• 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.
    • Smart sustainable bottle (SSB) system for E. coli based recombinant protein production.

      Li, Zhaopeng; Carstensen, Bettina; Rinas, Ursula; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2014)
      Recombinant proteins are usually required in laboratories interested in the protein but not in the production process itself. Thus, technical equipment which is easy to handle and straight forward protein production procedures are of great benefit to those laboratories. Companies selling single use cultivation bags and bioreactors are trying to satisfy at least part of these needs. However, single-use systems can contribute to major costs which might be acceptable when "good manufacturing practices" are required but not acceptable for most laboratories facing tight funding.
    • Structural Basis for Intrinsic Thermosensing by the Master Virulence Regulator RovA of Yersinia.

      Quade, Nick; Mendonca, Chriselle; Herbst, Katharina; Heroven, Ann Kathrin; Ritter, Christiane; Heinz, Dirk W; Dersch, Petra; From the Departments of Molecular Structural Biology and. (2012-10-19)
      Pathogens often rely on thermosensing to adjust virulence gene expression. In yersiniae, important virulence-associated traits are under the control of the master regulator RovA, which uses a built-in thermosensor to control its activity. Thermal upshifts encountered upon host entry induce conformational changes in the RovA dimer that attenuate DNA binding and render the protein more susceptible to proteolysis. Here, we report the crystal structure of RovA in the free and DNA-bound forms and provide evidence that thermo-induced loss of RovA activity is promoted mainly by a thermosensing loop in the dimerization domain and residues in the adjacent C-terminal helix. These determinants allow partial unfolding of the regulator upon an upshift to 37 °C. This structural distortion is transmitted to the flexible DNA-binding domain of RovA. RovA contacts mainly the DNA backbone in a low-affinity binding mode, which allows the immediate release of RovA from its operator sites. We also show that SlyA, a close homolog of RovA from Salmonella with a very similar structure, is not a thermosensor and remains active and stable at 37 °C. Strikingly, changes in only three amino acids, reflecting evolutionary replacements in SlyA, result in a complete loss of the thermosensing properties of RovA and prevent degradation. In conclusion, only minor alterations can transform a thermotolerant regulator into a thermosensor that allows adjustment of virulence and fitness determinants to their thermal environment.
    • Temperature-responsive in vitro RNA structurome of Yersinia pseudotuberculosis.

      Righetti, Francesco; Nuss, Aaron M; Twittenhoff, Christian; Beele, Sascha; Urban, Kristina; Will, Sebastian; Bernhart, Stephan H; Stadler, Peter F; Dersch, Petra; Narberhaus, Franz; et al. (2016-06-28)
      RNA structures are fundamentally important for RNA function. Dynamic, condition-dependent structural changes are able to modulate gene expression as shown for riboswitches and RNA thermometers. By parallel analysis of RNA structures, we mapped the RNA structurome of Yersinia pseudotuberculosis at three different temperatures. This human pathogen is exquisitely responsive to host body temperature (37 °C), which induces a major metabolic transition. Our analysis profiles the structure of more than 1,750 RNAs at 25 °C, 37 °C, and 42 °C. Average mRNAs tend to be unstructured around the ribosome binding site. We searched for 5'-UTRs that are folded at low temperature and identified novel thermoresponsive RNA structures from diverse gene categories. The regulatory potential of 16 candidates was validated. In summary, we present a dynamic bacterial RNA structurome and find that the expression of virulence-relevant functions in Y. pseudotuberculosis and reprogramming of its metabolism in response to temperature is associated with a restructuring of numerous mRNAs.
    • The Csr/Rsm system of Yersinia and related pathogens: A post-transcriptional strategy for managing virulence.

      Heroven, Ann Kathrin; Böhme, Katja; Dersch, Petra; Department of Molecular Infection Biology; Helmholtz Centre for Infection Research; Braunschweig, Germany; These authors contributed equally to this work. (2012-04-01)
      This review emphasizes the function and regulation of the Csr regulatory system in the human enteropathogen Yersinia pseudotuberculosis and compares its features with the homologous Csr/Rsm systems of related pathogens. The Csr/Rsm systems of eubacteria form a complex regulatory network in which redundant non-translated Csr/Rsm-RNAs bind the RNA-binding protein CsrA/RsmA, thereby preventing its interaction with mRNA targets. The Csr system is controlled by the BarA/UvrY-type of two-component sensor-regulator systems. Apart from that, common or pathogen-specific regulators control the abundance of the Csr components. The coordinate control of virulence factors and infection-linked physiological traits by the Csr/Rsm systems helps the pathogens to adapt individually to rapidly changing conditions to which they are exposed during the different stages of an infection. As Csr/Rsm function is relevant for full virulence, it represents a target suitable for antimicrobial drug development.
    • Thermosensing to adjust bacterial virulence in a fluctuating environment.

      Steinmann, Rebekka; Dersch, Petra; Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2013-01)
      The lifecycle of most microbial pathogens can be divided into two states: existence outside and inside their hosts. The sudden temperature upshift experienced upon entry from environmental or vector reservoirs into a warm-blooded host is one of the most crucial signals informing the pathogens to adjust virulence gene expression and their host-stress survival program. This article reviews the plethora of sophisticated strategies that bacteria have evolved to sense temperature, and outlines the molecular signal transduction mechanisms used to modulate synthesis of crucial virulence determinants. The molecular details of thermal control through conformational changes of DNA, RNA and proteins are summarized, complex and diverse thermosensing principles are introduced and their potential as drug targets or synthetic tools are discussed.
    • Tissue dual RNA-seq allows fast discovery of infection-specific functions and riboregulators shaping host-pathogen transcriptomes.

      Nuss, Aaron M; Beckstette, Michael; Pimenova, Maria; Schmühl, Carina; Opitz, Wiebke; Pisano, Fabio; Heroven, Ann Kathrin; Dersch, Petra; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-01-31)
      Pathogenic bacteria need to rapidly adjust their virulence and fitness program to prevent eradication by the host. So far, underlying adaptation processes that drive pathogenesis have mostly been studied in vitro, neglecting the true complexity of host-induced stimuli acting on the invading pathogen. In this study, we developed an unbiased experimental approach that allows simultaneous monitoring of genome-wide infection-linked transcriptional alterations of the host and colonizing extracellular pathogens. Using this tool for Yersinia pseudotuberculosis-infected lymphatic tissues, we revealed numerous alterations of host transcripts associated with inflammatory and acute-phase responses, coagulative activities, and transition metal ion sequestration, highlighting that the immune response is dominated by infiltrating neutrophils and elicits a mixed TH17/TH1 response. In consequence, the pathogen's response is mainly directed to prevent phagocytic attacks. Yersinia up-regulates the gene and expression dose of the antiphagocytic type III secretion system (T3SS) and induces functions counteracting neutrophil-induced ion deprivation, radical stress, and nutritional restraints. Several conserved bacterial riboregulators were identified that impacted this response. The strongest influence on virulence was found for the loss of the carbon storage regulator (Csr) system, which is shown to be essential for the up-regulation of the T3SS on host cell contact. In summary, our established approach provides a powerful tool for the discovery of infection-specific stimuli, induced host and pathogen responses, and underlying regulatory processes.
    • Toxoplasma gondii-induced neuronal alterations.

      Parlog, A; Schlüter, D; Dunay, I R; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2015-03)
      The zoonotic pathogen Toxoplasma gondii infects over 30% of the human population. The intracellular parasite can persist lifelong in the CNS within neurons modifying their function and structure, thus leading to specific behavioural changes of the host. In recent years, several in vitro studies and murine models have focused on the elucidation of these modifications. Furthermore, investigations of the human population have correlated Toxoplasma seropositivity with changes in neurological functions; however, the complex underlying mechanisms of the subtle behavioural alteration are still not fully understood. The parasites are able to induce direct modifications in the infected cells, for example by altering dopamine metabolism, by functionally silencing neurons as well as by hindering apoptosis. Moreover, indirect effects of the peripheral immune system and alterations of the immune status of the CNS, observed during chronic infection, might also contribute to changes in neuronal connectivity and synaptic plasticity. In this review, we will provide an overview and highlight recent advances, which describe changes in the neuronal function and morphology upon T. gondii infection.
    • Transcriptomic and Phenotypic Analysis Reveals New Functions for the Tat Pathway in Yersinia pseudotuberculosis.

      Avican, Ummehan; Beckstette, Michael; Heroven, Ann Kathrin; Lavander, Moa; Dersch, Petra; Forsberg, Åke; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-10-15)
      The twin-arginine translocation (Tat) system mediates the secretion of folded proteins that are identified via an N-terminal signal peptide in bacteria, plants, and archaea. Tat systems are associated with virulence in many bacterial pathogens, and our previous studies revealed that Tat-deficient Yersinia pseudotuberculosis was severely attenuated for virulence. Aiming to identify Tat-dependent pathways and phenotypes of relevance for in vivo infection, we analyzed the global transcriptome of parental and ΔtatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26°C and 37°C. The most significant changes in the transcriptome of the ΔtatC mutant were seen at 26°C during stationary-phase growth, and these included the altered expression of genes related to virulence, stress responses, and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat-dependent phenotypes, including decreased YadA expression, impaired growth under iron-limited and high-copper conditions, as well as acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypic defects observed in the Tat-deficient strain were generally more pronounced than those in mutants lacking the Tat substrate predicted to contribute to that specific function. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection.
    • Transcriptomic Profiling of Yersinia pseudotuberculosis Reveals Reprogramming of the Crp Regulon by Temperature and Uncovers Crp as a Master Regulator of Small RNAs.

      Nuss, Aaron M; Heroven, Ann Kathrin; Waldmann, Barbara; Reinkensmeier, Jan; Jarek, Michael; Beckstette, Michael; Dersch, Petra; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2015-02)
      One hallmark of pathogenic yersiniae is their ability to rapidly adjust their life-style and pathogenesis upon host entry. In order to capture the range, magnitude and complexity of the underlying gene control mechanisms we used comparative RNA-seq-based transcriptomic profiling of the enteric pathogen Y. pseudotuberculosis under environmental and infection-relevant conditions. We identified 1151 individual transcription start sites, multiple riboswitch-like RNA elements, and a global set of antisense RNAs and previously unrecognized trans-acting RNAs. Taking advantage of these data, we revealed a temperature-induced and growth phase-dependent reprogramming of a large set of catabolic/energy production genes and uncovered the existence of a thermo-regulated 'acetate switch', which appear to prime the bacteria for growth in the digestive tract. To elucidate the regulatory architecture linking nutritional status to virulence we also refined the CRP regulon. We identified a massive remodelling of the CRP-controlled network in response to temperature and discovered CRP as a transcriptional master regulator of numerous conserved and newly identified non-coding RNAs which participate in this process. This finding highlights a novel level of complexity of the regulatory network in which the concerted action of transcriptional regulators and multiple non-coding RNAs under control of CRP adjusts the control of Yersinia fitness and virulence to the requirements of their environmental and virulent life-styles.
    • The Type III Effector NleD from Enteropathogenic Escherichia coli Differentiates between Host Substrates p38 and JNK.

      Creuzburg, Kristina; Giogha, Cristina; Wong Fok Lung, Tania; Scott, Nichollas E; Mühlen, Sabrina; Hartland, Elizabeth L; Pearson, Jaclyn S; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-02)
      Enteropathogenic Escherichia coli (EPEC) is a gastrointestinal pathogen that utilizes a type III secretion system (T3SS) to inject an array of virulence effector proteins into host enterocytes to subvert numerous cellular processes for successful colonization and dissemination. The T3SS effector NleD is a 26-kDa zinc metalloprotease that is translocated into host enterocytes, where it directly cleaves and inactivates the mitogen-activated protein kinase signaling proteins JNK and p38. Here a library of 91 random transposon-based, in-frame, linker insertion mutants of NleD were tested for their ability to cleave JNK and p38 during transient transfection of cultured epithelial cells. Immunoblot analysis of p38 and JNK cleavage showed that 7 mutant derivatives of NleD no longer cleaved p38 but maintained the ability to cleave JNK. Site-directed mutation of specific regions surrounding the insertion sites within NleD revealed that a single amino acid, R203, was essential for cleavage of p38 but not JNK in a direct in vitro cleavage assay, in transiently transfected cells, or in EPEC-infected cells. Mass spectrometry analysis narrowed the cleavage region to within residues 187 and 213 of p38. Mutation of residue R203 within NleD to a glutamate residue abolished the cleavage of p38 and impaired the ability of NleD to inhibit AP-1-dependent gene transcription of a luciferase reporter. Furthermore, the R203 mutation abrogated the ability of NleD to dampen interleukin-6 production in EPEC-infected cells. Overall, this work provides greater insight into substrate recognition and specificity by the type III effector NleD.