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group leader: Prof. Dr. Vogel

Recent Submissions

  • A Review of the Multipronged Attack of Herpes Simplex Virus 1 on the Host Transcriptional Machinery.

    Hennig, Thomas; Djakovic, Lara; Dölken, Lars; Whisnant, Adam W; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (MDPI, 2021-09-14)
    Autophagy is an evolutionary conserved catabolic pathway that ensures the degradation of intracellular components. The autophagic pathway is regulated by autophagy-related (Atg) proteins that govern formation of double-membraned vesicles called autophagosomes. Autophagy deficiency in regulatory T (Treg) cells leads to increased apoptosis of these cells and to the development of autoimmune disorders, predominantly characterized by intestinal inflammation. Recently, RORγt-expressing Treg cells have been identified as key regulators of gut homeostasis, preventing intestinal immunopathology. To study the role of autophagy in RORγt+ Foxp3+ Treg cells, we generated mice lacking the essential component of the core autophagy machinery Atg5 in Foxp3+ cells. Atg5 deficiency in Treg cells led to a predominant intestinal inflammation. While Atg5-deficient Treg cells were reduced in peripheral lymphoid organs, the intestinal RORγt+ Foxp3+ subpopulation of Treg cells was most severely affected. Our data indicated that autophagy is essential to maintain the intestinal RORγt+ Foxp3+ Treg population, thereby protecting the mice from gut inflammatory disorders.
  • SPI2 T3SS effectors facilitate enterocyte apical to basolateral transmigration of -containing vacuoles .

    Fulde, Marcus; van Vorst, Kira; Zhang, Kaiyi; Westermann, Alexander J; Busche, Tobias; Huei, Yong Chiun; Welitschanski, Katharina; Froh, Isabell; Pägelow, Dennis; Plendl, Johanna; et al. (Taylor & Francis, 2021-09-20)
    Salmonella pathogenicity island (SPI) 2 type three secretion system (T3SS)-mediated effector molecules facilitate bacterial survival in phagocytes but their role in the intestinal epithelium in vivo remains ill-defined. Using our neonatal murine infection model in combination with SPI2 reporter technology and RNA-Seq of sorted primary enterocytes, we demonstrate expression of SPI2 effector molecules by intraepithelial Salmonella Typhimurium (S. Typhimurium). Contrary to expectation, immunostaining revealed that infection with SPI2 T3SS-mutants resulted in significantly enlarged intraepithelial Salmonella-containing vacuoles (SCV) with altered cellular positioning, suggesting impaired apical to basolateral transmigration. Also, infection with isogenic tagged S. Typhimurium strains revealed a reduced spread of intraepithelial SPI2 T3SS mutant S. Typhimurium to systemic body sites. These results suggest that SPI2 T3SS effector molecules contribute to enterocyte apical to basolateral transmigration of the SCV during the early stage of the infection.
  • Concatemeric Broccoli reduces mRNA stability and induces aggregates.

    Rink, Marco R; Baptista, Marisa A P; Flomm, Felix J; Hennig, Thomas; Whisnant, Adam W; Wolf, Natalia; Seibel, Jürgen; Dölken, Lars; Bosse, Jens B; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (PLOS, 2021-08-04)
    Fluorogenic aptamers are an alternative to established methodology for real-time imaging of RNA transport and dynamics. We developed Broccoli-aptamer concatemers ranging from 4 to 128 substrate-binding site repeats and characterized their behavior fused to an mCherry-coding mRNA in transient transfection, stable expression, and in recombinant cytomegalovirus infection. Concatemerization of substrate-binding sites increased Broccoli fluorescence up to a concatemer length of 16 copies, upon which fluorescence did not increase and mCherry signals declined. This was due to the combined effects of RNA aptamer aggregation and reduced RNA stability. Unfortunately, both cellular and cytomegalovirus genomes were unable to maintain and express high Broccoli concatemer copy numbers, possibly due to recombination events. Interestingly, negative effects of Broccoli concatemers could be partially rescued by introducing linker sequences in between Broccoli repeats warranting further studies. Finally, we show that even though substrate-bound Broccoli is easily photobleached, it can still be utilized in live-cell imaging by adapting a time-lapse imaging protocol.
  • RNA landscape of the emerging cancer-associated microbe Fusobacterium nucleatum.

    Ponath, Falk; Tawk, Caroline; Zhu, Yan; Barquist, Lars; Faber, Franziska; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Nature Research, 2021-07-08)
    Fusobacterium nucleatum, long known as a constituent of the oral microflora, has recently garnered renewed attention for its association with several different human cancers. The growing interest in this emerging cancer-associated bacterium contrasts with a paucity of knowledge about its basic gene expression features and physiological responses. As fusobacteria lack all established small RNA-associated proteins, post-transcriptional networks in these bacteria are also unknown. In the present study, using differential RNA-sequencing, we generate high-resolution global RNA maps for five clinically relevant fusobacterial strains-F. nucleatum subspecies nucleatum, animalis, polymorphum and vincentii, as well as F. periodonticum-for early, mid-exponential growth and early stationary phase. These data are made available in an online browser, and we use these to uncover fundamental aspects of fusobacterial gene expression architecture and a suite of non-coding RNAs. Developing a vector for functional analysis of fusobacterial genes, we discover a conserved fusobacterial oxygen-induced small RNA, FoxI, which serves as a post-transcriptional repressor of the major outer membrane porin FomA. Our findings provide a crucial step towards delineating the regulatory networks enabling F. nucleatum adaptation to different environments, which may elucidate how these bacteria colonize different compartments of the human body.
  • An RNA-centric global view of Clostridioides difficile reveals broad activity of Hfq in a clinically important gram-positive bacterium.

    Fuchs, Manuela; Lamm-Schmidt, Vanessa; Sulzer, Johannes; Ponath, Falk; Jenniches, Laura; Kirk, Joseph A; Fagan, Robert P; Barquist, Lars; Vogel, Jörg; Faber, Franziska; et al. (National Academy of Sciences, 2021-06-15)
    The gram-positive human pathogen Clostridioides difficile has emerged as the leading cause of antibiotic-associated diarrhea. However, little is known about the bacterium's transcriptome architecture and mechanisms of posttranscriptional control. Here, we have applied transcription start site and termination mapping to generate a single-nucleotide-resolution RNA map of C. difficile 5' and 3' untranslated regions, operon structures, and noncoding regulators, including 42 sRNAs. Our results indicate functionality of many conserved riboswitches and predict cis-regulatory RNA elements upstream of multidrug resistance (MDR)-type ATP-binding cassette (ABC) transporters and transcriptional regulators. Despite growing evidence for a role of Hfq in RNA-based gene regulation in C. difficile, the functions of Hfq-based posttranscriptional regulatory networks in gram-positive pathogens remain controversial. Using Hfq immunoprecipitation followed by sequencing of bound RNA species (RIP-seq), we identify a large cohort of transcripts bound by Hfq and show that absence of Hfq affects transcript stabilities and steady-state levels. We demonstrate sRNA expression during intestinal colonization by C. difficile and identify infection-related signals impacting its expression. As a proof of concept, we show that the utilization of the abundant intestinal metabolite ethanolamine is regulated by the Hfq-dependent sRNA CDIF630nc_085. Overall, our study lays the foundation for understanding clostridial riboregulation with implications for the infection process and provides evidence for a global role of Hfq in posttranscriptional regulation in a gram-positive bacterium.
  • The Zinc Finger Antiviral Protein ZAP Restricts Human Cytomegalovirus and Selectively Binds and Destabilizes Viral / Transcripts.

    Gonzalez-Perez, Ana Cristina; Stempel, Markus; Wyler, Emanuel; Urban, Christian; Piras, Antonio; Hennig, Thomas; Ganskih, Sabina; Wei, Yuanjie; Heim, Albert; Landthaler, Markus; et al. (ASM, 2021-05-04)
    Interferon-stimulated gene products (ISGs) play a crucial role in early infection control. The ISG zinc finger CCCH-type antiviral protein 1 (ZAP/ZC3HAV1) antagonizes several RNA viruses by binding to CG-rich RNA sequences, whereas its effect on DNA viruses is less well understood. Here, we decipher the role of ZAP in the context of human cytomegalovirus (HCMV) infection, a β-herpesvirus that is associated with high morbidity in immunosuppressed individuals and newborns. We show that expression of the two major isoforms of ZAP, ZAP-S and ZAP-L, is induced during HCMV infection and that both negatively affect HCMV replication. Transcriptome and proteome analyses demonstrated that the expression of ZAP results in reduced viral mRNA and protein levels and decelerates the progression of HCMV infection. Metabolic RNA labeling combined with high-throughput sequencing (SLAM-seq) revealed that most of the gene expression changes late in infection result from the general attenuation of HCMV. Furthermore, at early stages of infection, ZAP restricts HCMV by destabilizing a distinct subset of viral mRNAs, particularly those from the previously uncharacterized UL4-UL6 HCMV gene locus. Through enhanced cross-linking immunoprecipitation and sequencing analysis (eCLIP-seq), we identified the transcripts expressed from this HCMV locus as the direct targets of ZAP. Moreover, our data show that ZAP preferentially recognizes not only CG, but also other cytosine-rich sequences, thereby expanding its target specificity. In summary, this report is the first to reveal direct targets of ZAP during HCMV infection, which strongly indicates that transcripts from the UL4-UL6 locus may play an important role for HCMV replication.IMPORTANCE Viral infections have a large impact on society, leading to major human and economic losses and even global instability. So far, many viral infections, including human cytomegalovirus (HCMV) infection, are treated with a small repertoire of drugs, often accompanied by the occurrence of resistant mutants. There is no licensed HCMV vaccine in sight to protect those most at risk, particularly immunocompromised individuals or pregnant women who might otherwise transmit the virus to the fetus. Thus, the identification of novel intervention strategies is urgently required. In this study, we show that ZAP decelerates the viral gene expression cascade, presumably by selectively handpicking a distinct set of viral transcripts for degradation. Our study illustrates the potent role of ZAP as an HCMV restriction factor and sheds light on a possible role for UL4 and/or UL5 early during infection, paving a new avenue for the exploration of potential targets for novel therapies.
  • Amelioration of Cognitive and Behavioral Deficits after Traumatic Brain Injury in Coagulation Factor XII Deficient Mice.

    Stetter, Christian; Lopez-Caperuchipi, Simon; Hopp-Krämer, Sarah; Bieber, Michael; Kleinschnitz, Christoph; Sirén, Anna-Leena; Albert-Weißenberger, Christiane; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (MDPI, 2021-05-03)
    Based on recent findings that show that depletion of factor XII (FXII) leads to better posttraumatic neurological recovery, we studied the effect of FXII-deficiency on post-traumatic cognitive and behavioral outcomes in female and male mice. In agreement with our previous findings, neurological deficits on day 7 after weight-drop traumatic brain injury (TBI) were significantly reduced in FXII-/- mice compared to wild type (WT) mice. Also, glycoprotein Ib (GPIb)-positive platelet aggregates were more frequent in brain microvasculature of WT than FXII-/- mice 3 months after TBI. Six weeks after TBI, memory for novel object was significantly reduced in both female and male WT but not in FXII-/- mice compared to sham-operated mice. In the setting of automated home-cage monitoring of socially housed mice in IntelliCages, female WT mice but not FXII-/- mice showed decreased exploration and reacted negatively to reward extinction one month after TBI. Since neuroendocrine stress after TBI might contribute to trauma-induced cognitive dysfunction and negative emotional contrast reactions, we measured peripheral corticosterone levels and the ration of heart, lung, and spleen weight to bodyweight. Three months after TBI, plasma corticosterone levels were significantly suppressed in both female and male WT but not in FXII-/- mice, while the relative heart weight increased in males but not in females of both phenotypes when compared to sham-operated mice. Our results indicate that FXII deficiency is associated with efficient post-traumatic behavioral and neuroendocrine recovery.
  • 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.
  • In vivo targets of Salmonella FinO include a FinP-like small RNA controlling copy number of a cohabitating plasmid.

    El Mouali, Youssef; Gerovac, Milan; Mineikaitė, Raminta; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Oxford Academic, 2021-05-03)
    FinO-domain proteins represent an emerging family of RNA-binding proteins (RBPs) with diverse roles in bacterial post-transcriptional control and physiology. They exhibit an intriguing targeting spectrum, ranging from an assumed single RNA pair (FinP/traJ) for the plasmid-encoded FinO protein, to transcriptome-wide activity as documented for chromosomally encoded ProQ proteins. Thus, the shared FinO domain might bear an unusual plasticity enabling it to act either selectively or promiscuously on the same cellular RNA pool. One caveat to this model is that the full suite of in vivo targets of the assumedly highly selective FinO protein is unknown. Here, we have extensively profiled cellular transcripts associated with the virulence plasmid-encoded FinO in Salmonella enterica. While our analysis confirms the FinP sRNA of plasmid pSLT as the primary FinO target, we identify a second major ligand: the RepX sRNA of the unrelated antibiotic resistance plasmid pRSF1010. FinP and RepX are strikingly similar in length and structure, but not in primary sequence, and so may provide clues to understanding the high selectivity of FinO-RNA interactions. Moreover, we observe that the FinO RBP encoded on the Salmonella virulence plasmid controls the replication of a cohabitating antibiotic resistance plasmid, suggesting cross-regulation of plasmids on the RNA level.
  • The World of Stable Ribonucleoproteins and Its Mapping With Grad-Seq and Related Approaches.

    Gerovac, Milan; Vogel, Jörg; Smirnov, Alexandre; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Frontiers, 2021-04-07)
    Macromolecular complexes of proteins and RNAs are essential building blocks of cells. These stable supramolecular particles can be viewed as minimal biochemical units whose structural organization, i.e., the way the RNA and the protein interact with each other, is directly linked to their biological function. Whether those are dynamic regulatory ribonucleoproteins (RNPs) or integrated molecular machines involved in gene expression, the comprehensive knowledge of these units is critical to our understanding of key molecular mechanisms and cell physiology phenomena. Such is the goal of diverse complexomic approaches and in particular of the recently developed gradient profiling by sequencing (Grad-seq). By separating cellular protein and RNA complexes on a density gradient and quantifying their distributions genome-wide by mass spectrometry and deep sequencing, Grad-seq charts global landscapes of native macromolecular assemblies. In this review, we propose a function-based ontology of stable RNPs and discuss how Grad-seq and related approaches transformed our perspective of bacterial and eukaryotic ribonucleoproteins by guiding the discovery of new RNA-binding proteins and unusual classes of noncoding RNAs. We highlight some methodological aspects and developments that permit to further boost the power of this technique and to look for exciting new biology in understudied and challenging biological models.
  • 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.

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