• An Advanced Human Intestinal Coculture Model Reveals Compartmentalized Host and Pathogen Strategies during Infection.

      Schulte, Leon N; Schweinlin, Matthias; Westermann, Alexander J; Janga, Harshavardhan; Santos, Sara C; Appenzeller, Silke; Walles, Heike; Vogel, Jörg; Metzger, Marco; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (American Society for Microbiology (ASM), 2020-02-18)
      A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. Upon Salmonella infection, the model mimics human gastroenteritis, in that it restricts the pathogen to the epithelial compartment, an advantage over existing mouse models. Application of dual transcriptome sequencing to the Salmonella-infected model revealed the communication of epithelial, endothelial, monocytic, and natural killer cells among each other and with the pathogen. Our results suggest that Salmonella uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelium without accompanying alterations in the endothelial compartment. Our approach promises to reveal further human-specific infection strategies employed by Salmonella and other pathogens.IMPORTANCE Infection research routinely employs in vitro cell cultures or in vivo mouse models as surrogates of human hosts. Differences between murine and human immunity and the low level of complexity of traditional cell cultures, however, highlight the demand for alternative models that combine the in vivo-like properties of the human system with straightforward experimental perturbation. Here, we introduce a 3D tissue model comprising multiple cell types of the human intestinal barrier, a primary site of pathogen attack. During infection with the foodborne pathogen Salmonella enterica serovar Typhimurium, our model recapitulates human disease aspects, including pathogen restriction to the epithelial compartment, thereby deviating from the systemic infection in mice. Combination of our model with state-of-the-art genetics revealed Salmonella-mediated local manipulations of human immune responses, likely contributing to the establishment of the pathogen's infection niche. We propose the adoption of similar 3D tissue models to infection biology, to advance our understanding of molecular infection strategies employed by bacterial pathogens in their human host.
    • 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.
    • Amidochelocardin Overcomes Resistance Mechanisms Exerted on Tetracyclines and Natural Chelocardin.

      Hennessen, Fabienne; Miethke, Marcus; Zaburannyi, Nestor; Loose, Maria; Lukežič, Tadeja; Bernecker, Steffen; Hüttel, Stephan; Jansen, Rolf; Schmiedel, Judith; Fritzenwanker, Moritz; et al. (MDPI, 2020-09-18)
      The reassessment of known but neglected natural compounds is a vital strategy for providing novel lead structures urgently needed to overcome antimicrobial resistance. Scaffolds with resistance-breaking properties represent the most promising candidates for a successful translation into future therapeutics. Our study focuses on chelocardin, a member of the atypical tetracyclines, and its bioengineered derivative amidochelocardin, both showing broad-spectrum antibacterial activity within the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) panel. Further lead development of chelocardins requires extensive biological and chemical profiling to achieve favorable pharmaceutical properties and efficacy. This study shows that both molecules possess resistance-breaking properties enabling the escape from most common tetracycline resistance mechanisms. Further, we show that these compounds are potent candidates for treatment of urinary tract infections due to their in vitro activity against a large panel of multidrug-resistant uropathogenic clinical isolates. In addition, the mechanism of resistance to natural chelocardin was identified as relying on efflux processes, both in the chelocardin producer Amycolatopsis sulphurea and in the pathogen Klebsiella pneumoniae. Resistance development in Klebsiella led primarily to mutations in ramR, causing increased expression of the acrAB-tolC efflux pump. Most importantly, amidochelocardin overcomes this resistance mechanism, revealing not only the improved activity profile but also superior resistance-breaking properties of this novel antibacterial compound.
    • ANNOgesic: a Swiss army knife for the RNA-seq based annotation of bacterial/archaeal genomes.

      Yu, Sung-Huan; Vogel, Jörg; Förstner, Konrad U; HIRI, Helmoltz-Institut für RNA-basierteInfektionsforschung, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. (2018-09-01)
      To understand the gene regulation of an organism of interest, a comprehensive genome annotation is essential. While some features, such as coding sequences, can be computationally predicted with high accuracy based purely on the genomic sequence, others, such as promoter elements or noncoding RNAs, are harder to detect. RNA sequencing (RNA-seq) has proven to be an efficient method to identify these genomic features and to improve genome annotations. However, processing and integrating RNA-seq data in order to generate high-resolution annotations is challenging, time consuming, and requires numerous steps. We have constructed a powerful and modular tool called ANNOgesic that provides the required analyses and simplifies RNA-seq-based bacterial and archaeal genome annotation. It can integrate data from conventional RNA-seq and differential RNA-seq and predicts and annotates numerous features, including small noncoding RNAs, with high precision. The software is available under an open source license (ISCL) at https://pypi.org/project/ANNOgesic/.
    • Bacterial RNA Biology on a Genome Scale.

      Hör, Jens; Gorski, Stanislaw A; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. (2018-01-16)
      Bacteria are an exceedingly diverse group of organisms whose molecular exploration is experiencing a renaissance. While the classical view of bacterial gene expression was relatively simple, the emerging view is more complex, encompassing extensive post-transcriptional control involving riboswitches, RNA thermometers, and regulatory small RNAs (sRNAs) associated with the RNA-binding proteins CsrA, Hfq, and ProQ, as well as CRISPR/Cas systems that are programmed by RNAs. Moreover, increasing interest in members of the human microbiota and environmental microbial communities has highlighted the importance of understudied bacterial species with largely unknown transcriptome structures and RNA-based control mechanisms. Collectively, this creates a need for global RNA biology approaches that can rapidly and comprehensively analyze the RNA composition of a bacterium of interest. We review such approaches with a focus on RNA-seq as a versatile tool to investigate the different layers of gene expression in which RNA is made, processed, regulated, modified, translated, and turned over.
    • 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.
    • Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression.

      Parhi, Lishay; Alon-Maimon, Tamar; Sol, Asaf; Nejman, Deborah; Shhadeh, Amjad; Fainsod-Levi, Tanya; Yajuk, Olga; Isaacson, Batya; Abed, Jawad; Maalouf, Naseem; et al. (Nature Research, 2020-06-26)
      Fusobacterium nucleatum is an oral anaerobe recently found to be prevalent in human colorectal cancer (CRC) where it is associated with poor treatment outcome. In mice, hematogenous F. nucleatum can colonize CRC tissue using its lectin Fap2, which attaches to tumor-displayed Gal-GalNAc. Here, we show that Gal-GalNAc levels increase as human breast cancer progresses, and that occurrence of F. nucleatum gDNA in breast cancer samples correlates with high Gal-GalNAc levels. We demonstrate Fap2-dependent binding of the bacterium to breast cancer samples, which is inhibited by GalNAc. Intravascularly inoculated Fap2-expressing F. nucleatum ATCC 23726 specifically colonize mice mammary tumors, whereas Fap2-deficient bacteria are impaired in tumor colonization. Inoculation with F. nucleatum suppresses accumulation of tumor infiltrating T cells and promotes tumor growth and metastatic progression, the latter two of which can be counteracted by antibiotic treatment. Thus, targeting F. nucleatum or Fap2 might be beneficial during treatment of breast cancer.
    • The chlamydial deubiquitinase Cdu1 supports recruitment of Golgi vesicles to the inclusion.

      Auer, Daniela; Hügelschäffer, Sophie D; Fischer, Annette B; Rudel, Thomas; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Wiley, 2019-11-01)
      Chlamydia trachomatis is the main cause of sexually transmitted diseases worldwide. As obligate intracellular bacteria Chlamydia replicate in a membrane bound vacuole called inclusion and acquire nutrients for growth and replication from their host cells. However, like all intracellular bacteria, Chlamydia have to prevent eradication by the host's cell autonomous system. The chlamydial deubiquitinase Cdu1 is secreted into the inclusion membrane, facing the host cell cytosol where it deubiquitinates cellular proteins. Here we show that inactivation of Cdu1 causes a growth defect of C. trachomatis in primary cells. Moreover, ubiquitin and several autophagy receptors are recruited to the inclusion membrane of Cdu1-deficient Chlamydia. Interestingly, the growth defect of cdu1 mutants is not rescued when autophagy is prevented. We find reduced recruitment of Golgi vesicles to the inclusion of Cdu1 mutants indicating that vesicular trafficking is altered in bacteria without active deubiquitinase (DUB). Our work elucidates an important role of Cdu1 in the functional preservation of the chlamydial inclusion surface.
    • 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.
    • Concentration and composition dependent aggregation of Pluronic- and Poly-(2-oxazolin)-Efavirenz formulations in biorelevant media.

      Endres, Sebastian; Karaev, Emil; Hanio, Simon; Schlauersbach, Jonas; Kraft, Christian; Rasmussen, Tim; Luxenhofer, Robert; Böttcher, Bettina; Meinel, Lorenz; Pöppler, Ann-Christin; et al. (Elsevier, 2021-08-10)
      Many drugs and drug candidates are poorly water-soluble. Intestinal fluids play an important role in their solubilization. However, the interactions of intestinal fluids with polymer excipients, drugs and their formulations are not fully understood. Here, diffusion ordered spectroscopy (DOSY) and nuclear Overhauser effect spectroscopy (NOESY), complemented by cryo-TEM were employed to address this. Efavirenz (EFV) as model drug, the triblock copolymers Pluronic® F-127 (PF127) and poly(2-oxazoline) based pMeOx-b-pPrOzi-b-pMeOx (pOx/pOzi) and their respective formulations were studied in simulated fed-state intestinal fluid (FeSSIF). For the individual polymers, the bile interfering nature of PF127 was confirmed and pure pOx/pOzi was newly classified as non-interfering. A different and more complex behaviour was however observed if EFV was involved. PF127/EFV formulations in FeSSIF showed concentration dependent aggregation with separate colloids at low formulation concentrations, a merging of individual particles at the solubility limit of EFV in FeSSIF and joint aggregates above this concentration. In the case of pOx/pOzi/EFV formulations, coincident diffusion coefficients for pOx/pOzi, lipids and EFV indicate joint aggregates across the studied concentration range. This demonstrates that separate evaluation of polymers and drugs in biorelevant media is not sufficient and their mixtures need to be studied to learn about concentration and composition dependent behaviour.
    • Conditional Hfq Association with Small Noncoding RNAs in Pseudomonas aeruginosa Revealed through Comparative UV Cross-Linking Immunoprecipitation Followed by High-Throughput Sequencing.

      Chihara, Kotaro; Bischler, Thorsten; Barquist, Lars; Monzon, Vivian A; Noda, Naohiro; Vogel, Jörg; Tsuneda, Satoshi (2019-12-03)
      Bacterial small noncoding RNAs (sRNAs) play posttranscriptional regulatory roles in cellular responses to changing environmental cues and in adaptation to harsh conditions. Generally, the RNA-binding protein Hfq helps sRNAs associate with target mRNAs to modulate their translation and to modify global RNA pools depending on physiological state. Here, a combination of in vivo UV cross-linking immunoprecipitation followed by high-throughput sequencing (CLIP-seq) and total RNA-seq showed that Hfq interacts with different regions of the Pseudomonas aeruginosa transcriptome under planktonic versus biofilm conditions. In the present approach, P. aeruginosa Hfq preferentially interacted with repeats of the AAN triplet motif at mRNA 5' untranslated regions (UTRs) and sRNAs and U-rich sequences at rho-independent terminators. Further transcriptome analysis suggested that the association of sRNAs with Hfq is primarily a function of their expression levels, strongly supporting the notion that the pool of Hfq-associated RNAs is equilibrated by RNA concentration-driven cycling on and off Hfq. Overall, our combinatorial CLIP-seq and total RNA-seq approach highlights conditional sRNA associations with Hfq as a novel aspect of posttranscriptional regulation in P. aeruginosaIMPORTANCE The Gram-negative bacterium P. aeruginosa is ubiquitously distributed in diverse environments and can cause severe biofilm-related infections in at-risk individuals. Although the presence of a large number of putative sRNAs and widely conserved RNA chaperones in this bacterium implies the importance of posttranscriptional regulatory networks for environmental fluctuations, limited information is available regarding the global role of RNA chaperones such as Hfq in the P. aeruginosa transcriptome, especially under different environmental conditions. Here, we characterize Hfq-dependent differences in gene expression and biological processes in two physiological states: the planktonic and biofilm forms. A combinatorial comparative CLIP-seq and total RNA-seq approach uncovered condition-dependent association of RNAs with Hfq in vivo and expands the potential direct regulatory targets of Hfq in the P. aeruginosa transcriptome.
    • The conserved 3' UTR-derived small RNA NarS mediates mRNA crossregulation during nitrate respiration.

      Wang, Chuan; Chao, Yanjie; Matera, Gianluca; Gao, Qian; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Oxford Academic, 2019-12-21)
      Small noncoding RNAs (sRNAs) from mRNA 3' UTRs seem to present a previously unrecognized layer of bacterial post-transcriptional control whereby mRNAs influence each other's expression, independently of transcriptional control. Studies in Escherichia coli and Salmonella enterica showed that such sRNAs are natural products of RNase E-mediated mRNA decay and associate with major RNA-binding proteins (RBPs) such as Hfq and ProQ. If so, there must be additional sRNAs from mRNAs that accumulate only under specific physiological conditions. We test this prediction by characterizing candidate NarS that represents the 3' UTR of nitrate transporter NarK whose gene is silent during standard aerobic growth. We find that NarS acts by Hfq-dependent base pairing to repress the synthesis of the nitrite transporter, NirC, resulting in mRNA cross-regulation of nitrate and nitrite transporter genes. Interestingly, the NarS-mediated repression selectively targets the nirC cistron of the long nirBDC-cysG operon, an observation that we rationalize as a mechanism to protect the bacterial cytoplasm from excessive nitrite toxicity during anaerobic respiration with abundant nitrate. Our successful functional assignment of a 3' UTR sRNA from a non-standard growth condition supports the notion that mRNA crossregulation is more pervasive than currently appreciated.
    • The CRISPR/Cas system in Neisseria meningitidis affects bacterial adhesion to human nasopharyngeal epithelial cells.

      Heidrich, Nadja; Hagmann, Antony; Bauriedl, Saskia; Vogel, Jörg; Schoen, Christoph; HIRI, Helmoltz-Institut für RNA-basierteInfektionsforschung, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. (2018-07-30)
      Neisseria meningitidis, a commensal β-proteobacterium of the human nasopharynx, constitutes a worldwide leading cause of sepsis and epidemic meningitis. A recent genome-wide association study suggested an association of its type II-C CRISPR/Cas system with carriage and thus less invasive lineages. Here, we show that knock-out strains lacking the Cas9 protein are impaired in the adhesion to human nasopharyngeal cells which constitutes a central step in the pathogenesis of invasive meningococcal disease. Transcriptome sequencing data further suggest that meningococcal Cas9 does not affect the expression of surface adhesins but rather exerts its effect on cell adhesion in an indirect manner. Consequently, we speculate that the meningococcal CRISPR/Cas system exerts novel functions beyond its established role in defence against foreign DNA.
    • 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.
    • CRP-cAMP mediates silencing of Salmonella virulence at the post-transcriptional level.

      El Mouali, Youssef; Gaviria-Cantin, Tania; Sánchez-Romero, María Antonia; Gibert, Marta; Westermann, Alexander J; Vogel, Jörg; Balsalobre, Carlos; HIRI, Helmoltz-Institut für RNA-basierteInfektionsforschung, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. (2018-01-01)
      Invasion of epithelial cells by Salmonella enterica requires expression of genes located in the pathogenicity island I (SPI-1). The expression of SPI-1 genes is very tightly regulated and activated only under specific conditions. Most studies have focused on the regulatory pathways that induce SPI-1 expression. Here, we describe a new regulatory circuit involving CRP-cAMP, a widely established metabolic regulator, in silencing of SPI-1 genes under non-permissive conditions. In CRP-cAMP-deficient strains we detected a strong upregulation of SPI-1 genes in the mid-logarithmic growth phase. Genetic analyses revealed that CRP-cAMP modulates the level of HilD, the master regulator of Salmonella invasion. This regulation occurs at the post-transcriptional level and requires the presence of a newly identified regulatory motif within the hilD 3'UTR. We further demonstrate that in Salmonella the Hfq-dependent sRNA Spot 42 is under the transcriptional repression of CRP-cAMP and, when this transcriptional repression is relieved, Spot 42 exerts a positive effect on hilD expression. In vivo and in vitro assays indicate that Spot 42 targets, through its unstructured region III, the 3'UTR of the hilD transcript. Together, our results highlight the biological relevance of the hilD 3'UTR as a hub for post-transcriptional control of Salmonella invasion gene expression.
    • 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.