• 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.
    • The alarmones (p)ppGpp are part of the heat shock response of Bacillus subtilis.

      Schäfer, Heinrich; Beckert, Bertrand; Frese, Christian K; Steinchen, Wieland; Nuss, Aaron M; Beckstette, Michael; Hantke, Ingo; Driller, Kristina; Sudzinová, Petra; Krásný, Libor; et al. (PLOS, 2020-03-16)
      Bacillus subtilis cells are well suited to study how bacteria sense and adapt to proteotoxic stress such as heat, since temperature fluctuations are a major challenge to soil-dwelling bacteria. Here, we show that the alarmones (p)ppGpp, well known second messengers of nutrient starvation, are also involved in the heat stress response as well as the development of thermo-resistance. Upon heat-shock, intracellular levels of (p)ppGpp rise in a rapid but transient manner. The heat-induced (p)ppGpp is primarily produced by the ribosome-associated alarmone synthetase Rel, while the small alarmone synthetases RelP and RelQ seem not to be involved. Furthermore, our study shows that the generated (p)ppGpp pulse primarily acts at the level of translation, and only specific genes are regulated at the transcriptional level. These include the down-regulation of some translation-related genes and the up-regulation of hpf, encoding the ribosome-protecting hibernation-promoting factor. In addition, the alarmones appear to interact with the activity of the stress transcription factor Spx during heat stress. Taken together, our study suggests that (p)ppGpp modulates the translational capacity at elevated temperatures and thereby allows B. subtilis cells to respond to proteotoxic stress, not only by raising the cellular repair capacity, but also by decreasing translation to concurrently reduce the protein load on the cellular protein quality control system.
    • Adaptation to Photooxidative Stress: Common and Special Strategies of the Alphaproteobacteria and .

      Licht, Mathieu K; Nuss, Aaron M; Volk, Marcel; Konzer, Anne; Beckstette, Michael; Berghoff, Bork A; Klug, Gabriele; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-02-19)
      Photosynthetic bacteria have to deal with the risk of photooxidative stress that occurs in presence of light and oxygen due to the photosensitizing activity of (bacterio-) chlorophylls. Facultative phototrophs of the genus Rhodobacter adapt the formation of photosynthetic complexes to oxygen and light conditions, but cannot completely avoid this stress if environmental conditions suddenly change. R. capsulatus has a stronger pigmentation and faster switches to phototrophic growth than R. sphaeroides. However, its photooxidative stress response has not been investigated. Here, we compare both species by transcriptomics and proteomics, revealing that proteins involved in oxidation-reduction processes, DNA, and protein damage repair play pivotal roles. These functions are likely universal to many phototrophs. Furthermore, the alternative sigma factors RpoE and RpoHII are induced in both species, even though the genetic localization of the rpoE gene, the RpoE protein itself, and probably its regulon, are different. Despite sharing the same habitats, our findings also suggest individual strategies. The crtIB-tspO operon, encoding proteins for biosynthesis of carotenoid precursors and a regulator of photosynthesis, and cbiX, encoding a putative ferrochelatase, are induced in R. capsulatus. This specific response might support adaptation by maintaining high carotenoid-to-bacteriochlorophyll ratios and preventing the accumulation of porphyrin-derived photosensitizers.
    • Identification of antibiotics that diminish disease in a murine model of enterohemorrhagic infection.

      Mühlen, Sabrina; Ramming, Isabell; Pils, Marina C; Koeppel, Martin; Glaser, Jana; Leong, John; Flieger, Antje; Stecher, Bärbel; Dersch, Petra; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (American society for microbiology, 2020-02-03)
      Infections with enterohemorrhagic Escherichia coli (EHEC) cause disease ranging from mild diarrhea to hemolytic uremic syndrome (HUS) and are the most common cause of renal failure in children in high income countries. The severity of the disease derives from the release of Shiga toxins (Stx). The use of antibiotics to treat EHEC infections is generally avoided as it can result in increased stx expression. Here, we systematically tested different classes of antibiotics and found that their influence on stx expression and release varies significantly. We assessed a selection of these antibiotics in vivo using the Citrobacter rodentium φstx2dact mouse model and show that stx2d-inducing antibiotics resulted in weight loss and kidney damage despite clearing the infection. However, several non-Stx-inducing antibiotics cleared bacterial infection without causing Stx-mediated pathology. Our results suggest that these antibiotics could be useful in the treatment of EHEC-infected human patients and decrease the risk of HUS development.
    • 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.
    • Discovering Yersinia-Host Interactions by Tissue Dual RNA-Seq.

      Kusmierek, Maria; Heroven, Ann Kathrin; Beckstette, Michael; Nuss, Aaron M; Dersch, Petra; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer, 2019-01-01)
      A detailed knowledge about virulence-relevant genes, as well as where and when they are expressed during the course of an infection is required to obtain a comprehensive understanding of the complex host-pathogen interactions. The development of unbiased probe-independent RNA sequencing (RNA-seq) approaches has dramatically changed transcriptomics. It allows simultaneous monitoring of genome-wide, infection-linked transcriptional alterations of the host tissue and colonizing pathogens. Here, we provide a detailed protocol for the preparation and analysis of lymphatic tissue infected with the mainly extracellularly growing pathogen Yersinia pseudotuberculosis. This method can be used as a powerful tool for the discovery of Yersinia-induced host responses, colonization and persistence strategies of the pathogen, and underlying regulatory processes. Furthermore, we describe computational methods with which we analyzed obtained datasets.
    • 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.
    • 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.
    • Yersinia Pseudotuberculosis Modulates Regulatory T Cell Stability via Injection of Yersinia Outer Proteins in a Type III Secretion System-Dependent Manner.

      Elfiky, Ahmed; Bonifacius, Agnes; Pezoldt, Joern; Pasztoi, Maria; Chaoprasid, Paweena; Sadana, Pooja; El-Sherbeeny, Nagla; Hagras, Magda; Scrima, Andrea; Dersch, Petra; et al. (Akadémiai Kiadó, 2018-12-23)
      Adaptive immunity is essentially required to control acute infection with enteropathogenic
    • 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.
    • 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.
    • 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.
    • The Yersinia pseudotuberculosis Cpx envelope stress system contributes to transcriptional activation of rovM.

      Thanikkal, Edvin J; Gahlot, Dharmender K; Liu, Junfa; Fredriksson Sundbom, Marcus; Gurung, Jyoti M; Ruuth, Kristina; Francis, Monika K; Obi, Ikenna R; Thompson, Karl M; Chen, Shiyun; et al. (2018-12-06)
      The Gram-negative enteropathogen Yersinia pseudotuberculosis possesses a number of regulatory systems that detect cell envelope damage caused by noxious extracytoplasmic stresses. The CpxA sensor kinase and CpxR response regulator two-component regulatory system is one such pathway. Active Cpx signalling upregulates various factors designed to repair and restore cell envelope integrity. Concomitantly, this pathway also down-regulates key determinants of virulence. In Yersinia, cpxA deletion accumulates high levels of phosphorylated CpxR (CpxR~P). Accumulated CpxR~P directly repressed rovA expression and this limited expression of virulence-associated processes. A second transcriptional regulator, RovM, also negatively regulates rovA expression in response to nutrient stress. Hence, this study aimed to determine if CpxR~P can influence rovA expression through control of RovM levels. We determined that the active CpxR~P isoform bound to the promoter of rovM and directly induced its expression, which naturally associated with a concurrent reduction in rovA expression. Site-directed mutagenesis of the CpxR~P binding sequence in the rovM promoter region desensitised rovM expression to CpxR~P. These data suggest that accumulated CpxR~P inversely manipulates the levels of two global transcriptional regulators, RovA and RovM, and this would be expected to have considerable influence on Yersinia pathophysiology and metabolism.
    • 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.
    • 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
    • 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 invasin D protein fromYersinia pseudotuberculosisselectively binds the Fab region of host antibodies and affects colonization of the intestine.

      Sadana, Pooja; Geyer, Rebecca; Pezoldt, Joern; Helmsing, Saskia; Huehn, Jochen; Hust, Michael; Dersch, Petra; Scrima, Andrea; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-03-13)
      Yersinia pseudotuberculosis is a Gram-negative bacterium and zoonotic pathogen responsible for a wide range of diseases, ranging from mild diarrhea, enterocolitis, lymphatic adenitis to persistent local inflammation. TheY. pseudotuberculosisinvasin D (InvD) molecule belongs to the invasin (InvA)-type autotransporter proteins, but its structure and function remain unknown. In this study, we present the first crystal structure of InvD, analyzed its expression and function in a murine infection model, and identified its target molecule in the host. We found that InvD is induced at 37°C and expressed in vivo2-4 days after infection, indicating that InvD is a virulence factor. During infection, InvD was expressed in all parts of the intestinal tract, but not in deeper lymphoid tissues. The crystal structure of the C-terminal adhesion domain of InvD revealed a distinct Ig-related fold, that, apart from the canonical β-sheets, comprises various modifications of and insertions into the Ig-core structure. We identified the Fab fragment of host-derived IgG/IgA antibodies as the target of the adhesion domain. Phage display panning and flow cytometry data further revealed that InvD exhibits a preferential binding specificity toward antibodies with VH3/VK1 variable domains and that it is specifically recruited to a subset of B cells. This finding suggests that InvD modulates Ig functions in the intestine and affects direct interactions with a subset of cell surface-exposed B-cell receptors. In summary, our results provide extensive insights into the structure of InvD and its specific interaction with the target molecule in the host.
    • 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.
    • Identification of a Distinct Substrate-binding Domain in the Bacterial Cysteine Methyltransferase Effectors NleE and OspZ.

      Zhang, Ying; Mühlen, Sabrina; Oates, Clare V; Pearson, Jaclyn S; Hartland, Elizabeth L; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
      The type III secretion system effector protein NleE from enteropathogenic Escherichia coli plays a key role in the inhibition of NF-κB activation during infection. NleE inactivates the ubiquitin chain binding activity of host proteins TAK1-binding proteins 2 and 3 (TAB2 and TAB3) by modifying the Npl4 zinc finger domain through S-adenosyl methionine-dependent cysteine methylation. Using yeast two-hybrid protein interaction studies, we found that a conserved region between amino acids 34 and 52 of NleE, in particular the motif (49)GITR(52), was critical for TAB2 and TAB3 binding. NleE mutants lacking (49)GITR(52) were unable to methylate TAB3, and wild type NleE but not NleE(49AAAA52) where each of GITR was replaced with alanine restored the ability of an nleE mutant to inhibit IL-8 production during infection. Another NleE target, ZRANB3, also associated with NleE through the (49)GITR(52) motif. Ectopic expression of an N-terminal fragment of NleE (NleE(34-52)) in HeLa cells showed competitive inhibition of wild type NleE in the suppression of IL-8 secretion during enteropathogenic E. coli infection. Similar results were observed for the NleE homologue OspZ from Shigella flexneri 6 that also bound TAB3 through the (49)GITR(52) motif and decreased IL-8 transcription through modification of TAB3. In summary, we have identified a unique substrate-binding motif in NleE and OspZ that is required for the ability to inhibit the host inflammatory response.
    • Increased plasmid copy number is essential for Yersinia T3SS function and virulence.

      Wang, He; Avican, Kemal; Fahlgren, Anna; Erttmann, Saskia F; Nuss, Aaron M; Dersch, Petra; Fallman, Maria; Edgren, Tomas; Wolf-Watz, Hans; Helmholtz-Zentrum für Infektionsfoschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2016-07-29)
      Pathogenic bacteria have evolved numerous virulence mechanisms that are essential for establishing infections. The enterobacterium Yersinia uses a type III secretion system (T3SS) encoded by a 70-kilobase, low-copy, IncFII-class virulence plasmid. We report a novel virulence strategy in Y. pseudotuberculosis in which this pathogen up-regulates the plasmid copy number during infection. We found that an increased dose of plasmid-encoded genes is indispensable for virulence and substantially elevates the expression and function of the T3SS. Remarkably, we observed direct, tight coupling between plasmid replication and T3SS function. This regulatory pathway provides a framework for further exploration of the environmental sensing mechanisms of pathogenic bacteria.