli, Bruno; Meinel, Lorenz; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Elsevier, 2020-12-15)
    Poorly water-soluble drugs frequently solubilize into bile colloids and this natural mechanism is key for efficient bioavailability. We tested the impact of pharmaceutical polymers on this solubilization interplay using proton nuclear magnetic resonance spectroscopy, dynamic light scattering, and by assessing the flux across model membranes. Eudragit E, Soluplus, and a therapeutically used model polymer, Colesevelam, impacted the bile-colloidal geometry and molecular interaction. These polymer-induced changes reduced the flux of poorly water-soluble and bile interacting drugs (Perphenazine, Imatinib) but did not impact the flux of bile non-interacting Metoprolol. Non-bile interacting polymers (Kollidon VA 64, HPMC-AS) neither impacted the flux of colloid-interacting nor colloid-non-interacting drugs. These insights into the drug substance/polymer/bile colloid interplay potentially point towards a practical optimization parameter steering formulations to efficient bile-solubilization by rational polymer selection.
  • Intracellular Staphylococcus aureus Perturbs the Host Cell Ca+ Homeostasis To Promote Cell Death.

    Stelzner, Kathrin; Winkler, Ann-Cathrin; Liang, Chunguang; Boyny, Aziza; Ade, Carsten P; Dandekar, Thomas; Fraunholz, Martin J; Rudel, Thomas; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (ASM, 2020-12-15)
    The opportunistic human pathogen Staphylococcus aureus causes serious infectious diseases that range from superficial skin and soft tissue infections to necrotizing pneumonia and sepsis. While classically regarded as an extracellular pathogen, S. aureus is able to invade and survive within human cells. Host cell exit is associated with cell death, tissue destruction, and the spread of infection. The exact molecular mechanism employed by S. aureus to escape the host cell is still unclear. In this study, we performed a genome-wide small hairpin RNA (shRNA) screen and identified the calcium signaling pathway as being involved in intracellular infection. S. aureus induced a massive cytosolic Ca2+ increase in epithelial host cells after invasion and intracellular replication of the pathogen. This was paralleled by a decrease in endoplasmic reticulum Ca2+ concentration. Additionally, calcium ions from the extracellular space contributed to the cytosolic Ca2+ increase. As a consequence, we observed that the cytoplasmic Ca2+ rise led to an increase in mitochondrial Ca2+ concentration, the activation of calpains and caspases, and eventually to cell lysis of S. aureus-infected cells. Our study therefore suggests that intracellular S. aureus disturbs the host cell Ca2+ homeostasis and induces cytoplasmic Ca2+ overload, which results in both apoptotic and necrotic cell death in parallel or succession.IMPORTANCE Despite being regarded as an extracellular bacterium, the pathogen Staphylococcus aureus can invade and survive within human cells. The intracellular niche is considered a hideout from the host immune system and antibiotic treatment and allows bacterial proliferation. Subsequently, the intracellular bacterium induces host cell death, which may facilitate the spread of infection and tissue destruction. So far, host cell factors exploited by intracellular S. aureus to promote cell death are only poorly characterized. We performed a genome-wide screen and found the calcium signaling pathway to play a role in S. aureus invasion and cytotoxicity. The intracellular bacterium induces a cytoplasmic and mitochondrial Ca2+ overload, which results in host cell death. Thus, this study first showed how an intracellular bacterium perturbs the host cell Ca2+ homeostasis.
  • 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.
  • The SARS-CoV-2 RNA-protein interactome in infected human cells.

    Schmidt, Nora; Lareau, Caleb A; Keshishian, Hasmik; Ganskih, Sabina; Schneider, Cornelius; Hennig, Thomas; Melanson, Randy; Werner, Simone; Wei, Yuanjie; Zimmer, Matthias; et al. (Nature research, 2020-12-21)
    Characterizing the interactions that SARS-CoV-2 viral RNAs make with host cell proteins during infection can improve our understanding of viral RNA functions and the host innate immune response. Using RNA antisense purification and mass spectrometry, we identified up to 104 human proteins that directly and specifically bind to SARS-CoV-2 RNAs in infected human cells. We integrated the SARS-CoV-2 RNA interactome with changes in proteome abundance induced by viral infection and linked interactome proteins to cellular pathways relevant to SARS-CoV-2 infections. We demonstrated by genetic perturbation that cellular nucleic acid-binding protein (CNBP) and La-related protein 1 (LARP1), two of the most strongly enriched viral RNA binders, restrict SARS-CoV-2 replication in infected cells and provide a global map of their direct RNA contact sites. Pharmacological inhibition of three other RNA interactome members, PPIA, ATP1A1, and the ARP2/3 complex, reduced viral replication in two human cell lines. The identification of host dependency factors and defence strategies as presented in this work will improve the design of targeted therapeutics against SARS-CoV-2.
  • Single-Nucleotide RNA Maps for the Two Major Nosocomial Pathogens Enterococcus faecalis and Enterococcus faecium

    Michaux, Charlotte; Hansen, Elisabeth E; Jenniches, Laura; Gerovac, Milan; Barquist, Lars; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Frontiers, 2020-11-25)
    Enterococcus faecalis and faecium are two major representative clinical strains of the Enterococcus genus and are sadly notorious to be part of the top agents responsible for nosocomial infections. Despite their critical implication in worldwide public healthcare, essential and available resources such as deep transcriptome annotations remain poor, which also limits our understanding of post-transcriptional control small regulatory RNA (sRNA) functions in these bacteria. Here, using the dRNA-seq technique in combination with ANNOgesic analysis, we successfully mapped and annotated transcription start sites (TSS) of both E. faecalis V583 and E. faecium AUS0004 at single nucleotide resolution. Analyzing bacteria in late exponential phase, we capture ~40% (E. faecalis) and 43% (E. faecium) of the annotated protein-coding genes, determine 5' and 3' UTR (untranslated region) length, and detect instances of leaderless mRNAs. The transcriptome maps revealed sRNA candidates in both bacteria, some found in previous studies and new ones. Expression of candidate sRNAs is being confirmed under biologically relevant environmental conditions. This comprehensive global TSS mapping atlas provides a valuable resource for RNA biology and gene expression analysis in the Enterococci. It can be accessed online at www.helmholtz-hiri.de/en/datasets/enterococcus through an instance of the genomic viewer JBrowse.
  • Triple RNA-Seq Reveals Synergy in a Human Virus-Fungus Co-infection Model.

    Seelbinder, Bastian; Wallstabe, Julia; Marischen, Lothar; Weiss, Esther; Wurster, Sebastian; Page, Lukas; Löffler, Claudia; Bussemer, Lydia; Schmitt, Anna-Lena; Wolf, Thomas; et al. (Elsevier (Cell Press), 2020-11-17)
    High-throughput RNA sequencing (RNA-seq) is routinely applied to study diverse biological processes; however, when performed separately on interacting organisms, systemic noise intrinsic to RNA extraction, library preparation, and sequencing hampers the identification of cross-species interaction nodes. Here, we develop triple RNA-seq to simultaneously detect transcriptomes of monocyte-derived dendritic cells (moDCs) infected with the frequently co-occurring pulmonary pathogens Aspergillus fumigatus and human cytomegalovirus (CMV). Comparing expression patterns after co-infection with those after single infections, our data reveal synergistic effects and mutual interferences between host responses to the two pathogens. For example, CMV attenuates the fungus-mediated activation of pro-inflammatory cytokines through NF-κB (nuclear factor κB) and NFAT (nuclear factor of activated T cells) cascades, while A. fumigatus impairs viral clearance by counteracting viral nucleic acid-induced activation of type I interferon signaling. Together, the analytical power of triple RNA-seq proposes molecular hubs in the differential moDC response to fungal/viral single infection or co-infection that contribute to our understanding of the etiology and, potentially, clearance of post-transplant infections.

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