• Anti-virulence Strategies to Target Bacterial Infections.

      Mühlen, Sabrina; Dersch, Petra; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
      Resistance of important bacterial pathogens to common antimicrobial therapies and the emergence of multidrug-resistant bacteria are increasing at an alarming rate and constitute one of our greatest challenges in the combat of bacterial infection and accompanied diseases. The current shortage of effective drugs, lack of successful prevention measures and only a few new antibiotics in the clinical pipeline demand the development of novel treatment options and alternative antimicrobial therapies. Our increasing understanding of bacterial virulence strategies and the induced molecular pathways of the infectious disease provides novel opportunities to target and interfere with crucial pathogenicity factors or virulence-associated traits of the bacteria while bypassing the evolutionary pressure on the bacterium to develop resistance. In the past decade, numerous new bacterial targets for anti-virulence therapies have been identified, and structure-based tailoring of intervention strategies and screening assays for small-molecule inhibitors of such pathways were successfully established. In this chapter, we will take a closer look at the bacterial virulence-related factors and processes that present promising targets for anti-virulence therapies, recently discovered inhibitory substances and their promises and discuss the challenges, and problems that have to be faced.
    • Aspherical and Spherical InvA497-Functionalized Nanocarriers for Intracellular Delivery of Anti-Infective Agents.

      Castoldi, Arianna; Empting, Martin; De Rossi, Chiara; Mayr, Karsten; Dersch, Petra; Hartmann, Rolf; Müller, Rolf; Gordon, Sarah; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer, 2018-12-05)
      The objective of this work was to evaluate the potential of polymeric spherical and aspherical invasive nanocarriers, loaded with antibiotic, to access and treat intracellular bacterial infections. Aspherical nanocarriers were prepared by stretching of spherical precursors, and both aspherical and spherical nanocarriers were surface-functionalized with the invasive protein InvA497. The relative uptake of nanocarriers into HEp-2 epithelial cells was then assessed. Nanocarriers were subsequently loaded with a preparation of the non-permeable antibiotic gentamicin, and tested for their ability to treat HEp-2 cells infected with the enteroinvasive bacterium Shigella flexneri. InvA497-functionalized nanocarriers of both spherical and aspherical shape showed a significantly improved rate and extent of uptake into HEp-2 cells in comparison to non-functionalized nanocarriers. Functionalized and antibiotic-loaded nanocarriers demonstrated a dose dependent killing of intracellular S. flexneri. A slight but significant enhancement of intracellular bacterial killing was also observed with aspherical as compared to spherical functionalized nanocarriers at the highest tested concentration. InvA497-functionalized, polymer-based nanocarriers were able to efficiently deliver a non-permeable antibiotic across host cell membranes to affect killing of intracellular bacteria. Functionalized nanocarriers with an aspherical shape showed an interesting future potential for intracellular infection therapy.
    • Bacterial invasion factors: Tools for crossing biological barriers and drug delivery?

      Kochut, Annika; Dersch, Petra; Department of Molecular Infection Biology, Helmholtz Center for Infection Research, Braunschweig, Germany. (2013-06)
      The oral route is the preferential route of drug delivery in humans. However, effective delivery through the gastrointestinal tract is often hampered by the low permeability of the intestinal epithelium. One possibility to overcome this problem is the encapsulation of drugs inside nanoparticulate systems, containing targeting moieties with cell invasive properties. The bioinvasive features of the delivery system could be provided by the attachment of bacterial invasion factors, which promote efficient uptake into host cells and mediate rapid transcytosis of the pathogen through the intestinal epithelium. This review gives an overview of bacterial invasion systems. The molecular structure and function of suitable bacterial invasins, their relative values as targeting agents and possible pitfalls of their use are described. The potential of bioinvasive drug delivery systems is mainly presented on the basis of the well-characterized Yersinia invasin protein, which enters M cells to gain access to subepithelial layers of the gastrointestinal tract, but alternative approaches and future prospects for oral drug delivery are also discussed.
    • A bacterial secreted translocator hijacks riboregulators to control type III secretion in response to host cell contact.

      Kusmierek, Maria; Hoßmann, Jörn; Witte, Rebekka; Opitz, Wiebke; Vollmer, Ines; Volk, Marcel; Heroven, Ann Kathrin; Wolf-Watz, Hans; Dersch, Petra; HZI, Helmholtz -Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (PLOS, 2019-06-01)
      Numerous Gram-negative pathogens use a Type III Secretion System (T3SS) to promote virulence by injecting effector proteins into targeted host cells, which subvert host cell processes. Expression of T3SS and the effectors is triggered upon host cell contact, but the underlying mechanism is poorly understood. Here, we report a novel strategy of Yersinia pseudotuberculosis in which this pathogen uses a secreted T3SS translocator protein (YopD) to control global RNA regulators. Secretion of the YopD translocator upon host cell contact increases the ratio of post-transcriptional regulator CsrA to its antagonistic small RNAs CsrB and CsrC and reduces the degradosome components PNPase and RNase E levels. This substantially elevates the amount of the common transcriptional activator (LcrF) of T3SS/Yop effector genes and triggers the synthesis of associated virulence-relevant traits. The observed hijacking of global riboregulators allows the pathogen to coordinate virulence factor expression and also readjusts its physiological response upon host cell contact.
    • Bioorthogonal metabolic glycoengineering of human larynx carcinoma (HEp-2) cells targeting sialic acid.

      Homann, Arne; Qamar, Riaz-Ul; Serim, Sevnur; Dersch, Petra; Seibel, Jürgen; University of Würzburg, Department of Organic Chemistry, Am Hubland, 97074 Würzburg, Germany. (2010)
      Sialic acids are located at the termini of mammalian cell-surface glycostructures, which participate in essential interaction processes including adhesion of pathogens prior to infection and immunogenicity. Here we present the synthesis and bioorthogonal metabolic incorporation of the sialic acid analogue N-(1-oxohex-5-ynyl)neuraminic acid (Neu5Hex) into the cell-surface glycocalyx of a human larynx carcinoma cell line (HEp-2) and its fluorescence labelling by click chemistry.
    • Cell invasion of Yersinia pseudotuberculosis by invasin and YadA requires protein kinase C, phospholipase C-gamma1 and Akt kinase.

      Uliczka, Frank; Kornprobst, Tina; Eitel, Julia; Schneider, Daniela; Dersch, Petra; Institut für Mikrobiologie, Technische Universität Braunschweig, 38106 Braunschweig, Germany. (2009-12)
      The outer membrane proteins YadA and invasin of Yersinia pseudotuberculosis promote invasion into mammalian cells through beta(1)-integrins and trigger the production of interleukin (IL)-8. FAK, c-Src and the PI3 kinase were previously found to be important for both YadA- and invasin-promoted uptake. Here, we demonstrate that two different downstream effectors of PI3 kinase, Akt and phospholipase Cgamma1 are required for efficient cell invasion. Inhibition of Akt or phospholipase C-gamma (PLC-gamma)1 by pharmaceutical agents as well as reduced expression of the isoforms Akt1 and Akt2, and of PLC-gamma1 by RNA interference decreased entry of YadA- and Inv-expressing bacteria significantly. In addition, we report that the conventional protein kinases C (PKC)alpha and -beta, positioned downstream of PLC-gamma1, are activated upon Inv- or YadA-promoted cell entry. They colocalize with intracellular bacteria and their depletion by siRNA treatment also resulted in a strong reduction of cell entry. In contrast, neither Akt nor PLC-gamma1, and the PKCs are essential for YadA- and Inv-mediated IL-8 synthesis and release. We conclude that YadA and invasin of Y. pseudotuberculosis both trigger similar signal transduction pathways during integrin-mediated phagocytosis into epithelial cells, which lead to the activation of Akt, PLC-gamma1, PKCalpha and -beta downstream of PI3 kinase, separate from the MAPK-dependent pathway that triggers IL-8 production.
    • Common and divergent features in transcriptional control of the homologous small RNAs GlmY and GlmZ in Enterobacteriaceae.

      Göpel, Yvonne; Lüttmann, Denise; Heroven, Ann Kathrin; Reichenbach, Birte; Dersch, Petra; Görke, Boris; Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August-University, Grisebachstrasse 8, 37077 Göttingen, Germany. (2011-03)
      Small RNAs GlmY and GlmZ compose a cascade that feedback-regulates synthesis of enzyme GlmS in Enterobacteriaceae. Here, we analyzed the transcriptional regulation of glmY/glmZ from Yersinia pseudotuberculosis, Salmonella typhimurium and Escherichia coli, as representatives for other enterobacterial species, which exhibit similar promoter architectures. The GlmY and GlmZ sRNAs of Y. pseudotuberculosis are transcribed from σ(54)-promoters that require activation by the response regulator GlrR through binding to three conserved sites located upstream of the promoters. This also applies to glmY/glmZ of S. typhimurium and glmY of E. coli, but as a difference additional σ(70)-promoters overlap the σ(54)-promoters and initiate transcription at the same site. In contrast, E. coli glmZ is transcribed from a single σ(70)-promoter. Thus, transcription of glmY and glmZ is controlled by σ(54) and the two-component system GlrR/GlrK (QseF/QseE) in Y. pseudotuberculosis and presumably in many other Enterobacteria. However, in a subset of species such as E. coli this relationship is partially lost in favor of σ(70)-dependent transcription. In addition, we show that activity of the σ(54)-promoter of E. coli glmY requires binding of the integration host factor to sites upstream of the promoter. Finally, evidence is provided that phosphorylation of GlrR increases its activity and thereby sRNA expression.
    • Comparative Transcriptomic Profiling of Yersinia enterocolitica O:3 and O:8 Reveals Major Expression Differences of Fitness- and Virulence-Relevant Genes Indicating Ecological Separation.

      Schmühl, Carina; Beckstette, Michael; Heroven, Ann Kathrin; Bunk, Boyke; Spröer, Cathrin; McNally, Alan; Overmann, Jörg; Dersch, Petra; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
      Yersinia enterocolitica is a zoonotic pathogen and an important cause of bacterial gastrointestinal infections in humans. Large-scale population genomic analyses revealed genetic and phenotypic diversity of this bacterial species, but little is known about the differences in the transcriptome organization, small RNA (sRNA) repertoire, and transcriptional output. Here, we present the first comparative high-resolution transcriptome analysis of Y. enterocolitica strains representing highly pathogenic phylogroup 2 (serotype O:8) and moderately pathogenic phylogroup 3 (serotype O:3) grown under four infection-relevant conditions. Our transcriptome sequencing (RNA-seq) approach revealed 1,299 and 1,076 transcriptional start sites and identified strain-specific sRNAs that could contribute to differential regulation among the phylogroups. Comparative transcriptomics further uncovered major gene expression differences, in particular, in the temperature-responsive regulon. Multiple virulence-relevant genes are differentially regulated between the two strains, supporting an ecological separation of phylogroups with certain niche-adapted properties. Strong upregulation of the ystA enterotoxin gene in combination with constitutive high expression of cell invasion factor InvA further showed that the toxicity of recent outbreak O:3 strains has increased. Overall, our report provides new insights into the specific transcriptome organization of phylogroups 2 and 3 and reveals gene expression differences contributing to the substantial phenotypic differences that exist between the lineages. IMPORTANCE Yersinia enterocolitica is a major diarrheal pathogen and is associated with a large range of gut-associated diseases. Members of this species have evolved into different phylogroups with genotypic variations. We performed the first characterization of the Y. enterocolitica transcriptional landscape and tracked the consequences of the genomic variations between two different pathogenic phylogroups by comparing their RNA repertoire, promoter usage, and expression profiles under four different virulence-relevant conditions. Our analysis revealed major differences in the transcriptional outputs of the closely related strains, pointing to an ecological separation in which one is more adapted to an environmental lifestyle and the other to a mostly mammal-associated lifestyle. Moreover, a variety of pathoadaptive alterations, including alterations in acid resistance genes, colonization factors, and toxins, were identified which affect virulence and host specificity. This illustrates that comparative transcriptomics is an excellent approach to discover differences in the functional output from closely related genomes affecting niche adaptation and virulence, which cannot be directly inferred from DNA sequences.
    • Complete genome sequence and description of Salinispira pacifica gen. nov., sp. nov., a novel spirochaete isolated form a hypersaline microbial mat

      Ben Hania, Wajdi; Joseph, Manon; Schumann, Peter; Bunk, Boyke; Fiebig, Anne; Spröer, Cathrin; Klenk, Hans-Peter; Fardeau, Marie-Laure; Spring, Stefan (2015-02-09)
      Abstract During a study of the anaerobic microbial community of a lithifying hypersaline microbial mat of Lake 21 on the Kiritimati atoll (Kiribati Republic, Central Pacific) strain L21-RPul-D2T was isolated. The closest phylogenetic neighbor was Spirochaeta africana Z-7692T that shared a 16S rRNA gene sequence identity value of 90% with the novel strain and thus was only distantly related. A comprehensive polyphasic study including determination of the complete genome sequence was initiated to characterize the novel isolate. Cells of strain L21-RPul-D2T had a size of 0.2 – 0.25 × 8–9 μm, were helical, motile, stained Gram-negative and produced an orange carotenoid-like pigment. Optimal conditions for growth were 35°C, a salinity of 50 g/l NaCl and a pH around 7.0. Preferred substrates for growth were carbohydrates and a few carboxylic acids. The novel strain had an obligate fermentative metabolism and produced ethanol, acetate, lactate, hydrogen and carbon dioxide during growth on glucose. Strain L21-RPul-D2T was aerotolerant, but oxygen did not stimulate growth. Major cellular fatty acids were C14:0, iso-C15:0, C16:0 and C18:0. The major polar lipids were an unidentified aminolipid, phosphatidylglycerol, an unidentified phospholipid and two unidentified glycolipids. Whole-cell hydrolysates contained L-ornithine as diagnostic diamino acid of the cell wall peptidoglycan. The complete genome sequence was determined and annotated. The genome comprised one circular chromosome with a size of 3.78 Mbp that contained 3450 protein-coding genes and 50 RNA genes, including 2 operons of ribosomal RNA genes. The DNA G + C content was determined from the genome sequence as 51.9 mol%. There were no predicted genes encoding cytochromes or enzymes responsible for the biosynthesis of respiratory lipoquinones. Based on significant differences to the uncultured type species of the genus Spirochaeta, S. plicatilis, as well as to any other phylogenetically related cultured species it is suggested to place strain L21-RPul-D2T (=DSM 27196T = JCM 18663T) in a novel species and genus, for which the name Salinispira pacifica gen. nov., sp. nov. is proposed.
    • Concerted actions of a thermo-labile regulator and a unique intergenic RNA thermosensor control Yersinia virulence.

      Böhme, Katja; Steinmann, Rebekka; Kortmann, Jens; Seekircher, Stephanie; Heroven, Ann Kathrin; Berger, Evelin; Pisano, Fabio; Thiermann, Tanja; Wolf-Watz, Hans; Narberhaus, Franz; et al. (2012-02)
      Expression of all Yersinia pathogenicity factors encoded on the virulence plasmid, including the yop effector and the ysc type III secretion genes, is controlled by the transcriptional activator LcrF in response to temperature. Here, we show that a protein- and RNA-dependent hierarchy of thermosensors induce LcrF synthesis at body temperature. Thermally regulated transcription of lcrF is modest and mediated by the thermo-sensitive modulator YmoA, which represses transcription from a single promoter located far upstream of the yscW-lcrF operon at moderate temperatures. The transcriptional response is complemented by a second layer of temperature-control induced by a unique cis-acting RNA element located within the intergenic region of the yscW-lcrF transcript. Structure probing demonstrated that this region forms a secondary structure composed of two stemloops at 25°C. The second hairpin sequesters the lcrF ribosomal binding site by a stretch of four uracils. Opening of this structure was favored at 37°C and permitted ribosome binding at host body temperature. Our study further provides experimental evidence for the biological relevance of an RNA thermometer in an animal model. Following oral infections in mice, we found that two different Y. pseudotuberculosis patient isolates expressing a stabilized thermometer variant were strongly reduced in their ability to disseminate into the Peyer's patches, liver and spleen and have fully lost their lethality. Intriguingly, Yersinia strains with a destabilized version of the thermosensor were attenuated or exhibited a similar, but not a higher mortality. This illustrates that the RNA thermometer is the decisive control element providing just the appropriate amounts of LcrF protein for optimal infection efficiency.
    • Contribution of the Cpx envelope stress system to metabolism and virulence regulation in Salmonella enterica serovar Typhimurium.

      Subramaniam, Sivaraman; Müller, Volker S; Hering, Nina A; Mollenkopf, Hans; Becker, Daniel; Heroven, Ann Kathrin; Dersch, Petra; Pohlmann, Anne; Tedin, Karsten; Porwollik, Steffen; et al. (PLOS, 2019-01-01)
      The Cpx-envelope stress system regulates the expression of virulence factors in many Gram-negative pathogens. In Salmonella enterica serovar Typhimurium deletion of the sensor kinase CpxA but not of the response regulator CpxR results in the down regulation of the key regulator for invasion, HilA encoded by the Salmonella pathogenicity island 1 (SPI-1). Here, we provide evidence that cpxA deletion interferes with dephosphorylation of CpxR resulting in increased levels of active CpxR and consequently in misregulation of target genes. 14 potential operons were identified to be under direct control of CpxR. These include the virulence determinants ecotin, the omptin PgtE, and the SPI-2 regulator SsrB. The Tat-system and the PocR regulator that together promote anaerobic respiration of tetrathionate on 1,2-propanediol are also under direct CpxR control. Notably, 1,2-propanediol represses hilA expression. Thus, our work demonstrates for the first time the involvement of the Cpx system in a complex network mediating metabolism and virulence function.
    • Crp Induces Switching of the CsrB and CsrC RNAs in Yersinia pseudotuberculosis and Links Nutritional Status to Virulence.

      Heroven, Ann Kathrin; Sest, Maike; Pisano, Fabio; Scheb-Wetzel, Matthias; Steinmann, Rebekka; Böhme, Katja; Klein, Johannes; Münch, Richard; Schomburg, Dietmar; Dersch, Petra; et al. (2012)
      Colonization of the intestinal tract and dissemination into deeper tissues by the enteric pathogen Yersinia pseudotuberculosis demands expression of a special set of virulence factors important for the initiation and the persistence of the infection. In this study we demonstrate that many virulence-associated functions are coregulated with the carbohydrate metabolism. This link is mediated by the carbon storage regulator (Csr) system, including the regulatory RNAs CsrB and CsrC, and the cAMP receptor protein (Crp), which both control virulence gene expression in response to the nutrient composition of the medium. Here, we show that Crp regulates the synthesis of both Csr RNAs in an opposite manner. A loss of the crp gene resulted in a strong upregulation of CsrB synthesis, whereas CsrC levels were strongly reduced leading to downregulation of the virulence regulator RovA. Switching of the Csr RNA involves Crp-mediated repression of the response regulator UvrY which activates csrB transcription. To elucidate the regulatory links between virulence and carbon metabolism, we performed comparative metabolome, transcriptome, and phenotypic microarray analyses and found that Crp promotes oxidative catabolism of many different carbon sources, whereas fermentative patterns of metabolism are favored when crp is deleted. Mouse infection experiments further demonstrated that Crp is pivotal for a successful Y. pseudotuberculosis infection. In summary, placement of the Csr system and important virulence factors under control of Crp enables this pathogen to link its nutritional status to virulence in order to optimize biological fitness and infection efficiency through the infectious life cycle.
    • The Cytotoxic Necrotizing Factor of Yersinia pseudotuberculosis (CNFY) Enhances Inflammation and Yop Delivery during Infection by Activation of Rho GTPases.

      Schweer, Janina; Kulkarni, Devesha; Kochut, Annika; Pezoldt, Joern; Pisano, Fabio; Pils, Marina C; Genth, Harald; Huehn, Jochen; Dersch, Petra; Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2013-11)
      Some isolates of Yersinia pseudotuberculosis produce the cytotoxic necrotizing factor (CNFY), but the functional consequences of this toxin for host-pathogen interactions during the infection are unknown. In the present study we show that CNFY has a strong influence on virulence. We demonstrate that the CNFY toxin is thermo-regulated and highly expressed in all colonized lymphatic tissues and organs of orally infected mice. Most strikingly, we found that a cnfY knock-out variant of a naturally toxin-expressing Y. pseudotuberculosis isolate is strongly impaired in its ability to disseminate into the mesenteric lymph nodes, liver and spleen, and has fully lost its lethality. The CNFY toxin contributes significantly to the induction of acute inflammatory responses and to the formation of necrotic areas in infected tissues. The analysis of the host immune response demonstrated that presence of CNFY leads to a strong reduction of professional phagocytes and natural killer cells in particular in the spleen, whereas loss of the toxin allows efficient tissue infiltration of these immune cells and rapid killing of the pathogen. Addition of purified CNFY triggers formation of actin-rich membrane ruffles and filopodia, which correlates with the activation of the Rho GTPases, RhoA, Rac1 and Cdc42. The analysis of type III effector delivery into epithelial and immune cells in vitro and during the course of the infection further demonstrated that CNFY enhances the Yop translocation process and supports a role for the toxin in the suppression of the antibacterial host response. In summary, we highlight the importance of CNFY for pathogenicity by showing that this toxin modulates inflammatory responses, protects the bacteria from attacks of innate immune effectors and enhances the severity of a Yersinia infection.
    • DegS and RseP Homologous Proteases Are Involved in Singlet Oxygen Dependent Activation of RpoE in Rhodobacter sphaeroides.

      Nuss, Aaron M; Adnan, Fazal; Weber, Lennart; Berghoff, Bork A; Glaeser, Jens; Klug, Gabriele; Institute of Microbiology and Molecular Biology, Giessen University, Giessen, Germany ; Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2013)
      Singlet oxygen ((1)O2) is the main agent of photooxidative stress and is generated by photosensitizers as (bacterio)chlorophylls. It leads to the damage of cellular macromolecules and therefore photosynthetic organisms have to mount an adaptive response to (1)O2 formation. A major player of the photooxidative stress response in Rhodobacter sphaeroides is the alternative sigma factor RpoE, which is inactivated under non-stress conditions by its cognate anti-sigma factor ChrR. By using random mutagenesis we identified RSP_1090 to be required for full activation of the RpoE response under (1)O2 stress, but not under organic peroxide stress. In this study we show that both RSP_1090 and RSP_1091 are required for full resistance towards (1)O2. Moreover, we revealed that the DegS and RseP homologs RSP_3242 and RSP_2710 contribute to (1)O2 resistance and promote ChrR proteolysis. The RpoE signaling pathway in R. sphaeroides is therefore highly similar to that of Escherichia coli, although very different anti-sigma factors control RpoE activity. Based on the acquired results, the current model for RpoE activation in response to (1)O2 exposure in R. sphaeroides was extended.
    • A direct link between the global regulator PhoP and the Csr regulon in Y. pseudotuberculosis through the small regulatory RNA CsrC.

      Nuss, Aaron M; Schuster, Franziska; Kathrin Heroven, Ann; Heine, Wiebke; Pisano, Fabio; Dersch, Petra; Department of Molecular Infection Biology; Helmholtz Centre for Infection Research; Braunschweig, Germany. (2014-05)
      In this study we investigated the influence of the global response regulator PhoP on the complex regulatory cascade controlling expression of early stage virulence genes of Yersinia pseudotuberculosis via the virulence regulator RovA. Our analysis revealed the following novel features: (1) PhoP activates expression of the CsrC RNA in Y. pseudotuberculosis, leading to activation of RovA synthesis through the CsrABC-RovM cascade, (2) activation of csrC transcription is direct and PhoP is shown to bind to two separate PhoP box-like sites, (3) PhoP-mediated activation results in transcription from two different promoters closely downstream of the PhoP binding sites, leading to two distinct CsrC RNAs, and (4) the stability of the CsrC RNAs differs significantly between the Y. pseudotuberculosis strains YPIII and IP32953 due to a 20 nucleotides insertion in CsrC(IP32953), which renders the transcript more susceptible to degradation. In summary, our study showed that PhoP-mediated influence on the regulatory cascade controlling the Csr system and RovA in Y. pseudotuberculosis varies within the species, suggesting that the Csr system is a focal point to readjust and adapt the genus to different hosts and reservoirs.
    • Discovering RNA-Based Regulatory Systems for Virulence.

      Knittel, Vanessa; Vollmer, Ines; Volk, Marcel; Dersch, Petra; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-01-01)
      The genus Yersinia includes three human pathogenic species, Yersinia pestis, the causative agent of the bubonic and pneumonic plague, and enteric pathogens Y. enterocolitica and Y. pseudotuberculosis that cause a number of gut-associated diseases. Over the past years a large repertoire of RNA-based regulatory systems has been discovered in these pathogens using different RNA-seq based approaches. Among them are several conserved or species-specific RNA-binding proteins, regulatory and sensory RNAs as well as various RNA-degrading enzymes. Many of them were shown to control the expression of important virulence-relevant factors and have a very strong impact on Yersinia virulence. The precise targets, the molecular mechanism and their role for Yersinia pathogenicity is only known for a small subset of identified genus- or species-specific RNA-based control elements. However, the ongoing development of new RNA-seq based methods and data analysis methods to investigate the synthesis, composition, translation, decay, and modification of RNAs in the bacterial cell will help us to generate a more comprehensive view of Yersinia RNA biology in the near future
    • Filamentous fungi in good shape: Microparticles for tailor-made fungal morphology and enhanced enzyme production.

      Driouch, Habib; Roth, Andreas; Dersch, Petra; Wittmann, Christoph; Institute of Biochemical Engineering, Technische Universität Braunschweig, Germany. (2011-03-01)
      Filamentous fungi such as Aspergillus niger are important biocatalysts for industrial production of various enzymes as well as organic acids or antibiotics. In suspended culture these microorganisms exhibit a complex morphology which typically has a strong influence on their production properties. In this regard, we have recently shown that the addition of inorganic micro particles to the culture medium is a straightforward and elegant approach to precisely tame fungal morphology. For A. niger a full range of morphological forms from pellets with different diameters to free mycelium could be adjusted by supplementation with talc powder. Aluminium oxide particles similarly affected morphology, showing that this effect is largely independent of the chemical particle composition. Exemplified for different recombinant A. niger strains enzyme production could be strongly enhanced by the addition of microparticles. This was demonstrated for the production of fructofuranosidase, an important high-value biocatalyst for pre-biotic fructo-oligosaccharides, by recombinant A. niger. In a microparticle enhanced fed-batch process, a highly productive mycelium could be achieved. The enzyme titre of 2800 U/mL finally reached was more then tenfold higher then that of any other process reported so far. Here we provide additional insights into the novel production process. This includes the confirmation of the highly selective production of the target enzyme fructofuranosidase using MALDI-TOF MS analysis. Moreover, we show that the obtained enzyme suspension can be efficiently used with minimal pre-treatment for the biosynthesis of short chain fructooligosaccharides of the inulin type, such as 1-kestose and 1-nystose, prebiotics with substantial commercial interest. In particular, these compounds are highly attractive for human consumption, since they have been shown to reduce the risk of colon cancer. In summary, the use of microparticles opens a new avenue of engineering fungal morphology into the desired form for specific production processes.
    • Human and animal isolates of Yersinia enterocolitica show significant serotype-specific colonization and host-specific immune defense properties.

      Schaake, Julia; Kronshage, Malte; Uliczka, Frank; Rohde, Manfred; Knuuti, Tobias; Strauch, Eckhard; Fruth, Angelika; Wos-Oxley, Melissa; Dersch, Petra; Dept. of molecular infection biology, Helmholtz Centre for infection biology, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2013-11)
      Yersinia enterocolitica is a human pathogen that is ubiquitous in livestock, especially pigs. The bacteria are able to colonize the intestinal tract of a variety of mammalian hosts, but the severity of induced gut-associated diseases (yersiniosis) differs significantly between hosts. To gain more information about the individual virulence determinants that contribute to colonization and induction of immune responses in different hosts, we analyzed and compared the interactions of different human- and animal-derived isolates of serotypes O:3, O:5,27, O:8, and O:9 with murine, porcine, and human intestinal cells and macrophages. The examined strains exhibited significant serotype-specific cell binding and entry characteristics, but adhesion and uptake into different host cells were not host specific and were independent of the source of the isolate. In contrast, survival and replication within macrophages and the induced proinflammatory response differed between murine, porcine, and human macrophages, suggesting a host-specific immune response. In fact, similar levels of the proinflammatory cytokine macrophage inflammatory protein 2 (MIP-2) were secreted by murine bone marrow-derived macrophages with all tested isolates, but the equivalent interleukin-8 (IL-8) response of porcine bone marrow-derived macrophages was strongly serotype specific and considerably lower in O:3 than in O:8 strains. In addition, all tested Y. enterocolitica strains caused a considerably higher level of secretion of the anti-inflammatory cytokine IL-10 by porcine than by murine macrophages. This could contribute to limiting the severity of the infection (in particular of serotype O:3 strains) in pigs, which are the primary reservoir of Y. enterocolitica strains pathogenic to humans.
    • Hypoxia Decreases Invasin-Mediated Yersinia enterocolitica Internalization into Caco-2 Cells.

      Zeitouni, Nathalie E; Dersch, Petra; Naim, Hassan Y; von Köckritz-Blickwede, Maren; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2016)
      Yersinia enterocolitica is a major cause of human yersiniosis, with enterocolitis being a typical manifestation. These bacteria can cross the intestinal mucosa, and invade eukaryotic cells by binding to host β1 integrins, a process mediated by the bacterial effector protein invasin. This study examines the role of hypoxia on the internalization of Y. enterocolitica into intestinal epithelial cells, since the gastrointestinal tract has been shown to be physiologically deficient in oxygen levels (hypoxic), especially in cases of infection and inflammation. We show that hypoxic pre-incubation of Caco-2 cells resulted in significantly decreased bacterial internalization compared to cells grown under normoxia. This phenotype was absent after functionally blocking host β1 integrins as well as upon infection with an invasin-deficient Y. enterocolitica strain. Furthermore, downstream phosphorylation of the focal adhesion kinase was also reduced under hypoxia after infection. In good correlation to these data, cells grown under hypoxia showed decreased protein levels of β1 integrins at the apical cell surface whereas the total protein level of the hypoxia inducible factor (HIF-1) alpha was elevated. Furthermore, treatment of cells with the HIF-1 α stabilizer dimethyloxalylglycine (DMOG) also reduced invasion and decreased β1 integrin protein levels compared to control cells, indicating a potential role for HIF-1α in this process. These results suggest that hypoxia decreases invasin-integrin-mediated internalization of Y. enterocolitica into intestinal epithelial cells by reducing cell surface localization of host β1 integrins.