• Lead-seq: transcriptome-wide structure probing in vivo using lead(II) ions.

      Twittenhoff, Christian; Brandenburg, Vivian B; Righetti, Francesco; Nuss, Aaron M; Mosig, Axel; Dersch, Petra; Narberhaus, Franz; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Oxfford University Press, 2020-05-28)
      The dynamic conformation of RNA molecules within living cells is key to their function. Recent advances in probing the RNA structurome in vivo, including the use of SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension) or kethoxal reagents or DMS (dimethyl sulfate), provided unprecedented insights into the architecture of RNA molecules in the living cell. Here, we report the establishment of lead probing in a global RNA structuromics approach. In order to elucidate the transcriptome-wide RNA landscape in the enteric pathogen Yersinia pseudotuberculosis, we combined lead(II) acetate-mediated cleavage of single-stranded RNA regions with high-throughput sequencing. This new approach, termed 'Lead-seq', provides structural information independent of base identity. We show that the method recapitulates secondary structures of tRNAs, RNase P RNA, tmRNA, 16S rRNA and the rpsT 5'-untranslated region, and that it reveals global structural features of mRNAs. The application of Lead-seq to Y. pseudotuberculosis cells grown at two different temperatures unveiled the first temperature-responsive in vivo RNA structurome of a bacterial pathogen. The translation of candidate genes derived from this approach was confirmed to be temperature regulated. Overall, this study establishes Lead-seq as complementary approach to interrogate intracellular RNA structures on a global scale.
    • Local application of bacteria improves safety of Salmonella-mediated tumor therapy and retains advantages of systemic infection.

      Kocijancic, Dino; Felgner, Sebastian; Schauer, Tim; Frahm, Michael; Heise, Ulrike; Zimmermann, Kurt; Erhardt, Marc; Weiss, Siegfried; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-06-07)
      Cancer is a devastating disease and a large socio-economic burden. Novel therapeutic solutions are on the rise, although a cure remains elusive. Application of microorganisms represents an ancient therapeutic strategy, lately revoked and refined via simultaneous attenuation and amelioration of pathogenic properties. Salmonella Typhimurium has prevailed in preclinical development. Yet, using virulent strains for systemic treatment might cause severe side effects. In the present study, we highlight a modified strain based on Salmonella Typhimurium UK-1 expressing hexa-acylated Lipid A. We corroborate improved anti-tumor properties of this strain and investigate to which extent an intra-tumoral (i.t.) route of infection could help improve safety and retain advantages of systemic intravenous (i.v.) application. Our results show that i.t. infection exhibits therapeutic efficacy against CT26 and F1.A11 tumors similar to a systemic route of inoculation. Moreover, i.t. application allows extensive dose titration without compromising tumor colonization. Adverse colonization of healthy organs was generally reduced via i.t. infection and accompanied by less body weight loss of the murine host. Despite local application, adjuvanticity remained, and a CT26-specific CD8+ T cell response was effectively stimulated. Most interestingly, also secondary tumors could be targeted with this strategy, thereby extending the unique tumor targeting ability of Salmonella. The i.t. route of inoculation may reap the benefits of systemic infection and aid in safety assurance while directing potency of an oncolytic vector to where it is most needed, namely the primary tumor.
    • Loss of CNFY toxin-induced inflammation drives Yersinia pseudotuberculosis into persistency.

      Heine, Wiebke; Beckstette, Michael; Heroven, Ann Kathrin; Thiemann, Sophie; Heise, Ulrike; Nuss, Aaron Mischa; Pisano, Fabio; Strowig, Till; Dersch, Petra; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-02)
      Gastrointestinal infections caused by enteric yersiniae can become persistent and complicated by relapsing enteritis and severe autoimmune disorders. To establish a persistent infection, the bacteria have to cope with hostile surroundings when they transmigrate through the intestinal epithelium and colonize underlying gut-associated lymphatic tissues. How the bacteria gain a foothold in the face of host immune responses is poorly understood. Here, we show that the CNFY toxin, which enhances translocation of the antiphagocytic Yop effectors, induces inflammatory responses. This results in extensive tissue destruction, alteration of the intestinal microbiota and bacterial clearance. Suppression of CNFY function, however, increases interferon-γ-mediated responses, comprising non-inflammatory antimicrobial activities and tolerogenesis. This process is accompanied by a preterm reprogramming of the pathogen's transcriptional response towards persistence, which gives the bacteria a fitness edge against host responses and facilitates establishment of a commensal-type life style.
    • Management of a cluster of Clostridium difficile infections among patients with osteoarticular infections.

      Färber, Jacqueline; Illiger, Sebastian; Berger, Fabian; Gärtner, Barbara; von Müller, Lutz; Lohmann, Christoph H; Bauer, Katja; Grabau, Christina; Zibolka, Stefanie; Schlüter, Dirk; et al. (2017)
      Here we describe a cluster of hospital-acquired Clostridium difficile infections (CDI) among 26 patients with osteoarticular infections. The aim of the study was to define the source of C. difficile and to evaluate the impact of general infection control measures and antibiotic stewardship on the incidence of CDI.
    • Metabolome and transcriptome-wide effects of the carbon storage regulator A in enteropathogenic Escherichia coli.

      Berndt, Volker; Beckstette, Michael; Volk, Marcel; Dersch, Petra; Brönstrup, Mark; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer-Nature, 2019-01-15)
      The carbon storage regulator A (CsrA) is a conserved global regulatory system known to control central carbon pathways, biofilm formation, motility, and pathogenicity. The aim of this study was to characterize changes in major metabolic pathways induced by CsrA in human enteropathogenic Escherichia coli (EPEC) grown under virulence factor-inducing conditions. For this purpose, the metabolomes and transcriptomes of EPEC and an isogenic ∆csrA mutant derivative were analyzed by untargeted mass spectrometry and RNA sequencing, respectively. Of the 159 metabolites identified from untargeted GC/MS and LC/MS data, 97 were significantly (fold change ≥ 1.5; corrected p-value ≤ 0.05) regulated between the knockout and the wildtype strain. A lack of csrA led to an accumulation of fructose-6-phosphate (F6P) and glycogen synthesis pathway products, whereas metabolites in lower glycolysis and the citric acid cycle were downregulated. Associated pathways from the citric acid cycle like aromatic amino acid and siderophore biosynthesis were also negatively influenced. The nucleoside salvage pathways were featured by an accumulation of nucleosides and nucleobases, and a downregulation of nucleotides. In addition, a pronounced downregulation of lyso-lipid metabolites was observed. A drastic change in the morphology in the form of vesicle-like structures of the ∆csrA knockout strain was visible by electron microscopy. Colanic acid synthesis genes were strongly (up to 50 fold) upregulated, and the abundance of colanic acid was 3 fold increased according to a colorimetric assay. The findings expand the scope of pathways affected by the csrA regulon and emphasize its importance as a global regulator.
    • Molekulare Thermometer zur Steuerung der Virulenz von Yersinia

      Heroven, Ann Kathrin; Dersch, Petra; Dept. of molecular infectionbiology, Helmholtz Centre for infection research, Inhoffenstr. 7, D38124 Braunschweig, Germany. (2014-09-23)
    • Monitoring of gene expression in bacteria during infections using an adaptable set of bioluminescent, fluorescent and colorigenic fusion vectors.

      Uliczka, Frank; Pisano, Fabio; Kochut, Annika; Opitz, Wiebke; Herbst, Katharina; Stolz, Tatjana; Dersch, Petra; Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Lower Saxony, Germany. (2011)
      A family of versatile promoter-probe plasmids for gene expression analysis was developed based on a modular expression plasmid system (pZ). The vectors contain different replicons with exchangeable antibiotic cassettes to allow compatibility and expression analysis on a low-, midi- and high-copy number basis. Suicide vector variants also permit chromosomal integration of the reporter fusion and stable vector derivatives can be used for in vivo or in situ expression studies under non-selective conditions. Transcriptional and translational fusions to the reporter genes gfp(mut3.1), amCyan, dsRed2, luxCDABE, phoA or lacZ can be constructed, and presence of identical multiple cloning sites in the vector system facilitates the interchange of promoters or reporter genes between the plasmids of the series. The promoter of the constitutively expressed gapA gene of Escherichia coli was included to obtain fluorescent and bioluminescent expression constructs. A combination of the plasmids allows simultaneous detection and gene expression analysis in individual bacteria, e.g. in bacterial communities or during mouse infections. To test our vector system, we analyzed and quantified expression of Yersinia pseudotuberculosis virulence genes under laboratory conditions, in association with cells and during the infection process.
    • Natural Killer Cells Mediate Protection against Yersinia pseudotuberculosis in the Mesenteric Lymph Nodes.

      Rosenheinrich, Maik; Heine, Wiebke; Schmühl, Carina M; Pisano, Fabio; Dersch, Petra; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2015)
      Natural killer cells play a crucial role in the initial defense against bacterial pathogens. The crosstalk between host cells infected with intracellular pathogens and NK cells has been studied intensively, but not much attention has been given to characterize the role of NK cells in the response to extracellular bacterial pathogens such as yersiniae. In this study we used antibody-mediated NK cell depletion to address the importance of this immune cell type in controlling a Y. pseudotuberculosis infection. Analysis of the bacterial counts was used to follow the infection and flow cytometry was performed to characterize the composition and dynamic of immune cells. Depletion of NK cells led to higher bacterial loads within the mesenteric lymph nodes. We further show that in particular CD11b+ CD27+ NK cells which express higher levels of the activation marker CD69 increase within the mesenteric lymph nodes during a Y. pseudotuberculosis infection. Moreover, in response to the activation NK cells secrete higher levels of IFNy, which in turn triggers the production of the proinflammatory cytokine TNFα. These results suggest, that NK cells aid in the clearance of Y. pseudotuberculosis infections mainly by triggering the expression of proinflammatory cytokines manipulating the host immune response.
    • A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger.

      Roth, Andreas H F J; Dersch, Petra; Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, 38106, Braunschweig, Germany. (2010-03)
      A set of different integrative expression vectors for the intracellular production of recombinant proteins with or without affinity tag in Aspergillus niger was developed. Target genes can be expressed under the control of the highly efficient, constitutive pkiA promoter or the novel sucrose-inducible promoter of the beta-fructofuranosidase (sucA) gene of A. niger in the presence or absence of alternative carbon sources. All expression plasmids contain an identical multiple cloning sequence that allows parallel construction of N- or C-terminally His6- and StrepII-tagged versions of the target proteins. Production of two heterologous model proteins, the green fluorescence protein and the Thermobifida fusca hydrolase, proved the functionality of the vector system. Efficient production and easy detection of the target proteins as well as their fast purification by a one-step affinity chromatography, using the His6- or StrepII-tag sequence, was demonstrated.
    • Novel type of pilus associated with a Shiga-toxigenic E. coli hybrid pathovar conveys aggregative adherence and bacterial virulence.

      Lang, Christina; Fruth, Angelika; Holland, Gudrun; Laue, Michael; Mühlen, Sabrina; Dersch, Petra; Flieger, Antje; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer-Nature, 2018-12-05)
      A large German outbreak in 2011 was caused by a locus of enterocyte effacement (LEE)-negative enterohemorrhagic E. coli (EHEC) strain of the serotype O104:H4. This strain harbors markers that are characteristic of both EHEC and enteroaggregative E. coli (EAEC), including aggregative adhesion fimbriae (AAF) genes. Such rare EHEC/EAEC hybrids are highly pathogenic due to their possession of a combination of genes promoting severe toxicity and aggregative adhesion. We previously identified novel EHEC/EAEC hybrids and observed that one strain exhibited aggregative adherence but had no AAF genes. In this study, a genome sequence analysis showed that this strain belongs to the genoserotype O23:H8, MLST ST26, and harbors a 5.2 Mb chromosome and three plasmids. One plasmid carries some EAEC marker genes, such as aatA and genes with limited protein homology (11-61%) to those encoding the bundle-forming pilus (BFP) of enteropathogenic E. coli. Due to significant protein homology distance to known pili, we designated these as aggregate-forming pili (AFP)-encoding genes and the respective plasmid as pAFP. The afp operon was arranged similarly to the operon of BFP genes but contained an additional gene, afpA2, which is homologous to afpA. The deletion of the afp operon, afpA, or a nearby gene (afpR) encoding an AraC-like regulator, but not afpA2, led to a loss of pilin production, piliation, bacterial autoaggregation, and importantly, a >80% reduction in adhesion and cytotoxicity toward epithelial cells. Gene sets similar to the afp operon were identified in a variety of aatA-positive but AAF-negative intestinal pathogenic E. coli. In summary, we characterized widely distributed and novel fimbriae that are essential for aggregative adherence and cytotoxicity in a LEE-negative Shiga-toxigenic hybrid.
    • Persistence of Toxoplasma gondii in the central nervous system: a fine-tuned balance between the parasite, the brain and the immune system.

      Blanchard, N; Dunay, I R; Schlüter, D; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2015-03)
      Upon infection of humans and animals with Toxoplasma gondii, the parasites persist as intraneuronal cysts that are controlled, but not eliminated by the immune system. In particular, intracerebral T cells are crucial in the control of T. gondii infection and are supported by essential functions from other leukocyte populations. Additionally, brain-resident cells including astrocytes, microglia and neurons contribute to the intracerebral immune response by the production of cytokines, chemokines and expression of immunoregulatory cell surface molecules, such as major histocompatibility (MHC) antigens. However, the in vivo behaviour of these individual cell populations, specifically their interaction during cerebral toxoplasmosis, remains to be elucidated. We discuss here what is known about the function of T cells, recruited myeloid cells and brain-resident cells, with particular emphasis on the potential cross-regulation of these cell populations, in governing cerebral toxoplasmosis.
    • Phenotypic heterogeneity: a bacterial virulence strategy.

      Weigel, W A; Dersch, P; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-02-01)
      Growing knowledge of the complexity of the host-pathogen interactions during the course of an infection revealed an amazing variability of bacterial pathogens within the same host tissue site. This heterogeneity in bacterial populations is either the result of a different bacterial response to a slightly divergent tissue microenvironment or is caused by a genetic circuit in which small endogenous fluctuations in a small number of transcription factors drive gene expression in combination with a positive feedback loop. As a result host-pathogen encounters can have different outcomes in individual cells, which enables bet-hedging and/or a co-operative behavior that enhance bacterial fitness and virulence, drive different host responses and promote resistance of small subpopulations to antibiotic treatment. This has a strong impact on the progression and control of the infection, which must be considered for the development of successful antimicrobial therapies.
    • The pyruvate-tricarboxylic acid cycle node: a focal point of virulence control in the enteric pathogen Yersinia pseudotuberculosis.

      Bücker, René; Heroven, Ann Kathrin; Becker, Judith; Dersch, Petra; Wittmann, Christoph; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2014-10-24)
      Despite our increasing knowledge of the specific pathogenicity factors in bacteria, the contribution of metabolic processes to virulence is largely unknown. Here, we elucidate a tight connection between pathogenicity and core metabolism in the enteric pathogen Yersinia pseudotuberculosis by integrated transcriptome and [(13)C]fluxome analysis of the wild type and virulence-regulator mutants. During aerobic growth on glucose, Y. pseudotuberculosis reveals an unusual flux distribution with a high level of secreted pyruvate. The absence of the transcriptional and post-transcriptional regulators RovA, CsrA, and Crp strongly perturbs the fluxes of carbon core metabolism at the level of pyruvate metabolism and the tricarboxylic acid (TCA) cycle, and these perturbations are accompanied by transcriptional changes in the corresponding enzymes. Knock-outs of regulators of this metabolic branch point and of its central enzyme, pyruvate kinase (ΔpykF), result in mutants with significantly reduced virulence in an oral mouse infection model. In summary, our work identifies the pyruvate-TCA cycle node as a focal point for controlling the host colonization and virulence of Yersinia.
    • Receptor-Interacting Protein Kinase-2 Inhibition by CYLD Impairs Antibacterial Immune Responses in Macrophages.

      Wex, Katharina; Schmid, Ursula; Just, Sissy; Wang, Xu; Wurm, Rebecca; Naumann, Michael; Schlüter, Dirk; Nishanth, Gopala; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2015)
      Upon infection with intracellular bacteria, nucleotide oligomerization domain protein 2 recognizes bacterial muramyl dipeptide and binds, subsequently, to receptor-interacting serine/threonine kinase 2 (RIPK2), which activates immune responses via the nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) and extracellular signal-regulated kinase (ERK) pathways. Activation of RIPK2 depends on its K63 ubiquitination by E3 ligases, whereas the deubiquitinating enzyme A20 counter regulates RIPK2 activity by cleaving K63-polyubiquitin chains from RIPK2. Here, we newly identify the deubiquitinating enzyme CYLD as a new inhibitor of RIPK2. We show that CYLD binds to and removes K63-polyubiquitin chains from RIPK2 in Listeria monocytogenes (Lm) infected murine bone marrow-derived macrophages. CYLD-mediated K63 deubiquitination of RIPK2 resulted in an impaired activation of both NF-κB and ERK1/2 pathways, reduced production of proinflammatory cytokines interleukin-6 (IL-6), IL-12, anti-listerial reactive oxygen species (ROS) and nitric oxide (NO), and, finally, impaired pathogen control. In turn, RIPK2 inhibition by siRNA prevented activation of NF-κB and ERK1/2 and completely abolished the protective effect of CYLD deficiency with respect to the production of IL-6, NO, ROS, and pathogen control. Noteworthy, CYLD also inhibited autophagy of Listeria in a RIPK2-ERK1/2-dependent manner. The protective function of CYLD deficiency was dependent on interferon gamma (IFN-γ) prestimulation of infected macrophages. Interestingly, the reduced NF-κB activation in CYLD-expressing macrophages limited the protective effect of IFN-γ by reducing NF-κB-dependent signal transducers and activators of transcription-1 (STAT1) activation. Taken together, our study identifies CYLD as an important inhibitor of RIPK2-dependent antibacterial immune responses in macrophages.
    • Reprogramming of Yersinia from virulent to persistent mode revealed by complex in vivo RNA-seq analysis.

      Avican, Kemal; Fahlgren, Anna; Huss, Mikael; Heroven, Ann Kathrin; Beckstette, Michael; Dersch, Petra; Fällman, Maria (2015-01)
      We recently found that Yersinia pseudotuberculosis can be used as a model of persistent bacterial infections. We performed in vivo RNA-seq of bacteria in small cecal tissue biopsies at early and persistent stages of infection to determine strategies associated with persistence. Comprehensive analysis of mixed RNA populations from infected tissues revealed that Y. pseudotuberculosis undergoes transcriptional reprogramming with drastic down-regulation of T3SS virulence genes during persistence when the pathogen resides within the cecum. At the persistent stage, the expression pattern in many respects resembles the pattern seen in vitro at 26oC, with for example, up-regulation of flagellar genes and invA. These findings are expected to have impact on future rationales to identify suitable bacterial targets for new antibiotics. Other genes that are up-regulated during persistence are genes involved in anaerobiosis, chemotaxis, and protection against oxidative and acidic stress, which indicates the influence of different environmental cues. We found that the Crp/CsrA/RovA regulatory cascades influence the pattern of bacterial gene expression during persistence. Furthermore, arcA, fnr, frdA, and wrbA play critical roles in persistence. Our findings suggest a model for the life cycle of this enteropathogen with reprogramming from a virulent to an adapted phenotype capable of persisting and spreading by fecal shedding.
    • RNA Regulators: Formidable Modulators of Yersinia Virulence.

      Nuss, Aaron M; Heroven, Ann Kathrin; Dersch, Petra; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-01)
      A large repertoire of RNA-based regulatory mechanisms, including a plethora of cis- and trans-acting noncoding RNAs (ncRNAs), sensory RNA elements, regulatory RNA-binding proteins, and RNA-degrading enzymes have been uncovered lately as key players in the regulation of metabolism, stress responses, and virulence of the genus Yersinia. Many of them are strictly controlled in response to fluctuating environmental conditions sensed during the course of the infection, and certain riboregulators have already been shown to be crucial for virulence. Some of them are highly conserved among the family Enterobacteriaceae, while others are genus-, species-, or strain-specific and could contribute to the difference in Yersinia pathogenicity. Importantly, the analysis of Yersinia riboregulators has not only uncovered crucial elements and regulatory mechanisms governing host-pathogen interactions, it also revealed exciting new venues for the design of novel anti-infectives.
    • An RNA thermometer dictates production of a secreted bacterial toxin.

      Twittenhoff, Christian; Heroven, Ann Kathrin; Mühlen, Sabrina; Dersch, Petra; Narberhaus, Franz; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (PLOS, 2020-01-01)
      Frequent transitions of bacterial pathogens between their warm-blooded host and external reservoirs are accompanied by abrupt temperature shifts. A temperature of 37°C serves as reliable signal for ingestion by a mammalian host, which induces a major reprogramming of bacterial gene expression and metabolism. Enteric Yersiniae are Gram-negative pathogens accountable for self-limiting gastrointestinal infections. Among the temperature-regulated virulence genes of Yersinia pseudotuberculosis is cnfY coding for the cytotoxic necrotizing factor (CNFY), a multifunctional secreted toxin that modulates the host's innate immune system and contributes to the decision between acute infection and persistence. We report that the major determinant of temperature-regulated cnfY expression is a thermo-labile RNA structure in the 5'-untranslated region (5'-UTR). Various translational gene fusions demonstrated that this region faithfully regulates translation initiation regardless of the transcription start site, promoter or reporter strain. RNA structure probing revealed a labile stem-loop structure, in which the ribosome binding site is partially occluded at 25°C but liberated at 37°C. Consistent with translational control in bacteria, toeprinting (primer extension inhibition) experiments in vitro showed increased ribosome binding at elevated temperature. Point mutations locking the 5'-UTR in its 25°C structure impaired opening of the stem loop, ribosome access and translation initiation at 37°C. To assess the in vivo relevance of temperature control, we used a mouse infection model. Y. pseudotuberculosis strains carrying stabilized RNA thermometer variants upstream of cnfY were avirulent and attenuated in their ability to disseminate into mesenteric lymph nodes and spleen. We conclude with a model, in which the RNA thermometer acts as translational roadblock in a two-layered regulatory cascade that tightly controls provision of the CNFY toxin during acute infection. Similar RNA structures upstream of various cnfY homologs suggest that RNA thermosensors dictate the production of secreted toxins in a wide range of pathogens.
    • RNA-based mechanisms of virulence control in Enterobacteriaceae.

      Heroven, Ann Kathrin; Nuss, Aaron M; Dersch, Petra; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-07-21)
      Enteric pathogens of the family Enterobacteriaceae colonize various niches within animals and humans in which they compete with intestinal commensals and are attacked by the host immune system. To survive these hostile environments they possess complex, multilayer regulatory networks that coordinate the control of virulence factors, host-adapted metabolic functions and stress resistance. An important part of these intricate control networks are RNA-based control systems that enable the pathogen to fine-tune its responses. Recent next-generation sequencing approaches revealed a large repertoire of conserved and species-specific riboregulators, including numerous cis- and trans-acting non-coding RNAs, sensory RNA elements (RNA thermometers, riboswitches), regulatory RNA-binding proteins and RNA degrading enzymes which regulate colonization factors, toxins, host defense processes and virulence-relevant physiological and metabolic processes. All of which are important cues for pathogens to sense and respond to fluctuating conditions during the infection. This review covers infection-relevant riboregulators of E. coli, Salmonella, Shigella and Yersinia, highlights their versatile regulatory mechanisms, complex target regulons and functions, and discusses emerging topics and future challenges to fully understand and exploit RNA-based control to combat bacterial infections.
    • Roles of Regulatory RNAs for Antibiotic Resistance in Bacteria and Their Potential Value as Novel Drug Targets.

      Dersch, Petra; Khan, Muna A; Mühlen, Sabrina; Görke, Boris; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017)
      The emergence of antibiotic resistance mechanisms among bacterial pathogens increases the demand for novel treatment strategies. Lately, the contribution of non-coding RNAs to antibiotic resistance and their potential value as drug targets became evident. RNA attenuator elements in mRNA leader regions couple expression of resistance genes to the presence of the cognate antibiotic. Trans-encoded small RNAs (sRNAs) modulate antibiotic tolerance by base-pairing with mRNAs encoding functions important for resistance such as metabolic enzymes, drug efflux pumps, or transport proteins. Bacteria respond with extensive changes of their sRNA repertoire to antibiotics. Each antibiotic generates a unique sRNA profile possibly causing downstream effects that may help to overcome the antibiotic challenge. In consequence, regulatory RNAs including sRNAs and their protein interaction partners such as Hfq may prove useful as targets for antimicrobial chemotherapy. Indeed, several compounds have been developed that kill bacteria by mimicking ligands for riboswitches controlling essential genes, demonstrating that regulatory RNA elements are druggable targets. Drugs acting on sRNAs are considered for combined therapies to treat infections. In this review, we address how regulatory RNAs respond to and establish resistance to antibiotics in bacteria. Approaches to target RNAs involved in intrinsic antibiotic resistance or virulence for chemotherapy will be discussed.
    • RovC - a novel type of hexameric transcriptional activator promoting type VI secretion gene expression.

      Knittel, Vanessa; Sadana, Pooja; Seekircher, Stephanie; Stolle, Anne-Sophie; Körner, Britta; Volk, Marcel; Jeffries, Cy M; Svergun, Dmitri I; Heroven, Ann Kathrin; Scrima, Andrea; et al. (PLOS, 2020-09-23)
      Type VI secretion systems (T6SSs) are complex macromolecular injection machines which are widespread in Gram-negative bacteria. They are involved in host-cell interactions and pathogenesis, required to eliminate competing bacteria, or are important for the adaptation to environmental stress conditions. Here we identified regulatory elements controlling the T6SS4 of Yersinia pseudotuberculosis and found a novel type of hexameric transcription factor, RovC. RovC directly interacts with the T6SS4 promoter region and activates T6SS4 transcription alone or in cooperation with the LysR-type regulator RovM. A higher complexity of regulation was achieved by the nutrient-responsive global regulator CsrA, which controls rovC expression on the transcriptional and post-transcriptional level. In summary, our work unveils a central mechanism in which RovC, a novel key activator, orchestrates the expression of the T6SS weapons together with a global regulator to deploy the system in response to the availability of nutrients in the species' native environment.