• Identification of a Novel LysR-Type Transcriptional Regulator in Staphylococcus aureus That Is Crucial for Secondary Tissue Colonization during Metastatic Bloodstream Infection.

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

      Zeeb, Marius; Kerrinnes, Tobias; Cicin-Sain, Luka; Guzman, Carlos A; Puppe, Wolfram; Schulz, Thomas F; Peters, Annette; Berger, Klaus; Castell, Stefanie; Karch, André; et al. (Springer, 2020-07-09)
      Immunostimulation by chronic infection has been linked to an increased risk for different non-communicable diseases, which in turn are leading causes of death in high- and middle-income countries. Thus, we investigated if a positive serostatus for pathogens responsible for common chronic infections is individually or synergistically related to reduced overall survival in community dwelling elderly. We used data of 365 individuals from the German MEMO (Memory and Morbidity in Augsburg Elderly) cohort study with a median age of 73 years at baseline and a median follow-up of 14 years. We examined the effect of a positive serostatus at baseline for selected pathogens associated with chronic infections (Helicobacter pylori, Borrelia burgdorferi sensu lato, Toxoplasma gondii, cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1/2, and human herpesvirus 6) on all-cause mortality with multivariable parametric survival models. We found a reduced survival time in individuals with a positive serostatus for Helicobacter pylori (accelerated failure time (AFT) - 15.92, 95% CI - 29.96; - 1.88), cytomegalovirus (AFT - 22.81, 95% CI - 36.41; - 9.22) and Borrelia burgdorferi sensu lato (AFT - 25.25, 95% CI - 43.40; - 7.10), after adjusting for potential confounders. The number of infectious agents an individual was seropositive for had a linear effect on all-cause mortality (AFT per additional infection - 12.42 95% CI - 18.55; - 6.30). Our results suggest an effect of seropositivity for Helicobacter pylori, cytomegalovirus, and Borrelia burgdorferi sensu lato on all-cause mortality in older community dwelling individuals. Further research with larger cohorts and additional biomarkers is required, to assess mediators and molecular pathways of this effect.
    • 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.
    • 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.
    • Increasing storage stability of freeze-dried plasma using trehalose.

      Brogna, Raffaele; Oldenhof, Harriëtte; Sieme, Harald; Figueiredo, Constança; Kerrinnes, Tobias; Wolkers, Willem F; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (PLOS, 2020-06-11)
      Preservation of blood plasma in the dried state would facilitate long-term storage and transport at ambient temperatures, without the need of to use liquid nitrogen tanks or freezers. The aim of this study was to investigate the feasibility of dry preservation of human plasma, using sugars as lyoprotectants, and evaluate macromolecular stability of plasma components during storage. Blood plasma from healthy donors was freeze dried using 0-10% glucose, sucrose, or trehalose, and stored at various temperatures. Differential scanning calorimetry was used to measure the glass transition temperatures of freeze-dried samples. Protein aggregation, the overall protein secondary structure, and oxidative damage were studied under different storage conditions. Differential scanning calorimetry measurements showed that plasma freeze-dried with glucose, sucrose and trehalose have glass transition temperatures of respectively 72±3.4°C, 46±11°C, 15±2.4°C. It was found that sugars diminish freeze-drying induced protein aggregation in a dose-dependent manner, and that a 10% (w/v) sugar concentration almost entirely prevents protein aggregation. Protein aggregation after rehydration coincided with relatively high contents of β-sheet structures in the dried state. Trehalose reduced the rate of protein aggregation during storage at elevated temperatures, and plasma that is freeze- dried plasma with trehalose showed a reduced accumulation of reactive oxygen species and protein oxidation products during storage. In conclusion, freeze-drying plasma with trehalose provides an attractive alternative to traditional cryogenic preservation
    • Improved bacterial RNA-seq by Cas9-based depletion of ribosomal RNA reads.

      Prezza, Gianluca; Heckel, Tobias; Dietrich, Sascha; Homberger, Christina; Westermann, Alexander J; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Cold Spring Harbor Laboratory Press, 2020-04-28)
      A major challenge for RNA-seq analysis of gene expression is to achieve sufficient coverage of informative nonribosomal transcripts. In eukaryotic samples, this is typically achieved by selective oligo(dT)-priming of messenger RNAs to exclude ribosomal RNA (rRNA) during cDNA synthesis. However, this strategy is not compatible with prokaryotes in which functional transcripts are generally not polyadenylated. To overcome this, we adopted DASH (depletion of abundant sequences by hybridization), initially developed for eukaryotic cells, to improve both the sensitivity and depth of bacterial RNA-seq. DASH uses the Cas9 nuclease to remove unwanted cDNA sequences prior to library amplification. We report the design, evaluation, and optimization of DASH experiments for standard bacterial short-read sequencing approaches, including software for automated guide RNA (gRNA) design for Cas9-mediated cleavage in bacterial rDNA sequences. Using these gRNA pools, we effectively removed rRNA reads (56%-86%) in RNA-seq libraries from two different model bacteria, the Gram-negative pathogen Salmonella enterica and the anaerobic gut commensal Bacteroides thetaiotaomicron DASH works robustly, even with subnanogram amounts of input RNA. Its efficiency, high sensitivity, ease of implementation, and low cost (∼$5 per sample) render DASH an attractive alternative to rRNA removal protocols, in particular for material-constrained studies where conventional ribodepletion techniques fail.
    • Grad-seq in a Gram-positive bacterium reveals exonucleolytic sRNA activation in competence control.

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

      Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Wiley, 2020-03-17)
      Our body is colonized by a vast array of bacteria the sum of which forms our microbiota. The gut alone harbors >1,000 bacterial species. An understanding of their individual or synergistic contributions to human health and disease demands means to interfere with their functions on the species level. Most of the currently available antibiotics are broad-spectrum, thus too unspecific for a selective depletion of a single species of interest from the microbiota. Programmable RNA antibiotics in the form of short antisense oligonucleotides (ASOs) promise to achieve precision manipulation of bacterial communities. These ASOs are coupled to small peptides that carry them inside the bacteria to silence mRNAs of essential genes, for example, to target antibiotic-resistant pathogens as an alternative to standard antibiotics. There is already proof-of-principle with diverse bacteria, but many open questions remain with respect to true species specificity, potential off-targeting, choice of peptides for delivery, bacterial resistance mechanisms and the host response. While there is unlikely a one-fits-all solution for all microbiome species, I will discuss how recent progress in bacterial RNA biology may help to accelerate the development of programmable RNA antibiotics for microbiome editing and other applications.
    • 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.
    • Herpes simplex virus blocks host transcription termination via the bimodal activities of ICP27.

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

      Gerovac, Milan; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Elsevier BV, 2019-12)
      acterial pathogens secrete effector proteins to manipulate host signaling proteins and cellular structures. In this issue of Cell Host & Microbe, Pagliuso et al. (2019) propose an effector mechanism in Listeria monocytogenes whereby an RNA-binding protein associates with bacterial RNA that stimulates RIG-I (retinoic acid inducible gene I)-based innate immunity in the host cytosol.
    • 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.
    • The Major RNA-Binding Protein ProQ Impacts Virulence Gene Expression in Salmonella enterica Serovar Typhimurium.

      Westermann, Alexander J; Venturini, Elisa; Sellin, Mikael E; Förstner, Konrad U; Hardt, Wolf-Dietrich; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Amercan Society of Microbiology, 2019-01-02)
      FinO domain proteins such as ProQ of the model pathogen
    • scSLAM-seq reveals core features of transcription dynamics in single cells.

      Erhard, Florian; Baptista, Marisa A P; Krammer, Tobias; Hennig, Thomas; Lange, Marius; Arampatzi, Panagiota; Jürges, Christopher S; Theis, Fabian J; Saliba, Antoine-Emmanuel; Dölken, Lars; et al. (Springer-Nature, 2019-01-01)
      Single-cell RNA sequencing (scRNA-seq) has highlighted the important role of intercellular heterogeneity in phenotype variability in both health and disease1. However, current scRNA-seq approaches provide only a snapshot of gene expression and convey little information on the true temporal dynamics and stochastic nature of transcription. A further key limitation of scRNA-seq analysis is that the RNA profile of each individual cell can be analysed only once. Here we introduce single-cell, thiol-(SH)-linked alkylation of RNA for metabolic labelling sequencing (scSLAM-seq), which integrates metabolic RNA labelling2, biochemical nucleoside conversion3 and scRNA-seq to record transcriptional activity directly by differentiating between new and old RNA for thousands of genes per single cell. We use scSLAM-seq to study the onset of infection with lytic cytomegalovirus in single mouse fibroblasts. The cell-cycle state and dose of infection deduced from old RNA enable dose-response analysis based on new RNA. scSLAM-seq thereby both visualizes and explains differences in transcriptional activity at the single-cell level. Furthermore, it depicts 'on-off' switches and transcriptional burst kinetics in host gene expression with extensive gene-specific differences that correlate with promoter-intrinsic features (TBP-TATA-box interactions and DNA methylation). Thus, gene-specific, and not cell-specific, features explain the heterogeneity in transcriptomes between individual cells and the transcriptional response to perturbations.
    • Salmonella persisters undermine host immune defenses during antibiotic treatment.

      Stapels, Daphne A C; Hill, Peter W S; Westermann, Alexander J; Fisher, Robert A; Thurston, Teresa L; Saliba, Antoine-Emmanuel; Blommestein, Isabelle; Vogel, Jörg; Helaine, Sophie; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (American Association for the Advancement of Science, 2018-12-07)
      Many bacterial infections are hard to treat and tend to relapse, possibly due to the presence of antibiotic-tolerant persisters. In vitro, persister cells appear to be dormant. After uptake of Salmonella species by macrophages, nongrowing persisters also occur, but their physiological state is poorly understood. In this work, we show that Salmonella persisters arising during macrophage infection maintain a metabolically active state. Persisters reprogram macrophages by means of effectors secreted by the Salmonella pathogenicity island 2 type 3 secretion system. These effectors dampened proinflammatory innate immune responses and induced anti-inflammatory macrophage polarization. Such reprogramming allowed nongrowing Salmonella cells to survive for extended periods in their host. Persisters undermining host immune defenses might confer an advantage to the pathogen during relapse once antibiotic pressure is relieved.
    • Long Noncoding RNA SSR42 Controls Staphylococcus aureus Alpha-Toxin Transcription in Response to Environmental Stimuli.

      Horn, Jessica; Klepsch, Maximilian; Manger, Michelle; Wolz, Christiane; Rudel, Thomas; Fraunholz, Martin; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (2018-11-15)
      Staphylococcus aureus is a human pathogen causing a variety of diseases by versatile expression of a large set of virulence factors that most prominently features the cytotoxic and hemolytic pore-forming alpha-toxin. Expression of alpha-toxin is regulated by an intricate network of transcription factors. These include two-component systems sensing quorum and environmental signals as well as regulators reacting to the nutritional status of the pathogen. We previously identified the repressor of surface proteins (Rsp) as a virulence regulator. Acute cytotoxicity and hemolysis are strongly decreased in rsp mutants, which are characterized by decreased transcription of toxin genes as well as loss of transcription of a 1,232- nucleotide (nt)-long noncoding RNA (ncRNA), SSR42. Here, we show that SSR42 is the effector of Rsp in transcription regulation of the alpha-toxin gene, hla. SSR42 transcription is enhanced after exposure of S. aureus to subinhibitory concentrations of oxacillin which thus leads to an SSR42-dependent increase in hemolysis. Aside from Rsp, SSR42 transcription is under the control of additional global regulators, such as CodY, AgrA, CcpE, and B, but is positioned upstream of the two-component system SaeRS in the regulatory cascade leading to alpha-toxin production. Thus, alpha-toxin expression depends on two long ncRNAs, SSR42 and RNAIII, which control production of the cytolytic toxin on the transcriptional and translational levels, respectively, with SSR42 as an important regulator of SaeRS-dependent S. aureus toxin production in response to environmental and metabolic signals. IMPORTANCE Staphylococcus aureus is a major cause of life-threatening infections. The bacterium expresses alpha-toxin, a hemolysin and cytotoxin responsible for many of the pathologies of S. aureus. Alpha-toxin production is enhanced by subinhibitory concentrations of antibiotics. Here, we show that this process is dependent on the long noncoding RNA, SSR42. Further, SSR42 itself is regulated by several global regulators, thereby integrating environmental and nutritional signals that modulate hemolysis of the pathogen.
    • Stress-induced host membrane remodeling protects from infection by non-motile bacterial pathogens.

      Tawk, Caroline; Nigro, Giulia; Rodrigues Lopes, Ines; Aguilar, Carmen; Lisowski, Clivia; Mano, Miguel; Sansonetti, Philippe; Vogel, Jörg; Eulalio, Ana; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (2018-11-02)
      While mucosal inflammation is a major source of stress during enteropathogen infection, it remains to be fully elucidated how the host benefits from this environment to clear the pathogen. Here, we show that host stress induced by different stimuli mimicking inflammatory conditions strongly reduces the binding of Shigella flexneri to epithelial cells. Mechanistically, stress activates acid sphingomyelinase leading to host membrane remodeling. Consequently, knockdown or pharmacological inhibition of the acid sphingomyelinase blunts the stress-dependent inhibition of Shigella binding to host cells. Interestingly, stress caused by intracellular Shigella replication also results in remodeling of the host cell membrane, in vitro and in vivo, which precludes re-infection by this and other non-motile pathogens. In contrast, Salmonella Typhimurium overcomes the shortage of permissive entry sites by gathering effectively at the remaining platforms through its flagellar motility. Overall, our findings reveal host membrane remodeling as a novel stress-responsive cell-autonomous defense mechanism that protects epithelial cells from infection by non-motile bacterial pathogens.
    • RNA-binding proteins in bacteria.

      Holmqvist, Erik; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Springer Nature, 2018-10-01)
      RNA-binding proteins (RBPs) are central to most if not all cellular processes, dictating the fate of virtually all RNA molecules in the cell. Starting with pioneering work on ribosomal proteins, studies of bacterial RBPs have paved the way for molecular studies of RNA-protein interactions. Work over the years has identified major RBPs that act on cellular transcripts at the various stages of bacterial gene expression and that enable their integration into post-transcriptional networks that also comprise small non-coding RNAs. Bacterial RBP research has now entered a new era in which RNA sequencing-based methods permit mapping of RBP activity in a truly global manner in vivo. Moreover, the soaring interest in understudied members of host-associated microbiota and environmental communities is likely to unveil new RBPs and to greatly expand our knowledge of RNA-protein interactions in bacteria.
    • 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/.