@article{MaudetSourisceDraginetal.2013, author = {Maudet, Claire and Sourisce, Ad{\`e}le and Dragin, Lo{\"i}c and Lahouassa, Hichem and Rain, Jean-Christopher and Bouaziz, Serge and Ramirez, Bertha C{\´e}cilia and Margottin-Goguet, Florence}, title = {HIV-1 Vpr Induces the Degradation of ZIP and sZIP, Adaptors of the NuRD Chromatin Remodeling Complex, by Hijacking DCAF1/VprBP}, series = {PLOS ONE}, volume = {8}, journal = {PLOS ONE}, number = {10}, issn = {1932-6203}, doi = {10.1371/journal.pone.0077320}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-128316}, pages = {e77320}, year = {2013}, abstract = {The Vpr protein from type 1 and type 2 Human Immunodeficiency Viruses (HIV-1 and HIV-2) is thought to inactivate several host proteins through the hijacking of the DCAF1 adaptor of the Cul4A ubiquitin ligase. Here, we identified two transcriptional regulators, ZIP and sZIP, as Vpr-binding proteins degraded in the presence of Vpr. ZIP and sZIP have been shown to act through the recruitment of the NuRD chromatin remodeling complex. Strikingly, chromatin is the only cellular fraction where Vpr is present together with Cul4A ubiquitin ligase subunits. Components of the NuRD complex and exogenous ZIP and sZIP were also associated with this fraction. Several lines of evidence indicate that Vpr induces ZIP and sZIP degradation by hijacking DCAF1: (i) Vpr induced a drastic decrease of exogenously expressed ZIP and sZIP in a dose-dependent manner, (ii) this decrease relied on the proteasome activity, (iii) ZIP or sZIP degradation was impaired in the presence of a DCAF1-binding deficient Vpr mutant or when DCAF1 expression was silenced. Vpr-mediated ZIP and sZIP degradation did not correlate with the growth-related Vpr activities, namely G2 arrest and G2 arrest-independent cytotoxicity. Nonetheless, infection with HIV-1 viruses expressing Vpr led to the degradation of the two proteins. Altogether our results highlight the existence of two host transcription factors inactivated by Vpr. The role of Vpr-mediated ZIP and sZIP degradation in the HIV-1 replication cycle remains to be deciphered.}, language = {en} } @article{FellerThomKochetal.2013, author = {Feller, Tatjana and Thom, Pascal and Koch, Natalie and Spiegel, Holger and Addai-Mensah, Otchere and Fischer, Rainer and Reimann, Andreas and Pradel, Gabriele and Fendel, Rolf and Schillberg, Stefan and Scheuermayer, Matthias and Schinkel, Helga}, title = {Plant-Based Production of Recombinant Plasmodium Surface Protein Pf38 and Evaluation of its Potential as a Vaccine Candidate}, series = {PLOS ONE}, volume = {8}, journal = {PLOS ONE}, number = {11}, issn = {1932-6203}, doi = {10.1371/journal.pone.0079920}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-128221}, pages = {e79920}, year = {2013}, abstract = {Pf38 is a surface protein of the malarial parasite Plasmodium falciparum. In this study, we produced and purified recombinant Pf38 and a fusion protein composed of red fluorescent protein and Pf38 (RFP-Pf38) using a transient expression system in the plant Nicotiana benthamiana. To our knowledge, this is the first description of the production of recombinant Pf38. To verify the quality of the recombinant Pf38, plasma from semi-immune African donors was used to confirm specific binding to Pf38. ELISA measurements revealed that immune responses to Pf38 in this African subset were comparable to reactivities to AMA-1 and \(MSP1_{19}\). Pf38 and RFP-Pf38 were successfully used to immunise mice, although titres from these mice were low (on average 1:11.000 and 1:39.000, respectively). In immune fluorescence assays, the purified IgG fraction from the sera of immunised mice recognised Pf38 on the surface of schizonts, gametocytes, macrogametes and zygotes, but not sporozoites. Growth inhibition assays using \(\alpha Pf38\) antibodies demonstrated strong inhibition \((\geq 60 \\% ) \) of the growth of blood-stage P. falciparum. The development of zygotes was also effectively inhibited by \(\alpha Pf38\) antibodies, as determined by the zygote development assay. Collectively, these results suggest that Pf38 is an interesting candidate for the development of a malaria vaccine.}, language = {en} } @phdthesis{Westermann2014, author = {Westermann, Alexander J.}, title = {Dual RNA-seq of pathogen and host}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-112462}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {The infection of a eukaryotic host cell by a bacterial pathogen is one of the most intimate examples of cross-kingdom interactions in biology. Infection processes are highly relevant from both a basic research as well as a clinical point of view. Sophisticated mechanisms have evolved in the pathogen to manipulate the host response and vice versa host cells have developed a wide range of anti-microbial defense strategies to combat bacterial invasion and clear infections. However, it is this diversity and complexity that makes infection research so challenging to technically address as common approaches have either been optimized for bacterial or eukaryotic organisms. Instead, methods are required that are able to deal with the often dramatic discrepancy between host and pathogen with respect to various cellular properties and processes. One class of cellular macromolecules that exemplify this host-pathogen heterogeneity is given by their transcriptomes: Bacterial transcripts differ from their eukaryotic counterparts in many aspects that involve both quantitative and qualitative traits. The entity of RNA transcripts present in a cell is of paramount interest as it reflects the cell's physiological state under the given condition. Genome-wide transcriptomic techniques such as RNA-seq have therefore been used for single-organism analyses for several years, but their applicability has been limited for infection studies. The present work describes the establishment of a novel transcriptomic approach for infection biology which we have termed "Dual RNA-seq". Using this technology, it was intended to shed light particularly on the contribution of non-protein-encoding transcripts to virulence, as these classes have mostly evaded previous infection studies due to the lack of suitable methods. The performance of Dual RNA-seq was evaluated in an in vitro infection model based on the important facultative intracellular pathogen Salmonella enterica serovar Typhimurium and different human cell lines. Dual RNA-seq was found to be capable of capturing all major bacterial and human transcript classes and proved reproducible. During the course of these experiments, a previously largely uncharacterized bacterial small non-coding RNA (sRNA), referred to as STnc440, was identified as one of the most strongly induced genes in intracellular Salmonella. Interestingly, while inhibition of STnc440 expression has been previously shown to cause a virulence defect in different animal models of Salmonellosis, the underlying molecular mechanisms have remained obscure. Here, classical genetics, transcriptomics and biochemical assays proposed a complex model of Salmonella gene expression control that is orchestrated by this sRNA. In particular, STnc440 was found to be involved in the regulation of multiple bacterial target mRNAs by direct base pair interaction with consequences for Salmonella virulence and implications for the host's immune response. These findings exemplify the scope of Dual RNA-seq for the identification and characterization of novel bacterial virulence factors during host infection.}, subject = {Transkriptomanalyse}, language = {en} } @article{BerghoffKonzerManketal.2013, author = {Berghoff, Bork A. and Konzer, Anne and Mank, Nils N. and Looso, Mario and Rische, Tom and F{\"o}rstner, Konrad U. and Kr{\"u}ger, Marcus and Klug, Gabriele}, title = {Integrative "Omics"-Approach Discovers Dynamic and Regulatory Features of Bacterial Stress Responses}, series = {PLOS Genetics}, volume = {9}, journal = {PLOS Genetics}, number = {6}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1003576}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-127587}, pages = {e1003576}, year = {2013}, abstract = {Bacteria constantly face stress conditions and therefore mount specific responses to ensure adaptation and survival. Stress responses were believed to be predominantly regulated at the transcriptional level. In the phototrophic bacterium Rhodobacter sphaeroides the response to singlet oxygen is initiated by alternative sigma factors. Further adaptive mechanisms include post-transcriptional and post-translational events, which have to be considered to gain a deeper understanding of how sophisticated regulation networks operate. To address this issue, we integrated three layers of regulation: (1) total mRNA levels at different time-points revealed dynamics of the transcriptome, (2) mRNAs in polysome fractions reported on translational regulation (translatome), and (3) SILAC-based mass spectrometry was used to quantify protein abundances (proteome). The singlet oxygen stress response exhibited highly dynamic features regarding short-term effects and late adaptation, which could in part be assigned to the sigma factors RpoE and RpoH2 generating distinct expression kinetics of corresponding regulons. The occurrence of polar expression patterns of genes within stress-inducible operons pointed to an alternative of dynamic fine-tuning upon stress. In addition to transcriptional activation, we observed significant induction of genes at the post-transcriptional level (translatome), which identified new putative regulators and assigned genes of quorum sensing to the singlet oxygen stress response. Intriguingly, the SILAC approach explored the stress-dependent decline of photosynthetic proteins, but also identified 19 new open reading frames, which were partly validated by RNA-seq. We propose that comparative approaches as presented here will help to create multi-layered expression maps on the system level ("expressome"). Finally, intense mass spectrometry combined with RNA-seq might be the future tool of choice to re-annotate genomes in various organisms and will help to understand how they adapt to alternating conditions.}, language = {en} } @article{MuellerWindhofMaximovetal.2013, author = {M{\"u}ller, Sara and Windhof, Indra M. and Maximov, Vladimir and Jurkowski, Tomasz and Jeltsch, Albert and F{\"o}rstner, Konrad U. and Sharma, Cynthia M. and Gr{\"a}f, Ralph and Nellen, Wolfgang}, title = {Target recognition, RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA)}, series = {Nucleic Acids Research}, volume = {41}, journal = {Nucleic Acids Research}, number = {18}, issn = {1362-4962}, doi = {10.1093/nar/gkt634}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123149}, pages = {8615-8627}, year = {2013}, abstract = {Although the DNA methyltransferase 2 family is highly conserved during evolution and recent reports suggested a dual specificity with stronger activity on transfer RNA (tRNA) than DNA substrates, the biological function is still obscure. We show that the Dictyostelium discoideum Dnmt2-homologue DnmA is an active tRNA methyltransferase that modifies C38 in \(tRNA^{Asp(GUC)}\) in vitro and in vivo. By an ultraviolet-crosslinking and immunoprecipitation approach, we identified further DnmA targets. This revealed specific tRNA fragments bound by the enzyme and identified \(tRNA^{Glu(CUC/UUC)}\) and \(tRNA^{Gly(GCC)}\) as new but weaker substrates for both human Dnmt2 and DnmA in vitro but apparently not in vivo. Dnmt2 enzymes form transient covalent complexes with their substrates. The dynamics of complex formation and complex resolution reflect methylation efficiency in vitro. Quantitative PCR analyses revealed alterations in dnmA expression during development, cell cycle and in response to temperature stress. However, dnmA expression only partially correlated with tRNA methylation in vivo. Strikingly, dnmA expression in the laboratory strain AX2 was significantly lower than in the NC4 parent strain. As expression levels and binding of DnmA to a target in vivo are apparently not necessarily accompanied by methylation, we propose an additional biological function of DnmA apart from methylation.}, language = {en} } @article{GholamiChenBelinetal.2013, author = {Gholami, Sepideh and Chen, Chun-Hao and Belin, Laurence J. and Lou, Emil and Fujisawa, Sho and Antonacci, Caroline and Carew, Amanda and Chen, Nanhai G. and De Brot, Marina and Zanzonico, Pat B. and Szalay, Aladar A. and Fong, Yuman}, title = {Vaccinia virus GLV-1h153 is a novel agent for detection and effective local control of positive surgical margins for breast cancer}, series = {Breast Cancer Research}, volume = {15}, journal = {Breast Cancer Research}, number = {R26}, doi = {10.1186/bcr3404}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-122140}, year = {2013}, abstract = {Introduction: Surgery is currently the definitive treatment for early-stage breast cancer. However, the rate of positive surgical margins remains unacceptably high. The human sodium iodide symporter (hNIS) is a naturally occurring protein in human thyroid tissue, which enables cells to concentrate radionuclides. The hNIS has been exploited to image and treat thyroid cancer. We therefore investigated the potential of a novel oncolytic vaccinia virus GLV1h-153 engineered to express the hNIS gene for identifying positive surgical margins after tumor resection via positron emission tomography (PET). Furthermore, we studied its role as an adjuvant therapeutic agent in achieving local control of remaining tumors in an orthotopic breast cancer model. Methods: GLV-1h153, a replication-competent vaccinia virus, was tested against breast cancer cell lines at various multiplicities of infection (MOIs). Cytotoxicity and viral replication were determined. Mammary fat pad tumors were generated in athymic nude mice. To determine the utility of GLV-1h153 in identifying positive surgical margins, 90\% of the mammary fat pad tumors were surgically resected and subsequently injected with GLV-1h153 or phosphate buffered saline (PBS) in the surgical wound. Serial Focus 120 microPET images were obtained six hours post-tail vein injection of approximately 600 mu Ci of I-124-iodide. Results: Viral infectivity, measured by green fluorescent protein (GFP) expression, was time-and concentrationdependent. All cell lines showed less than 10\% of cell survival five days after treatment at an MOI of 5. GLV-1h153 replicated efficiently in all cell lines with a peak titer of 27 million viral plaque forming units (PFU) ( < 10,000-fold increase from the initial viral dose) by Day 4. Administration of GLV-1h153 into the surgical wound allowed positive surgical margins to be identified via PET scanning. In vivo, mean volume of infected surgically resected residual tumors four weeks after treatment was 14 mm(3) versus 168 mm(3) in untreated controls (P < 0.05). Conclusions: This is the first study to our knowledge to demonstrate a novel vaccinia virus carrying hNIS as an imaging tool in identifying positive surgical margins of breast cancers in an orthotopic murine model. Moreover, our results suggest that GLV-1h153 is a promising therapeutic agent in achieving local control for positive surgical margins in resected breast tumors.}, language = {en} } @article{NeumannOhlsenDonatetal.2015, author = {Neumann, Yvonne and Ohlsen, Knut and Donat, Stefanie and Engelmann, Susanne and Kusch, Harald and Albrecht, Dirk and Cartron, Michael and Hurd, Alexander and Foster, Simon J.}, title = {The effect of skin fatty acids on Staphylococcus aureus}, series = {Archives of Microbiology}, volume = {197}, journal = {Archives of Microbiology}, doi = {10.1007/s00203-014-1048-1}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121657}, pages = {245-67}, year = {2015}, abstract = {Staphylococcus aureus is a commensal of the human nose and skin. Human skin fatty acids, in particular cis-6-hexadecenoic acid (C-6-H), have high antistaphylococcal activity and can inhibit virulence determinant production. Here, we show that sub-MIC levels of C-6-H result in induction of increased resistance. The mechanism(s) of C-6-H activity was investigated by combined transcriptome and proteome analyses. Proteome analysis demonstrated a pleiotropic effect of C-6-H on virulence determinant production. In response to C-6-H, transcriptomics revealed altered expression of over 500 genes, involved in many aspects of virulence and cellular physiology. The expression of toxins (hla, hlb, hlgBC) was reduced, whereas that of host defence evasion components (cap, sspAB, katA) was increased. In particular, members of the SaeRS regulon had highly reduced expression, and the use of specific mutants revealed that the effect on toxin production is likely mediated via SaeRS}, language = {en} } @article{VembarScherfSiegel2014, author = {Vembar, Shruti S. and Scherf, Artur and Siegel, T. Nicolai}, title = {Noncoding RNAs as emerging regulators of Plasmodium falciparum virulence gene expression}, series = {Current Opinion in Microbiology}, volume = {20}, journal = {Current Opinion in Microbiology}, number = {100}, issn = {1369-5274}, doi = {10.1016/j.mib.2014.06.013}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121416}, pages = {153-61}, year = {2014}, abstract = {The eukaryotic unicellular pathogen Plasmodium falciparum tightly regulates gene expression, both during development and in adaptation to dynamic host environments. This regulation is evident in the mutually exclusive expression of members of clonally variant virulence multigene families. While epigenetic regulators have been selectively identified at active or repressed virulence genes, their specific recruitment remains a mystery. In recent years, noncoding RNAs (ncRNAs) have emerged as lynchpins of eukaryotic gene regulation; by binding to epigenetic regulators, they provide target specificity to otherwise non-specific enzyme complexes. Not surprisingly, there is great interest in understanding the role of ncRNA in P. falciparum, in particular, their contribution to the mutually exclusive expression of virulence genes. The current repertoire of P. falciparum ncRNAs includes, but is not limited to, subtelomeric ncRNAs, virulence gene-associated ncRNAs and natural antisense RNA transcripts. Continued improvement in high-throughput sequencing methods is sure to expand this repertoire. Here, we summarize recent advances in P. falciparum ncRNA biology, with an emphasis on ncRNA-mediated epigenetic modes of gene regulation.}, language = {en} } @article{JaegerFoerstnerSharmaetal.2014, author = {J{\"a}ger, Dominik and F{\"o}rstner, Konrad U. and Sharma, Cynthia M. and Santangelo, Thomas J. and Reeve, John N.}, title = {Primary transcriptome map of the hyperthermophilic archaeon Thermococcus kodakarensis}, series = {BMC Genomics}, volume = {15}, journal = {BMC Genomics}, number = {684}, issn = {1471-2164}, doi = {10.1186/1471-2164-15-684}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-120966}, year = {2014}, abstract = {Background Prokaryotes have relatively small genomes, densely-packed with protein-encoding sequences. RNA sequencing has, however, revealed surprisingly complex transcriptomes and here we report the transcripts present in the model hyperthermophilic Archaeon, Thermococcus kodakarensis, under different physiological conditions. Results Sequencing cDNA libraries, generated from RNA isolated from cells under different growth and metabolic conditions has identified >2,700 sites of transcription initiation, established a genome-wide map of transcripts, and consensus sequences for transcription initiation and post-transcription regulatory elements. The primary transcription start sites (TSS) upstream of 1,254 annotated genes, plus 644 primary TSS and their promoters within genes, are identified. Most mRNAs have a 5'-untranslated region (5'-UTR) 10 to 50 nt long (median = 16 nt), but ~20\% have 5'-UTRs from 50 to 300 nt long and ~14\% are leaderless. Approximately 50\% of mRNAs contain a consensus ribosome binding sequence. The results identify TSS for 1,018 antisense transcripts, most with sequences complementary to either the 5'- or 3'-region of a sense mRNA, and confirm the presence of transcripts from all three CRISPR loci, the RNase P and 7S RNAs, all tRNAs and rRNAs and 69 predicted snoRNAs. Two putative riboswitch RNAs were present in growing but not in stationary phase cells. The procedure used is designed to identify TSS but, assuming that the number of cDNA reads correlates with transcript abundance, the results also provide a semi-quantitative documentation of the differences in T. kodakarensis genome expression under different growth conditions and confirm previous observations of substrate-dependent specific gene expression. Many previously unanticipated small RNAs have been identified, some with relative low GC contents (≤50\%) and sequences that do not fold readily into base-paired secondary structures, contrary to the classical expectations for non-coding RNAs in a hyperthermophile. Conclusion The results identify >2,700 TSS, including almost all of the primary sites of transcription initiation upstream of annotated genes, plus many secondary sites, sites within genes and sites resulting in antisense transcripts. The T. kodakarensis genome is small (~2.1 Mbp) and tightly packed with protein-encoding genes, but the transcriptomes established also contain many non-coding RNAs and predict extensive RNA-based regulation in this model Archaeon.}, language = {en} } @article{GlaserSchurigtSuzukietal.2015, author = {Glaser, Jan and Schurigt, Uta and Suzuki, Brian M. and Caffrey, Connor R. and Holzgrabe, Ulrike}, title = {Anti-Schistosomal Activity of Cinnamic Acid Esters: Eugenyl}, series = {Molecules}, volume = {20}, journal = {Molecules}, doi = {10.3390/molecules200610873}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-125712}, pages = {10873-10883}, year = {2015}, abstract = {Bornyl caffeate (1) was previously isolated by us from Valeriana (V.) wallichii rhizomes and identified as an anti-leishmanial substance. Here, we screened a small compound library of synthesized derivatives 1-30 for activity against schistosomula of Schistosoma (S.) mansoni. Compound 1 did not show any anti-schistosomal activity. However, strong phenotypic changes, including the formation of vacuoles, degeneration and death were observed after in vitro treatment with compounds 23 (thymyl cinnamate) and 27 (eugenyl cinnamate). Electron microscopy analysis of the induced vacuoles in the dying parasites suggests that 23 and 27 interfere with autophagy.}, language = {en} } @article{SchneiderKleinMielichSuessetal.2015, author = {Schneider, Johannes and Klein, Teresa and Mielich-S{\"u}ss, Benjamin and Koch, Gudrun and Franke, Christian and Kuipers, Oskar P. and Kov{\´a}cs, {\´A}kos T. and Sauer, Markus and Lopez, Daniel}, title = {Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium}, series = {PLoS Genetics}, volume = {11}, journal = {PLoS Genetics}, number = {4}, doi = {10.1371/journal.pgen.1005140}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-125577}, pages = {e1005140}, year = {2015}, abstract = {Lipid rafts are membrane microdomains specialized in the regulation of numerous cellular processes related to membrane organization, as diverse as signal transduction, protein sorting, membrane trafficking or pathogen invasion. It has been proposed that this functional diversity would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known. We recently discovered that bacterial membranes organize their signal transduction pathways in functional membrane microdomains (FMMs) that are structurally and functionally similar to the eukaryotic lipid rafts. In this report, we took advantage of the tractability of the prokaryotic model Bacillus subtilis to provide evidence for the coexistence of two distinct families of FMMs in bacterial membranes, displaying a distinctive distribution of proteins specialized in different biological processes. One family of microdomains harbors the scaffolding flotillin protein FloA that selectively tethers proteins specialized in regulating cell envelope turnover and primary metabolism. A second population of microdomains containing the two scaffolding flotillins, FloA and FloT, arises exclusively at later stages of cell growth and specializes in adaptation of cells to stationary phase. Importantly, the diversification of membrane microdomains does not occur arbitrarily. We discovered that bacterial cells control the spatio-temporal remodeling of microdomains by restricting the activation of FloT expression to stationary phase. This regulation ensures a sequential assembly of functionally specialized membrane microdomains to strategically organize signaling networks at the right time during the lifespan of a bacterium.}, language = {en} } @article{MasicValenciaHernandezHazraetal.2015, author = {Masic, Anita and Valencia Hernandez, Ana Maria and Hazra, Sudipta and Glaser, Jan and Holzgrabe, Ulrike and Hazra, Banasri and Schurigt, Uta}, title = {Cinnamic Acid Bornyl Ester Derivatives from Valeriana wallichii Exhibit Antileishmanial In Vivo Activity in Leishmania major-Infected BALB/c Mice}, series = {PLoS One}, volume = {10}, journal = {PLoS One}, number = {11}, doi = {10.1371/journal.pone.0142386}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-125354}, pages = {e0142386}, year = {2015}, abstract = {Human leishmaniasis covers a broad spectrum of clinical manifestations ranging from self-healing cutaneous leishmaniasis to severe and lethal visceral leishmaniasis caused among other species by Leishmania major or Leishmania donovani, respectively. Some drug candidates are in clinical trials to substitute current therapies, which are facing emerging drug-resistance accompanied with serious side effects. Here, two cinnamic acid bornyl ester derivatives (1 and 2) were assessed for their antileishmanial activity. Good selectivity and antileishmanial activity of bornyl 3-phenylpropanoate (2) in vitro prompted the antileishmanial assessment in vivo. For this purpose, BALB/c mice were infected with Leishmania major promastigotes and treated with three doses of 50 mg/kg/day of compound 2. The treatment prevented the characteristic swelling at the site of infection and correlated with reduced parasite burden. Transmitted light microscopy and transmission electron microscopy of Leishmania major promastigotes revealed that compounds 1 and 2 induce mitochondrial swelling. Subsequent studies on Leishmania major promastigotes showed the loss of mitochondrial transmembrane potential (ΔΨm) as a putative mode of action. As the cinnamic acid bornyl ester derivatives 1 and 2 had exhibited antileishmanial activity in vitro, and compound 2 in Leishmania major-infected BALB/c mice in vivo, they can be regarded as possible lead structures for the development of new antileishmanial therapeutic approaches.}, language = {en} } @article{FrankMarcudeOliveiraAlmeidaPetersenetal.2015, author = {Frank, Benjamin and Marcu, Ana and de Oliveira Almeida Petersen, Antonio Luis and Weber, Heike and Stigloher, Christian and Mottram, Jeremy C. and Scholz, Claus J{\"u}rgen and Schurigt, Uta}, title = {Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210}, series = {Parasites \& Vectors}, volume = {8}, journal = {Parasites \& Vectors}, number = {404}, doi = {10.1186/s13071-015-0974-3}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-124997}, year = {2015}, abstract = {Background Autophagy participates in innate immunity by eliminating intracellular pathogens. Consequently, numerous microorganisms have developed strategies to impair the autophagic machinery in phagocytes. In the current study, interactions between Leishmania major (L. m.) and the autophagic machinery of bone marrow-derived macrophages (BMDM) were analyzed. Methods BMDM were generated from BALB/c mice, and the cells were infected with L. m. promastigotes. Transmission electron microscopy (TEM) and electron tomography were used to investigate the ultrastructure of BMDM and the intracellular parasites. Affymetrix® chip analyses were conducted to identify autophagy-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The protein expression levels of autophagy related 5 (ATG5), BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), cathepsin E (CTSE), mechanistic target of rapamycin (MTOR), microtubule-associated proteins 1A/1B light chain 3B (LC3B), and ubiquitin (UB) were investigated through western blot analyses. BMDM were transfected with specific small interfering RNAs (siRNAs) against autophagy-related genes and with mimics or inhibitors of autophagy-associated miRNAs. The infection rates of BMDM were determined by light microscopy after a parasite-specific staining. Results The experiments demonstrated autophagy induction in BMDM after in vitro infection with L. m.. The results suggested a putative MTOR phosphorylation-dependent counteracting mechanism in the early infection phase and indicated that intracellular amastigotes were cleared by autophagy in BMDM in the late infection phase. Transcriptomic analyses and specific downregulation of protein expression with siRNAs suggested there is an association between the infection-specific over expression of BNIP3, as well as CTSE, and the autophagic activity of BMDM. Transfection with mimics of mmu-miR-101c and mmu-miR-129-5p, as well as with an inhibitor of mmu-miR-210-5p, demonstrated direct effects of the respective miRNAs on parasite clearance in L. m.-infected BMDM. Furthermore, Affymetrix® chip analyses revealed a complex autophagy-related RNA network consisting of differentially expressed mRNAs and miRNAs in BMDM, which indicates high glycolytic and inflammatory activity in the host macrophages. Conclusions Autophagy in L. m.-infected host macrophages is a highly regulated cellular process at both the RNA level and the protein level. Autophagy has the potential to clear parasites from the host. The results obtained from experiments with murine host macrophages could be translated in the future to develop innovative and therapeutic antileishmanial strategies for human patients.}, language = {en} } @article{AmichKrappmann2012, author = {Amich, Jorge and Krappmann, Sven}, title = {Deciphering metabolic traits of the fungal pathogen Aspergillus fumigatus: redundancy vs. essentiality}, series = {Frontiers in Microbiology}, volume = {3}, journal = {Frontiers in Microbiology}, doi = {10.3389/fmicb.2012.00414}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123669}, pages = {414}, year = {2012}, abstract = {Incidence rates of infections caused by environmental opportunistic fungi have risen over recent decades. Aspergillus species have emerged as serious threat for the immunecompromised, and detailed knowledge about virulence-determining traits is crucial for drug target identification. As a prime saprobe, A. fumigatus has evolved to efficiently adapt to various stresses and to sustain nutritional supply by osmotrophy, which is characterized by extracellular substrate digestion followed by efficient uptake of breakdown products that are then fed into the fungal primary metabolism. These intrinsic metabolic features are believed to be related with its virulence ability. The plethora of genes that encode underlying effectors has hampered their in-depth analysis with respect to pathogenesis. Recent developments in Aspergillus molecular biology allow conditional gene expression or comprehensive targeting of gene families to cope with redundancy. Furthermore, identification of essential genes that are intrinsically connected to virulence opens accurate perspectives for novel targets in antifungal therapy.}, language = {en} } @article{SiegelHonZhangetal.2014, author = {Siegel, T. Nicolai and Hon, Chung-Chau and Zhang, Qinfeng and Lopez-Rubio, Jose-Juan and Scheidig-Benatar, Christine and Martins, Rafeal M. and Sismeiro, Odile and Copp{\´e}e, Jean-Yves}, title = {Strand-specific RNA-Seq reveals widespread and developmentally regulated transcription of natural antisense transcripts in Plasmodium falciparum}, series = {BMC Genomics}, volume = {15}, journal = {BMC Genomics}, doi = {10.1186/1471-2164-15-150}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119892}, pages = {150}, year = {2014}, abstract = {Background Advances in high-throughput sequencing have led to the discovery of widespread transcription of natural antisense transcripts (NATs) in a large number of organisms, where these transcripts have been shown to play important roles in the regulation of gene expression. Likewise, the existence of NATs has been observed in Plasmodium but our understanding towards their genome-wide distribution remains incomplete due to the limited depth and uncertainties in the level of strand specificity of previous datasets. Results To gain insights into the genome-wide distribution of NATs in P. falciparum, we performed RNA-ligation based strand-specific RNA sequencing at unprecedented depth. Our data indicate that 78.3\% of the genome is transcribed during blood-stage development. Moreover, our analysis reveals significant levels of antisense transcription from at least 24\% of protein-coding genes and that while expression levels of NATs change during the intraerythrocytic developmental cycle (IDC), they do not correlate with the corresponding mRNA levels. Interestingly, antisense transcription is not evenly distributed across coding regions (CDSs) but strongly clustered towards the 3′-end of CDSs. Furthermore, for a significant subset of NATs, transcript levels correlate with mRNA levels of neighboring genes. Finally, we were able to identify the polyadenylation sites (PASs) for a subset of NATs, demonstrating that at least some NATs are polyadenylated. We also mapped the PASs of 3443 coding genes, yielding an average 3′ untranslated region length of 523 bp. Conclusions Our strand-specific analysis of the P. falciparum transcriptome expands and strengthens the existing body of evidence that antisense transcription is a substantial phenomenon in P. falciparum. For a subset of neighboring genes we find that sense and antisense transcript levels are intricately linked while other NATs appear to be regulated independently of mRNA transcription. Our deep strand-specific dataset will provide a valuable resource for the precise determination of expression levels as it separates sense from antisense transcript levels, which we find to often significantly differ. In addition, the extensive novel data on 3′ UTR length will allow others to perform searches for regulatory motifs in the UTRs and help understand post-translational regulation in P. falciparum.}, language = {en} } @article{AdelfingerGentschevdeGuibertetal.2014, author = {Adelfinger, Marion and Gentschev, Ivaylo and de Guibert, Julio Grimm and Weibel, Stephanie and Langbein-Laugwitz, Johanna and H{\"a}rtl, Barbara and Escobar, Hugo Murua and Nolte, Ingo and Chen, Nanhai G. and Aguilar, Richard J. and Yu, Yong A. and Zhang, Qian and Frentzen, Alexa and Szalay, Aladar A.}, title = {Evaluation of a New Recombinant Oncolytic Vaccinia Virus Strain GLV-5b451 for Feline Mammary Carcinoma Therapy}, series = {PLoS ONE}, volume = {9}, journal = {PLoS ONE}, number = {8}, doi = {10.1371/journal.pone.0104337}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119387}, pages = {e104337}, year = {2014}, abstract = {Virotherapy on the basis of oncolytic vaccinia virus (VACV) infection is a promising approach for cancer therapy. In this study we describe the establishment of a new preclinical model of feline mammary carcinoma (FMC) using a recently established cancer cell line, DT09/06. In addition, we evaluated a recombinant vaccinia virus strain, GLV-5b451, expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as an oncolytic agent against FMC. Cell culture data demonstrate that GLV-5b451 virus efficiently infected, replicated in and destroyed DT09/06 cancer cells. In the selected xenografts of FMC, a single systemic administration of GLV-5b451 led to significant inhibition of tumor growth in comparison to untreated tumor-bearing mice. Furthermore, tumor-specific virus infection led to overproduction of functional scAb GLAF-2, which caused drastic reduction of intratumoral VEGF levels and inhibition of angiogenesis. In summary, here we have shown, for the first time, that the vaccinia virus strains and especially GLV-5b451 have great potential for effective treatment of FMC in animal model.}, language = {en} } @article{LiWongNongetal.2014, author = {Li, Lei and Wong, Hin-chung and Nong, Wenyan and Cheung, Man Kit and Law, Patrick Tik Wan and Kam, Kai Man and Kwan, Hoi Shan}, title = {Comparative genomic analysis of clinical and environmental strains provides insight into the pathogenicity and evolution of Vibrio parahaemolyticus}, series = {BMC Genomics}, volume = {15}, journal = {BMC Genomics}, number = {1135}, issn = {1471-2164}, doi = {10.1186/1471-2164-15-1135}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-118080}, year = {2014}, abstract = {Background: Vibrio parahaemolyticus is a Gram-negative halophilic bacterium. Infections with the bacterium could become systemic and can be life-threatening to immunocompromised individuals. Genome sequences of a few clinical isolates of V. parahaemolyticus are currently available, but the genome dynamics across the species and virulence potential of environmental strains on a genome-scale have not been described before. Results: Here we present genome sequences of four V. parahaemolyticus clinical strains from stool samples of patients and five environmental strains in Hong Kong. Phylogenomics analysis based on single nucleotide polymorphisms revealed a clear distinction between the clinical and environmental isolates. A new gene cluster belonging to the biofilm associated proteins of V. parahaemolyticus was found in clincial strains. In addition, a novel small genomic island frequently found among clinical isolates was reported. A few environmental strains were found harboring virulence genes and prophage elements, indicating their virulence potential. A unique biphenyl degradation pathway was also reported. A database for V. parahaemolyticus (http://kwanlab.bio.cuhk.edu.hk/vp webcite) was constructed here as a platform to access and analyze genome sequences and annotations of the bacterium. Conclusions: We have performed a comparative genomics analysis of clinical and environmental strains of V. parahaemolyticus. Our analyses could facilitate understanding of the phylogenetic diversity and niche adaptation of this bacterium. "}, language = {en} } @article{ReynoldsCliffeFoerstneretal.2014, author = {Reynolds, David and Cliffe, Laura and F{\"o}rstner, Konrad U. and Hon, Chung-Chau and Siegel, T. Nicolai and Sabatini, Robert}, title = {Regulation of transcription termination by glucosylated hydroxymethyluracil, base J, in Leishmania major and Trypanosoma brucei}, series = {Nucleic Acids Research}, volume = {42}, journal = {Nucleic Acids Research}, number = {15}, doi = {10.1093/nar/gku714}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-117863}, pages = {9717-9729}, year = {2014}, abstract = {Base J, beta-d-glucosyl-hydroxymethyluracil, is an epigenetic modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. J is enriched at sites involved in RNA polymerase ( RNAP) II initiation and termination. Reduction of J in Leishmania tarentolae via growth in BrdU resulted in cell death and indicated a role of J in the regulation of RNAP II termination. To further explore J function in RNAP II termination among kinetoplastids and avoid indirect effects associated with BrdU toxicity and genetic deletions, we inhibited J synthesis in Leishmania major and Trypanosoma brucei using DMOG. Reduction of J in L. major resulted in genome-wide defects in transcription termination at the end of polycistronic gene clusters and the generation of antisense RNAs, without cell death. In contrast, loss of J in T. brucei did not lead to genome-wide termination defects; however, the loss of J at specific sites within polycistronic gene clusters led to altered transcription termination and increased expression of downstream genes. Thus, J regulation of RNAP II transcription termination genome-wide is restricted to Leishmania spp., while in T. brucei it regulates termination and gene expression at specific sites within polycistronic gene clusters.}, language = {en} } @article{JunGholamiSongetal.2014, author = {Jun, Kyong-Hwa and Gholami, Spedideh and Song, Tae-Jin and Au, Joyce and Haddad, Dana and Carson, Joshua and Chen, Chun-Hao and Mojica, Kelly and Zanzonico, Pat and Chen, Nanhai G. and Zhang, Qian and Szalay, Aladar and Fong, Yuman}, title = {A novel oncolytic viral therapy and imaging technique for gastric cancer using a genetically engineered vaccinia virus carrying the human sodium iodide symporter}, series = {Journal of Experimental \& Clinical Cancer Research}, volume = {33}, journal = {Journal of Experimental \& Clinical Cancer Research}, number = {2}, issn = {1756-9966}, doi = {10.1186/1756-9966-33-2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-117716}, year = {2014}, abstract = {Background: Gastric cancers have poor overall survival despite recent advancements in early detection methods, endoscopic resection techniques, and chemotherapy treatments. Vaccinia viral therapy has had promising therapeutic potential for various cancers and has a great safety profile. We investigated the therapeutic efficacy of a novel genetically-engineered vaccinia virus carrying the human sodium iodide symporter (hNIS) gene, GLV-1 h153, on gastric cancers and its potential utility for imaging with Tc-99m pertechnetate scintigraphy and I-124 positron emission tomography (PET). Methods: GLV-1 h153 was tested against five human gastric cancer cell lines using cytotoxicity and standard viral plaque assays. In vivo, subcutaneous flank tumors were generated in nude mice with human gastric cancer cells, MKN-74. Tumors were subsequently injected with either GLV-1 h153 or PBS and followed for tumor growth. Tc-99m pertechnetate scintigraphy and I-124 microPET imaging were performed. Results: GFP expression, a surrogate for viral infectivity, confirmed viral infection by 24 hours. At a multiplicity of infection (MOI) of 1, GLV-1 h153 achieved > 90\% cytotoxicity in MNK-74, OCUM-2MD3, and AGS over 9 days, and >70\% cytotoxicity in MNK-45 and TMK-1. In vivo, GLV-1 h153 was effective in treating xenografts (p < 0.001) after 2 weeks of treatment. GLV-1 h153-infected tumors were readily imaged by Tc-99m pertechnetate scintigraphy and I-124 microPET imaging 2 days after treatment. Conclusions: GLV-1 h153 is an effective oncolytic virus expressing the hNIS protein that can efficiently regress gastric tumors and allow deep-tissue imaging. These data encourages its continued investigation in clinical settings.}, language = {en} } @article{BielaszewskaSchillerLammersetal.2014, author = {Bielaszewska, Martina and Schiller, Roswitha and Lammers, Lydia and Bauwens, Andreas and Fruth, Angelika and Middendorf, Barbara and Schmidt, M. Alexander and Tarr, Phillip I. and Dobrindt, Ulrich and Karch, Helge and Mellmann, Alexander}, title = {Heteropathogenic virulence and phylogeny reveal phased pathogenic metamorphosis in Escherichia coli O2:H6}, series = {EMBO Molecular Medicine}, volume = {6}, journal = {EMBO Molecular Medicine}, number = {3}, issn = {1757-4684}, doi = {10.1002/emmm.201303133}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-117254}, pages = {347-357}, year = {2014}, abstract = {Extraintestinal pathogenic and intestinal pathogenic (diarrheagenic) Escherichia coli differ phylogenetically and by virulence profiles. Classic theory teaches simple linear descent in this species, where non-pathogens acquire virulence traits and emerge as pathogens. However, diarrheagenic Shiga toxin-producing E.coli (STEC) O2:H6 not only possess and express virulence factors associated with diarrheagenic and uropathogenic E.coli but also cause diarrhea and urinary tract infections. These organisms are phylogenetically positioned between members of an intestinal pathogenic group (STEC) and extraintestinal pathogenic E.coli. STEC O2:H6 is, therefore, a 'heteropathogen,' and the first such hybrid virulent E.coli identified. The phylogeny of these E.coli and the repertoire of virulence traits they possess compel consideration of an alternate view of pathogen emergence, whereby one pathogroup of E.coli undergoes phased metamorphosis into another. By understanding the evolutionary mechanisms of bacterial pathogens, rational strategies for counteracting their detrimental effects on humans can be developed.}, language = {en} } @article{NguyenMuellerParketal.2014, author = {Nguyen, Tu N. and M{\"u}ller, Laura S. M. and Park, Sung Hee and Siegel, T. Nicolai and G{\"u}nzl, Arthur}, title = {Promoter occupancy of the basal class I transcription factor A differs strongly between active and silent VSG expression sites in Trypanosoma brucei}, series = {Nucleic Acid Research}, volume = {42}, journal = {Nucleic Acid Research}, number = {5}, issn = {1362-4962}, doi = {10.1093/nar/gkt1301}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-117232}, pages = {3164-3176}, year = {2014}, abstract = {Monoallelic expression within a gene family is found in pathogens exhibiting antigenic variation and in mammalian olfactory neurons. Trypanosoma brucei, a lethal parasite living in the human bloodstream, expresses variant surface glycoprotein (VSG) from 1 of 15 bloodstream expression sites (BESs) by virtue of a multifunctional RNA polymerase I. The active BES is transcribed in an extranucleolar compartment termed the expression site body (ESB), whereas silent BESs, located elsewhere within the nucleus, are repressed epigenetically. The regulatory mechanisms, however, are poorly understood. Here we show that two essential subunits of the basal class I transcription factor A (CITFA) predominantly occupied the promoter of the active BES relative to that of a silent BES, a phenotype that was maintained after switching BESs in situ. In these experiments, high promoter occupancy of CITFA was coupled to high levels of both promoter-proximal RNA abundance and RNA polymerase I occupancy. Accordingly, fluorescently tagged CITFA-7 was concentrated in the nucleolus and the ESB. Because a ChIP-seq analysis found that along the entire BES, CITFA-7 is specifically enriched only at the promoter, our data strongly indicate that monoallelic BES transcription is activated by a mechanism that functions at the level of transcription initiation.}, language = {en} } @article{BischlerKopfVoss2014, author = {Bischler, Thorsten and Kopf, Matthias and Voss, Bjoern}, title = {Transcript mapping based on dRNA-seq data}, series = {BMC Bioinformatics}, volume = {15}, journal = {BMC Bioinformatics}, number = {122}, issn = {1471-2105}, doi = {10.1186/1471-2105-15-122}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-116663}, year = {2014}, abstract = {Background: RNA-seq and its variant differential RNA-seq (dRNA-seq) are today routine methods for transcriptome analysis in bacteria. While expression profiling and transcriptional start site prediction are standard tasks today, the problem of identifying transcriptional units in a genome-wide fashion is still not solved for prokaryotic systems. Results: We present RNASEG, an algorithm for the prediction of transcriptional units based on dRNA-seq data. A key feature of the algorithm is that, based on the data, it distinguishes between transcribed and un-transcribed genomic segments. Furthermore, the program provides many different predictions in a single run, which can be used to infer the significance of transcriptional units in a consensus procedure. We show the performance of our method based on a well-studied dRNA-seq data set for Helicobacter pylori. Conclusions: With our algorithm it is possible to identify operons and 5'- and 3'-UTRs in an automated fashion. This alleviates the need for labour intensive manual inspection and enables large-scale studies in the area of comparative transcriptomics.}, language = {en} } @article{WagnerVolkmerSharanetal.2014, author = {Wagner, Ines and Volkmer, Michael and Sharan, Malvika and Villaveces, Jose M. and Oswald, Felix and Surendranath, Vineeth and Habermann, Bianca H.}, title = {morFeus: a web-based program to detect remotely conserved orthologs using symmetrical best hits and orthology network scoring}, series = {BMC Bioinformatics}, volume = {15}, journal = {BMC Bioinformatics}, number = {263}, doi = {10.1186/1471-2105-15-263}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115590}, year = {2014}, abstract = {Background: Searching the orthologs of a given protein or DNA sequence is one of the most important and most commonly used Bioinformatics methods in Biology. Programs like BLAST or the orthology search engine Inparanoid can be used to find orthologs when the similarity between two sequences is sufficiently high. They however fail when the level of conservation is low. The detection of remotely conserved proteins oftentimes involves sophisticated manual intervention that is difficult to automate. Results: Here, we introduce morFeus, a search program to find remotely conserved orthologs. Based on relaxed sequence similarity searches, morFeus selects sequences based on the similarity of their alignments to the query, tests for orthology by iterative reciprocal BLAST searches and calculates a network score for the resulting network of orthologs that is a measure of orthology independent of the E-value. Detecting remotely conserved orthologs of a protein using morFeus thus requires no manual intervention. We demonstrate the performance of morFeus by comparing it to state-of-the-art orthology resources and methods. We provide an example of remotely conserved orthologs, which were experimentally shown to be functionally equivalent in the respective organisms and therefore meet the criteria of the orthology-function conjecture. Conclusions: Based on our results, we conclude that morFeus is a powerful and specific search method for detecting remotely conserved orthologs.}, language = {en} } @article{TalmanPrietoMarquesetal.2014, author = {Talman, Arthur M. and Prieto, Judith H. and Marques, Sara and Ubaida-Mohien, Ceereena and Lawniczak, Mara and Wass, Mark N. and Xu, Tao and Frank, Roland and Ecker, Andrea and Stanway, Rebecca S. and Krishna, Sanjeev and Sternberg, Michael J. E. and Christophides, Georges K. and Graham, David R. and Dinglasan, Rhoel R. and Yates, John R., III and Sinden, Robert E.}, title = {Proteomic analysis of the Plasmodium male gamete reveals the key role for glycolysis in flagellar motility}, series = {Malaria Journal}, volume = {13}, journal = {Malaria Journal}, number = {315}, doi = {10.1186/1475-2875-13-315}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115572}, year = {2014}, abstract = {Background: Gametogenesis and fertilization play crucial roles in malaria transmission. While male gametes are thought to be amongst the simplest eukaryotic cells and are proven targets of transmission blocking immunity, little is known about their molecular organization. For example, the pathway of energy metabolism that power motility, a feature that facilitates gamete encounter and fertilization, is unknown. Methods: Plasmodium berghei microgametes were purified and analysed by whole-cell proteomic analysis for the first time. Data are available via ProteomeXchange with identifier PXD001163. Results: 615 proteins were recovered, they included all male gamete proteins described thus far. Amongst them were the 11 enzymes of the glycolytic pathway. The hexose transporter was localized to the gamete plasma membrane and it was shown that microgamete motility can be suppressed effectively by inhibitors of this transporter and of the glycolytic pathway. Conclusions: This study describes the first whole-cell proteomic analysis of the malaria male gamete. It identifies glycolysis as the likely exclusive source of energy for flagellar beat, and provides new insights in original features of Plasmodium flagellar organization.}, language = {en} } @article{RemesBerghoffFoerstneretal.2014, author = {Remes, Bernhard and Berghoff, Bork A. and F{\"o}rstner, Konrad U. and Klug, Gabriele}, title = {Role of oxygen and the OxyR protein in the response to iron limitation in Rhodobacter sphaeroides}, series = {BMC Genomics}, volume = {15}, journal = {BMC Genomics}, number = {794}, issn = {1471-2164}, doi = {10.1186/1471-2164-15-794}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115357}, year = {2014}, abstract = {Background: High intracellular levels of unbound iron can contribute to the production of reactive oxygen species (ROS) via the Fenton reaction, while depletion of iron limits the availability of iron-containing proteins, some of which have important functions in defence against oxidative stress. Vice versa increased ROS levels lead to the damage of proteins with iron sulphur centres. Thus, organisms have to coordinate and balance their responses to oxidative stress and iron availability. Our knowledge of the molecular mechanisms underlying the co-regulation of these responses remains limited. To discriminate between a direct cellular response to iron limitation and indirect responses, which are the consequence of increased levels of ROS, we compared the response of the alpha-proteobacterium Rhodobacter sphaeroides to iron limitation in the presence or absence of oxygen. Results: One third of all genes with altered expression under iron limitation showed a response that was independent of oxygen availability. The other iron-regulated genes showed different responses in oxic or anoxic conditions and were grouped into six clusters based on the different expression profiles. For two of these clusters, induction in response to iron limitation under oxic conditions was dependent on the OxyR regulatory protein. An OxyR mutant showed increased ROS production and impaired growth under iron limitation. Conclusion: Some R. sphaeroides genes respond to iron limitation irrespective of oxygen availability. These genes therefore reflect a "core iron response" that is independent of potential ROS production under oxic, iron-limiting conditions. However, the regulation of most of the iron-responsive genes was biased by oxygen availability. Most strikingly, the OxyR-dependent activation of a subset of genes upon iron limitation under oxic conditions, including many genes with a role in iron metabolism, revealed that elevated ROS levels were an important trigger for this response. OxyR thus provides a regulatory link between the responses to oxidative stress and to iron limitation in R. sphaeroides.}, language = {en} } @article{LindgreenUmuLaietal.2014, author = {Lindgreen, Stinus and Umu, Sinan Uğur and Lai, Alicia Sook-Wei and Eldai, Hisham and Liu, Wenting and McGimpsey, Stephanie and Wheeler, Nicole E. and Biggs, Patrick J. and Thomson, Nick R. and Barquist, Lars and Poole, Anthony M. and Gardner, Paul P.}, title = {Robust Identification of Noncoding RNA from Transcriptomes Requires Phylogenetically-Informed Sampling}, series = {PLOS Computational Biology}, volume = {10}, journal = {PLOS Computational Biology}, number = {10}, doi = {10.1371/journal.pcbi.1003907}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115259}, pages = {e1003907}, year = {2014}, abstract = {Noncoding RNAs are integral to a wide range of biological processes, including translation, gene regulation, host-pathogen interactions and environmental sensing. While genomics is now a mature field, our capacity to identify noncoding RNA elements in bacterial and archaeal genomes is hampered by the difficulty of de novo identification. The emergence of new technologies for characterizing transcriptome outputs, notably RNA-seq, are improving noncoding RNA identification and expression quantification. However, a major challenge is to robustly distinguish functional outputs from transcriptional noise. To establish whether annotation of existing transcriptome data has effectively captured all functional outputs, we analysed over 400 publicly available RNA-seq datasets spanning 37 different Archaea and Bacteria. Using comparative tools, we identify close to a thousand highly-expressed candidate noncoding RNAs. However, our analyses reveal that capacity to identify noncoding RNA outputs is strongly dependent on phylogenetic sampling. Surprisingly, and in stark contrast to protein-coding genes, the phylogenetic window for effective use of comparative methods is perversely narrow: aggregating public datasets only produced one phylogenetic cluster where these tools could be used to robustly separate unannotated noncoding RNAs from a null hypothesis of transcriptional noise. Our results show that for the full potential of transcriptomics data to be realized, a change in experimental design is paramount: effective transcriptomics requires phylogeny-aware sampling.}, language = {en} } @article{RicoYepesRodriguezetal.2014, author = {Rico, Sergio and Yepes, Ana and Rodriguez, Hector and Santamaria, Jorge and Antoraz, Sergio and Krause, Eva M. and Diaz, Margarita and Santamaria, Ramon I.}, title = {Regulation of the AbrA1/A2 Two-Component System in Streptomyces coelicolor and the Potential of Its Deletion Strain as a Heterologous Host for Antibiotic Production}, series = {PLOS ONE}, volume = {9}, journal = {PLOS ONE}, number = {10}, issn = {1932-6203}, doi = {10.1371/journal.pone.0109844}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115151}, pages = {e109844}, year = {2014}, abstract = {The Two-Component System (TCS) AbrA1/A2 from Streptomyces coelicolor M145 is a negative regulator of antibiotic production and morphological differentiation. In this work we show that it is able to auto-regulate its expression, exerting a positive induction of its own operon promoter, and that its activation is dependent on the presence of iron. The overexpression of the abrA2 response regulator (RR) gene in the mutant DabrA1/A2 results in a toxic phenotype. The reason is an excess of phosphorylated AbrA2, as shown by phosphoablative and phosphomimetic AbrA2 mutants. Therefore, non-cognate histidine kinases (HKs) or small phospho-donors may be responsible for AbrA2 phosphorylation in vivo. The results suggest that in the parent strain S. coelicolor M145 the correct amount of phosphorylated AbrA2 is adjusted through the phosphorylation-dephosphorylation activity rate of the HK AbrA1. Furthermore, the ABC transporter system, which is part of the four-gene operon comprising AbrA1/A2, is necessary to de-repress antibiotic production in the TCS null mutant. Finally, in order to test the possible biotechnological applications of the DabrA1/A2 strain, we demonstrate that the production of the antitumoral antibiotic oviedomycin is duplicated in this strain as compared with the production obtained in the wild type, showing that this strain is a good host for heterologous antibiotic production. Thus, this genetically modified strain could be interesting for the biotechnology industry.}, language = {en} } @article{MoellerOverloeperFoerstneretal.2014, author = {M{\"o}ller, Philip and Overl{\"o}per, Aaron and F{\"o}rstner, Konrad U. and Wen, Tuan-Nan and Sharma, Cynthia M. and Lai, Erh-Min and Narberhaus, Franz}, title = {Profound Impact of Hfq on Nutrient Acquisition, Metabolism and Motility in the Plant Pathogen Agrobacterium tumefaciens}, series = {PLOS ONE}, volume = {9}, journal = {PLOS ONE}, number = {10}, doi = {10.1371/journal.pone.0110427}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114874}, pages = {e110427}, year = {2014}, abstract = {As matchmaker between mRNA and sRNA interactions, the RNA chaperone Hfq plays a key role in riboregulation of many bacteria. Often, the global influence of Hfq on the transcriptome is reflected by substantially altered proteomes and pleiotropic phenotypes in hfq mutants. Using quantitative proteomics and co-immunoprecipitation combined with RNA-sequencing (RIP-seq) of Hfq-bound RNAs, we demonstrate the pervasive role of Hfq in nutrient acquisition, metabolism and motility of the plant pathogen Agrobacterium tumefaciens. 136 of 2544 proteins identified by iTRAQ (isobaric tags for relative and absolute quantitation) were affected in the absence of Hfq. Most of them were associated with ABC transporters, general metabolism and motility. RIP-seq of chromosomally encoded Hfq 3xFlag revealed 1697 mRNAs and 209 non-coding RNAs (ncRNAs) associated with Hfq. 56 ncRNAs were previously undescribed. Interestingly, 55\% of the Hfq-bound ncRNAs were encoded antisense (as) to a protein-coding sequence suggesting that A. tumefaciens Hfq plays an important role in asRNA-target interactions. The exclusive enrichment of 296 mRNAs and 31 ncRNAs under virulence conditions further indicates a role for post-transcriptional regulation in A. tumefaciens-mediated plant infection. On the basis of the iTRAQ and RIP-seq data, we assembled a comprehensive model of the Hfq core regulon in A. tumefaciens.}, language = {en} } @article{ZukherNovikovaTikhonovetal.2014, author = {Zukher, Inna and Novikova, Maria and Tikhonov, Anton and Nesterchuk, Mikhail V. and Osterman, Ilya A. and Djordjevic, Marko and Sergiev, Petr V. and Sharma, Cynthia M. and Severinov, Konstantin}, title = {Ribosome-controlled transcription termination is essential for the production of antibiotic microcin C}, series = {Nucleic Acids Research}, volume = {42}, journal = {Nucleic Acids Research}, number = {19}, issn = {0305-1048}, doi = {10.1093/nar/gku880}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114839}, pages = {11891-11902}, year = {2014}, abstract = {Microcin C (McC) is a peptide-nucleotide antibiotic produced by Escherichia coli cells harboring a plasmid-borne operon mccABCDE. The heptapeptide MccA is converted into McC by adenylation catalyzed by the MccB enzyme. Since MccA is a substrate for MccB, a mechanism that regulates the MccA/MccB ratio likely exists. Here, we show that transcription from a promoter located upstream of mccA directs the synthesis of two transcripts: a short highly abundant transcript containing the mccA ORF and a longer minor transcript containing mccA and downstream ORFs. The short transcript is generated when RNA polymerase terminates transcription at an intrinsic terminator located in the intergenic region between the mccA and mccB genes. The function of this terminator is strongly attenuated by upstream mcc sequences. Attenuation is relieved and transcription termination is induced when ribosome binds to the mccA ORF. Ribosome binding also makes the mccA RNA exceptionally stable. Together, these two effects-ribosome induced transcription termination and stabilization of the message-account for very high abundance of the mccA transcript that is essential for McC production. The general scheme appears to be evolutionary conserved as ribosome-induced transcription termination also occurs in a homologous operon from Helicobacter pylori.}, language = {en} } @article{DimastrogiovanniFroehlichBandyraetal.2014, author = {Dimastrogiovanni, Daniela and Fr{\"o}hlich, Kathrin S. and Bandyra, Katarzyna J. and Bruce, Heather A. and Hohensee, Susann and Vogel, J{\"o}rg and Luisi, Ben F.}, title = {Recognition of the small regulatory RNA RydC by the bacterial Hfq protein}, series = {eLife}, volume = {3}, journal = {eLife}, number = {e05375}, issn = {2050-084X}, doi = {10.7554/eLife.05375}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114191}, year = {2014}, abstract = {Bacterial small RNAs (sRNAs) are key elements of regulatory networks that modulate gene expression. The sRNA RydC of Salmonella sp. and Escherichia coli is an example of this class of riboregulators. Like many other sRNAs, RydC bears a 'seed' region that recognises specific transcripts through base-pairing, and its activities are facilitated by the RNA chaperone Hfq. The crystal structure of RydC in complex with E. coli Hfq at 3.48 angstrom resolution illuminates how the protein interacts with and presents the sRNA for target recognition. Consolidating the protein-RNA complex is a host of distributed interactions mediated by the natively unstructured termini of Hfq. Based on the structure and other data, we propose a model for a dynamic effector complex comprising Hfq, small RNA, and the cognate mRNA target.}, language = {en} } @article{GlaserSchultheisHazraetal.2014, author = {Glaser, Jan and Schultheis, Martina and Hazra, Sudipta and Hazra, Banazri and Moll, Heidrun and Schurigt, Uta and Holzgrabe, Ulrike}, title = {Antileishmanial Lead Structures from Nature: Analysis of Structure-Activity Relationships of a Compound Library Derived from Caffeic Acid Bornyl Ester}, doi = {10.3390/molecules19021394}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-112835}, year = {2014}, abstract = {Bioassay-guided fractionation of a chloroform extract of Valeriana wallichii (V. wallichii) rhizomes lead to the isolation and identification of caffeic acid bornyl ester (1) as the active component against Leishmania major (L. major) promastigotes (IC50 = 48.8 µM). To investigate the structure-activity relationship (SAR), a library of compounds based on 1 was synthesized and tested in vitro against L. major and L. donovani promastigotes, and L. major amastigotes. Cytotoxicity was determined using a murine J774.1 cell line and bone marrow derived macrophages (BMDM). Some compounds showed antileishmanial activity in the concentration range of pentamidine and miltefosine which are the standard drugs in use. In the L. major amastigote assay compounds 15, 19 and 20 showed good activity with relatively low cytotoxicity against BMDM, resulting in acceptable selectivity indices. Molecules with adjacent phenolic hydroxyl groups exhibited elevated cytotoxicity against murine cell lines J774.1 and BMDM. The Michael system seems not to be essential for antileishmanial activity. Based on the results compound 27 can be regarded as new lead structure for further structure optimization}, language = {en} } @article{GorskiVogelSalibaetal.2014, author = {Gorski, Stanislaw A. and Vogel, J{\"o}rg and Saliba, Antoine-Emmanuel and Westermann, Alexander J.}, title = {Single-cell RNA-seq: advances and future challenges}, doi = {10.1093/nar/gku555}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-110993}, year = {2014}, abstract = {Phenotypically identical cells can dramatically vary with respect to behavior during their lifespan and this variation is reflected in their molecular composition such as the transcriptomic landscape. Singlecell transcriptomics using next-generation transcript sequencing (RNA-seq) is now emerging as a powerful tool to profile cell-to-cell variability on a genomic scale. Its application has already greatly impacted our conceptual understanding of diverse biological processes with broad implications for both basic and clinical research. Different single-cell RNAseq protocols have been introduced and are reviewed here - each one with its own strengths and current limitations. We further provide an overview of the biological questions single-cell RNA-seq has been used to address, the major findings obtained from such studies, and current challenges and expected future developments in this booming field.}, subject = {RNS}, language = {en} } @article{HofmannWeibelSzalay2014, author = {Hofmann, Elisabeth and Weibel, Stephanie and Szalay, Aladar A.}, title = {Combination treatment with oncolytic Vaccinia virus and cyclophosphamide results in synergistic antitumor effects in human lung adenocarcinoma bearing mice}, doi = {10.1186/1479-5876-12-197}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-110168}, year = {2014}, abstract = {Background The capacity of the recombinant Vaccinia virus GLV-1h68 as a single agent to efficiently treat different human or canine cancers has been shown in several preclinical studies. Currently, its human safety and efficacy are investigated in phase I/II clinical trials. In this study we set out to evaluate the oncolytic activity of GLV-1h68 in the human lung adenocarcinoma cell line PC14PE6-RFP in cell cultures and analyzed the antitumor potency of a combined treatment strategy consisting of GLV-1h68 and cyclophosphamide (CPA) in a mouse model of PC14PE6-RFP lung adenocarcinoma. Methods PC14PE6-RFP cells were treated in cell culture with GLV-1h68. Viral replication and cell survival were determined by plaque assays and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. Subcutaneously implanted PC14PE6-RFP xenografts were treated by systemic injection of GLV-1h68, CPA or a combination of both. Tumor growth and viral biodistribution were monitored and immune-related antigen profiling of tumor lysates was performed. Results GLV-1h68 efficiently infected, replicated in and lysed human PC14PE6-RFP cells in cell cultures. PC14PE6-RFP tumors were efficiently colonized by GLV-1h68 leading to much delayed tumor growth in PC14PE6-RFP tumor-bearing nude mice. Combination treatment with GLV-1h68 and CPA significantly improved the antitumor efficacy of GLV-1h68 and led to an increased viral distribution within the tumors. Pro-inflammatory cytokines and chemokines were distinctly elevated in tumors of GLV-1h68-treated mice. Factors expressed by endothelial cells or present in the blood were decreased after combination treatment. A complete loss in the hemorrhagic phenotype of the PC14PE6-RFP tumors and a decrease in the number of blood vessels after combination treatment could be observed. Conclusions CPA and GLV-1h68 have synergistic antitumor effects on PC14PE6-RFP xenografts. We strongly suppose that in the PC14PE6-RFP model the enhanced tumor growth inhibition achieved by combining GLV-1h68 with CPA is due to an effect on the vasculature rather than an immunosuppressive action of CPA. These results provide evidence to support further preclinical studies of combining GLV-1h68 and CPA in other highly angiogenic tumor models. Moreover, data presented here demonstrate that CPA can be combined successfully with GLV-1h68 based oncolytic virus therapy and therefore might be promising as combination therapy in human clinical trials.}, language = {en} } @inproceedings{FoerstnerHagedornKoltzenburgetal.2011, author = {F{\"o}rstner, Konrad and Hagedorn, Gregor and Koltzenburg, Claudia and Kubke, Fabiana and Mietchen, Daniel}, title = {Collaborative platforms for streamlining workflows in Open Science}, series = {Proceedings of the 6th Open Knowledge Conference}, booktitle = {Proceedings of the 6th Open Knowledge Conference}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-101678}, year = {2011}, abstract = {Despite the internet's dynamic and collaborative nature, scientists continue to produce grant proposals, lab notebooks, data files, conclusions etc. that stay in static formats or are not published online and therefore not always easily accessible to the interested public. Because of limited adoption of tools that seamlessly integrate all aspects of a research project (conception, data generation, data evaluation, peerreviewing and publishing of conclusions), much effort is later spent on reproducing or reformatting individual entities before they can be repurposed independently or as parts of articles. We propose that workflows - performed both individually and collaboratively - could potentially become more efficient if all steps of the research cycle were coherently represented online and the underlying data were formatted, annotated and licensed for reuse. Such a system would accelerate the process of taking projects from conception to publication stages and allow for continuous updating of the data sets and their interpretation as well as their integration into other independent projects. A major advantage of such work ows is the increased transparency, both with respect to the scientific process as to the contribution of each participant. The latter point is important from a perspective of motivation, as it enables the allocation of reputation, which creates incentives for scientists to contribute to projects. Such work ow platforms offering possibilities to fine-tune the accessibility of their content could gradually pave the path from the current static mode of research presentation into a more coherent practice of open science.}, language = {en} } @article{MietchenHagedornFoerstneretal.2011, author = {Mietchen, Daniel and Hagedorn, Gregor and F{\"o}rstner, Konrad U. and Kubke, M Fabiana and Koltzenburg, Claudia and Hahnel, Mark J. and Penev, Lyubomir}, title = {Wikis in scholarly publishing}, doi = {10.3233/ISU-2011-0621}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-87770}, year = {2011}, abstract = {Scientific research is a process concerned with the creation, collective accumulation, contextualization, updating and maintenance of knowledge. Wikis provide an environment that allows to collectively accumulate, contextualize, update and maintain knowledge in a coherent and transparent fashion. Here, we examine the potential of wikis as platforms for scholarly publishing. In the hope to stimulate further discussion, the article itself was drafted on Species-ID - a wiki that hosts a prototype for wiki-based scholarly publishing - where it can be updated, expanded or otherwise improved.}, subject = {Elektronisches Publizieren}, language = {en} } @article{SchrotenWolskePlogmannetal.1991, author = {Schroten, Horst and Wolske, Anja and Plogmann, Ricarda and Hanisch, Franz-Georg and Hacker, J{\"o}rg and Uhlenbr{\"u}ck, Gerhard and Wahn, Volker}, title = {Binding of cloned S-fimbriated E. coli to human buccal epithelial cells-different inhibition of binding by neonatal saliva and adult saliva.}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86291}, year = {1991}, abstract = {Investigations were carried out on the adhesion of cloned S-fimbriated E. coli, labelled with fluoresceinisothiocyanate (FITC) to human buccal epithelial cells. Fluorescence microscopic analysis revealed binding of bacteria to 75-95\% of epithelial cells. Inhibition experiments with fetuin, a 1-acid glycoprotein and N-acetyl neuraminic acid confirmed the specificity of bacterial binding to sialoglycoproteins. Further studies using saliva as an inhibitor resulted in a 4-5 times stronger binding inhibition by newborn saliva in comparison to adult saliva coinciding with a 4-5 times higher content of total N-acetyl neuraminic acid in samples of newborn saliva. In Western blot analysis sialoglycoprotein bands with a molecular weight >200 kD reacting with wheat germ agglutinin (WGA), were only identified in samples of newborn saliva. These bands are classified as mucins on account of molecular weight and staining. These data suggest that saliva mucins could represent a major defense mechanism against bacterial infections at a stage of ontogeny where the secretory IgAsystem is not yet developed.}, subject = {Escherichia coli}, language = {en} } @article{MorschhaeuserVetterKorhonenetal.1993, author = {Morschh{\"a}user, Joachim and Vetter, Viktoria and Korhonen, Timo and Uhlin, Bernt Eric and Hacker, J{\"o}rg}, title = {Regulation and binding properties of S fimbriae cloned from E. coli strains causing urinary tract infection and meningitis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86140}, year = {1993}, abstract = {S fimbriae are able to recognize receptor molecules containing sialic acid and are produced by pathogenic E. coli strains causing urinary tract infection and menigitis. In order to characterize the corresponding genetic determinant, termed S fimbrial adhesin ( sfa) gene duster, we have cloned the S-specific genes from a urinary pathogen and from a meningitis isolate. Nine genes are involved in the production of S fimbriae, two of these, sfaB and sfaC code for regulatory proteins being necessary for the expression of S fimbriae. Two promoters, PB and Pc, are located in front of these genes. Transcription of the sfa determinant is influenced by activation of the promotersvia SfaB and SfaC, the action of the H-NS protein and an RNaseE-specific mRNA processing. In addition, a third promoter, P A• located in front of the major subunit gene sfaA, can be activated under special circumstances. Four genes of the sfa determinant code for the subunit-specific proteins, SfaA (16 kda), SfaG (17 kda), SfaS (14 kda) and SfaH (29 kda). It was demonstrated that the protein SfaA is the major subunit protein while SfaS is identical to the sialic-acid-specific adhesin of S fimbriae. The introduction of specific mutations into sfaS revealed that a region of six amino acids of the adhesin which includes two lysine and one arginine residues is involved in the receptor specific interaction of S fimbriae. Additionally, it has been shown that SfaS is necessary for the induction of fimbriation while SfaH plays a role in the stringency of binding of S fimbriae to erythrocytes.}, subject = {Escherichia coli}, language = {en} } @article{TschaepeBenderOttetal.1992, author = {Tsch{\"a}pe, Helmut and Bender, Larisa and Ott, Manfred and Wittig, Walter and Hacker, J{\"o}rg}, title = {Restriction fragments length polymorphism and virulence pattern of the veterinary pathogen Escherichia coli O139:K82:H1}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86131}, year = {1992}, abstract = {Escherichia coli 0139: K82: H1 strains originating from outbreaks and single cases of oedema disease in pigs were characterized by their genomic restriction fragment length polymorphism (RFLP), their virulence pattern, and by the occurrence as well as the genomic distribution of the determinants for hemolysin (hly) and verotoxins (shiga-like toxins; sltI, sltII). Whereas the RFLPs revealed considerable variation among the E. coli 0139: K82: H1 isolates depending the origin and epidemic source of the strains, the virulence gene slt II was found to be present in nearly all strains in a particular chromosomal region. Similar to RFLPs, the plasmid profiles are useful for epidemiological analysis.}, subject = {Escherichia coli}, language = {en} } @article{HackerOttWintermeyeretal.1993, author = {Hacker, J{\"o}rg and Ott, Manfred and Wintermeyer, Eva and Ludwig, Birgit and Fischer, Gunter}, title = {Analysis of virulence factors of Legionella pneumophila.}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70620}, year = {1993}, abstract = {Legionella pneumophila, the causative agent of Legionnaires' disease is a facultative intracellular bacterium, which in the course of human infection multiplies in lung macrophages predominantly manifesting as pneumonia. The natural habitat of Legionella is found in sweet water reservoirs and man-made water systems. Virulent L. pneumophila spontaneously convert to an avirulent status at a high frequency. Genetic approaches have led to the identification of various L. pneumophila genes. The mip (macrophage infectivity potentiator) determinant remains at present the sole established virulence factor. The Mip protein exhibits activity of a peptidyl prolyl cis trans isomerase (PPiase), an enzyme which is able to bind the immunosuppressant FK506 and is involved in protein folding. The recently cloned major outer membrane protein (MOMP) could play a role in the uptake of legionellae by macrophages. Cellular models are useful in studying the intracellular replication of legionellae in eukaryotic cells. Human celllines and protozoan models are appropriate for this purpose. By using U 937 macrophage-like cells and Acanthamoeba castellanii as hosts, we could discriminate virulent and avirulent L. pneumophila variants since only the virulent strain was capable of intracellular growth at 37 oc. By using these systems we further demonstrated that a hemolytic factor cloned and characterized in our laboratory, legiolysin (lly), had no influence on the intracellular growth of L. pneumophila.}, subject = {Legionella pneumophila}, language = {en} } @article{HackerOttBlumetal.1992, author = {Hacker, J{\"o}rg and Ott, Manfred and Blum, Gabriele and Marre, Reinhard and Heesemann, J{\"u}rgen and Tsch{\"a}pe, Helmut and Goebel, Werner}, title = {Genetics of Escherichia coli uropathogenicity: Analysis of the O6:K15:H31 isolate 536}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71578}, year = {1992}, abstract = {E. coli strain 536 (06: K15: H31) isolated from a case of acute pyelonephritis, expresses S-fimbrial adhesins, P-related fimbriae, common type I fimbriae, and hemolysins. The respective chromosomally encoded determinants were cloned by constructing a genomic library of this strain. Furthermore, the strain produces the iron uptake substance, enterocheline, damages HeLa cells, and behaves in a serum-resistant mode. Genetic analysis of spontaneously arising non-hemolytic variants revealed that some of the virulence genes were physically linked to large unstable DNA regions, termed "pathogenicity islands", which were mapped in the respective positions on the E. coli K-12linkage map. By comparing the wild type strain and mutants in in vitro and in vivo assays, virulence features have been evaluated. In addition, a regulatory cross talk between adhesin determinants was found for the wild-type isolate. This particular mode of virulence regulation is missing in the mutant strain.}, subject = {Escherichia coli}, language = {en} } @article{ParkkinenHackerKorhonen1991, author = {Parkkinen, Jaakko and Hacker, J{\"o}rg and Korhonen, Timo K.}, title = {Enhancement of tissue plasminogen activator-catalyzed plasminogen activation by Escherichia coli S fimbriae associated with neonatal septicaemia and meningitis.}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71566}, year = {1991}, abstract = {The effect of Escherichia coli strains isolated from blood and cerebrospinal fluid of septic infants on plasminogen activation was studied. These strains typically carry a filamentous surface protein, S fimbria, that has formerly been shown to bind to endothelial cells and interact with plasminogen. The bacteria effectively promoted plasminogen activation by tissue plasminogen activator (t-PA) which was inhibited by e-aminocaproic acid. A recombinant strain expressing S fimbriae accelerated t-PAcatalyzed plasminogen activation to a similar extent as did the wild-type strains whereas the nonfimbriate recipient strain had no effect. After incubation with t-PA and plasminogen, the S-fimbriate strain displayed bacterium-bound plasmin activity whereas the nonfimbriate strain did not. Bacterium-associated plasmin generation was also observed with a strain expressing mutagenized S fimbriae that Iack the cell-binding subunit SfaS but not with a strain lacking the major subunit SfaA. Both t-PA and plasminogen bound to purified S fimbriae in a lysine-dependent manner and purified S fimbriae accelerated t-PA-catalyzed plasminogen activation. The results indicate that E. coli S fimbriae form a complex with t-PA and plasminogen which enhances the rate of plasminogen activation and generates bacterium-bound plasmin. This may promote bacterial invasion and persistence in tissues and contribute to the systemic activation of fibrinolysis in septicaemia.}, subject = {Escherichia coli}, language = {en} } @article{MorschhaeuserRamirezZavalaWeyleretal.2013, author = {Morschh{\"a}user, Joachim and Ram{\´i}rez-Zavala, Bernardo and Weyler, Michael and Gildor, Tsvia and Schmauch, Christian and Kornitzer, Daniel and Arkowitz, Robert}, title = {Activation of the Cph1-Dependent MAP Kinase Signaling Pathway Induces White-Opaque Switching in Candida albicans}, series = {PLoS Pathogens}, journal = {PLoS Pathogens}, doi = {10.1371/journal.ppat.1003696}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-97281}, year = {2013}, abstract = {Depending on the environmental conditions, the pathogenic yeast Candida albicans can undergo different developmental programs, which are controlled by dedicated transcription factors and upstream signaling pathways. C. albicans strains that are homozygous at the mating type locus can switch from the normal yeast form (white) to an elongated cell type (opaque), which is the mating-competent form of this fungus. Both white and opaque cells use the Ste11-Hst7-Cek1/Cek2 MAP kinase signaling pathway to react to the presence of mating pheromone. However, while opaque cells employ the transcription factor Cph1 to induce the mating response, white cells recruit a different downstream transcription factor, Tec1, to promote the formation of a biofilm that facilitates mating of opaque cells in the population. The switch from the white to the opaque cell form is itself induced by environmental signals that result in the upregulation of the transcription factor Wor1, the master regulator of white-opaque switching. To get insight into the upstream signaling pathways controlling the switch, we expressed all C. albicans protein kinases from a tetracycline-inducible promoter in a switching-competent strain. Screening of this library of strains showed that a hyperactive form of Ste11 lacking its N-terminal domain (Ste11ΔN467) efficiently stimulated white cells to switch to the opaque phase, a behavior that did not occur in response to pheromone. Ste11ΔN467-induced switching specifically required the downstream MAP kinase Cek1 and its target transcription factor Cph1, but not Cek2 and Tec1, and forced expression of Cph1 also promoted white-opaque switching in a Wor1-dependent manner. Therefore, depending on the activation mechanism, components of the pheromone-responsive MAP kinase pathway can be reconnected to stimulate an alternative developmental program, switching of white cells to the mating-competent opaque phase.}, language = {en} } @article{SharmaDugarHerbigetal.2013, author = {Sharma, Cynthia M. and Dugar, Gaurav and Herbig, Alexander and F{\"o}rstner, Konrad U. and Heidrich, Nadja and Reinhardt, Richard and Nieselt, Kay}, title = {High-Resolution Transcriptome Maps Reveal Strain-Specific Regulatory Features of Multiple Campylobacter jejuni Isolates}, series = {PLoS Genetics}, journal = {PLoS Genetics}, doi = {10.1371/journal.pgen.1003495}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-96610}, year = {2013}, abstract = {Campylobacter jejuni is currently the leading cause of bacterial gastroenteritis in humans. Comparison of multiple Campylobacter strains revealed a high genetic and phenotypic diversity. However, little is known about differences in transcriptome organization, gene expression, and small RNA (sRNA) repertoires. Here we present the first comparative primary transcriptome analysis based on the differential RNA-seq (dRNA-seq) of four C. jejuni isolates. Our approach includes a novel, generic method for the automated annotation of transcriptional start sites (TSS), which allowed us to provide genome-wide promoter maps in the analyzed strains. These global TSS maps are refined through the integration of a SuperGenome approach that allows for a comparative TSS annotation by mapping RNA-seq data of multiple strains into a common coordinate system derived from a whole-genome alignment. Considering the steadily increasing amount of RNA-seq studies, our automated TSS annotation will not only facilitate transcriptome annotation for a wider range of pro- and eukaryotes but can also be adapted for the analysis among different growth or stress conditions. Our comparative dRNA-seq analysis revealed conservation of most TSS, but also single-nucleotide-polymorphisms (SNP) in promoter regions, which lead to strain-specific transcriptional output. Furthermore, we identified strain-specific sRNA repertoires that could contribute to differential gene regulation among strains. In addition, we identified a novel minimal CRISPR-system in Campylobacter of the type-II CRISPR subtype, which relies on the host factor RNase III and a trans-encoded sRNA for maturation of crRNAs. This minimal system of Campylobacter, which seems active in only some strains, employs a unique maturation pathway, since the crRNAs are transcribed from individual promoters in the upstream repeats and thereby minimize the requirements for the maturation machinery. Overall, our study provides new insights into strain-specific transcriptome organization and sRNAs, and reveals genes that could modulate phenotypic variation among strains despite high conservation at the DNA level.}, language = {en} } @article{SzalayWeibelHofmannetal.2013, author = {Szalay, Aladar A and Weibel, Stephanie and Hofmann, Elisabeth and Basse-Luesebrink, Thomas Christian and Donat, Ulrike and Seubert, Carolin and Adelfinger, Marion and Gnamlin, Prisca and Kober, Christina and Frentzen, Alexa and Gentschev, Ivaylo and Jakob, Peter Michael}, title = {Treatment of malignant effusion by oncolytic virotherapy in an experimental subcutaneous xenograft model of lung cancer}, series = {Journal of Translational Medicine}, journal = {Journal of Translational Medicine}, doi = {doi:10.1186/1479-5876-11-106}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-96016}, year = {2013}, abstract = {Background Malignant pleural effusion (MPE) is associated with advanced stages of lung cancer and is mainly dependent on invasion of the pleura and expression of vascular endothelial growth factor (VEGF) by cancer cells. As MPE indicates an incurable disease with limited palliative treatment options and poor outcome, there is an urgent need for new and efficient treatment options. Methods In this study, we used subcutaneously generated PC14PE6 lung adenocarcinoma xenografts in athymic mice that developed subcutaneous malignant effusions (ME) which mimic pleural effusions of the orthotopic model. Using this approach monitoring of therapeutic intervention was facilitated by direct observation of subcutaneous ME formation without the need of sacrificing mice or special imaging equipment as in case of MPE. Further, we tested oncolytic virotherapy using Vaccinia virus as a novel treatment modality against ME in this subcutaneous PC14PE6 xenograft model of advanced lung adenocarcinoma. Results We demonstrated significant therapeutic efficacy of Vaccinia virus treatment of both advanced lung adenocarcinoma and tumor-associated ME. We attribute the efficacy to the virus-mediated reduction of tumor cell-derived VEGF levels in tumors, decreased invasion of tumor cells into the peritumoral tissue, and to viral infection of the blood vessel-invading tumor cells. Moreover, we showed that the use of oncolytic Vaccinia virus encoding for a single-chain antibody (scAb) against VEGF (GLAF-1) significantly enhanced mono-therapy of oncolytic treatment. Conclusions Here, we demonstrate for the first time that oncolytic virotherapy using tumor-specific Vaccinia virus represents a novel and promising treatment modality for therapy of ME associated with advanced lung cancer.}, subject = {Lungenkrebs}, language = {en} } @phdthesis{Froehlich2012, author = {Fr{\"o}hlich, Kathrin}, title = {Assigning functions to Hfq-dependent small RNAs in the model pathogen Salmonella Typhimurium}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85488}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Non-coding RNAs constitute a major class of regulators involved in bacterial gene expression. A group of riboregulators of heterogeneous size and shape referred to as small regulatory RNAs (sRNAs) control trans- or cis-encoded genes through direct base-pairing with their mRNAs. Although mostly inhibiting their target mRNAs, several sRNAs also induce gene expression. An important co-factor for sRNA activity is the RNA chaperone, Hfq, which is able to rearrange intramolecular secondary structures and to promote annealing of complementary RNA sequences. In addition, Hfq protects unpaired RNA from degradation by ribonucleases and thus increases sRNA stability. Co-immunoprecipitation of RNA with the Hfq protein, and further experimental as well as bioinformatical studies performed over the last decade suggested the presence of more than 150 different sRNAs in various Enterobacteria including Escherichia coli and Salmonellae. So-called core sRNAs are considered to fulfill central cellular activities as deduced from their high degree of conservation among different species. Approximately 25 core sRNAs have been implicated in gene regulation under a variety of environmental responses. However, for the majority of sRNAs, both the riboregulators' individual biological roles as well as modes of action remain to be elucidated. The current study aimed to define the cellular functions of the two highly conserved, Hfq-dependent sRNAs, SdsR and RydC, in the model pathogen Salmonella Typhimurium. SdsR had been known as one of the most abundant sRNAs during stationary growth phase in E. coli. Examination of the conservation patterns in the sdsR promoter region in combination with classic genetic analyses revealed SdsR as the first sRNA under direct transcriptional control of the alternative σ factor σS. In Salmonella, over-expression of SdsR down-regulates the synthesis of the major porin OmpD, and the interaction site in the ompD mRNA coding sequence was mapped by a 3'RACE-based approach. At the post-transcriptional level, expression of ompD is controlled by three additional sRNAs, but SdsR plays a specific role in porin regulation during the stringent response. Similarly, RydC, the second sRNA adressed in this study, was initially discovered in E. coli but appeared to be conserved in many related γ-proteobacteria. An interesting aspect of this Hfq-dependent sRNAs is its secondary structure involving a pseudo-knot configuration, while the 5' end remains single stranded. A transcriptomic approach combining RydC pulse-expression and scoring of global mRNA changes on microarrays was employed to identify the targets of this sRNA. RydC specifically activated expression of the longer of two versions of the cfa mRNA encoding for the phospholipid-modifying enzyme cyclopropane fatty acid synthase. Employing its conserved single-stranded 5' end, RydC acts as a positive regulator and masks a recognition site of the endoribonuclease, RNase E, in the cfa leader.}, subject = {Small RNA}, language = {en} } @phdthesis{Ngwa2013, author = {Ngwa, Che Julius}, title = {The mosquito midgut-specific stages of the malaria parasite as targets for transmission blocking interventions}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-83594}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Die Tropenkrankheit Malaria, wird durch eine Infektion mit einzelligen Parasiten der Gattung Plasmodium verursacht und durch den Stich der weiblichen Anopheles-M{\"u}cke von Mensch zu Mensch verbreitet. Dabei kann eine erfolgreiche {\"U}bertragung des Parasiten auf den Menschen nur dann stattfinden, wenn der Parasit seine sexuelle Entwicklungsphase im Mitteldarm der M{\"u}cke erfolgreich durchl{\"a}uft. Ziel dieser Arbeit war es daher, die Wechselwirkungen des Malariaparasiten im Mitteldarm der M{\"u}cke in Hinblick auf die Identifizierung m{\"o}glicher neuer transmissionsblockierender Strategien zu untersuchen. Der Zweck von transmissionsblockierende Strategien ist es, der Verbreitung der Malaria durch die M{\"u}cke entgegenzuwirken, indem die Entwicklung des Parasiten in der M{\"u}cke unterbunden und dadurch der Lebenszyklus des Parasiten unterbrochen wird. Der Schwerpunkt der vorliegenden Arbeit lag auf insgesamt drei Aspekten. Der erste Aspekt der Arbeit befasste sich mit der Wechselwirkung zwischen dem Para-siten und der mikrobiellen Darmflora der M{\"u}cke. Dabei sollte der m{\"o}gliche Einfluss des Parasiten auf die Darmflora untersucht werden und weiterf{\"u}hrend die potentielle Verwendung von Darmbakterien als Vehikel f{\"u}r die Herstellung paratransgener M{\"u}cken erforscht werden. Vergleichende16S-rRNA- und DGGE-Analysen an der Darmflora des asiatischen Malariavektors Anopheles stephensi zeigten eine deutliche Reduktion der mikrobiellen Diversit{\"a}t w{\"a}hrend der Entwicklung vom Ei zur adulten M{\"u}cke. Zudem konnte das gram-negative Bakterium Elizabethkingia meningoseptica, das sich stadien- und generations{\"u}bergreifend verbreitet, als dominante Darmspezies bei im Labor aufgezogenen weiblichen und m{\"a}nnlichen An. stephensi festgestellt werden. Die Dominanz von E. meningoseptica wurde zudem nicht durch die Aufnahme von infiziertem Blut oder einer ver{\"a}nderten Nahrung beeinflusst. F{\"u}r die Studien wurde sowohl der humanpathogene Parasit P. falciparum als auch der Nagermalariaerreger P. berghei verwendet. Weiterf{\"u}hrende Versuche zeigten, dass Extrakte von E. meningoseptica antibakterielle, antifungale und antiplasmodiale Aktivit{\"a}ten aufwiesen, die ein m{\"o}glicher Grund f{\"u}r die Dominanz dieser Spezies im Mitteldarm des Vektors waren. Isolate von E. meningoseptica sind im Labor kultivierbar; dadurch stellt das Bakterium einen potentiellen Kandidaten zur Generierung von paratransgenen Anopheles-M{\"u}cken dar. Ein zweites Ziel dieser Arbeit war es, m{\"o}gliche Unterschiede in der Genexpression von P. falciparum darzustellen, die in den ersten 30 Minuten nach dessen {\"U}bertragung auf die M{\"u}cke erfolgen. Dies hatte zum einen zum Zweck, die durch den Wirtswechsel hervorgerufenen Genregulationen besser zu verstehen, und bot zum anderen die M{\"o}glichkeit, neue Proteine zu identifizieren, die als potentielle transmissionsblockierende Ziele genutzt werden k{\"o}nnen. Mittels supression substractive hybridization (SSH) konnten insgesamt 126 Gene identifiziert werden, deren Expression sich w{\"a}hrend der Gametogenese ver{\"a}ndert. Die identifizierten Gene konnten einer Vielzahl von putativen Funktionen wie zum Beispiel in der Signaltransduktion (17,5\%), im Zellzyklus (14,3\%) oder im Zytoskelett (8,7\%) zugeordnet werden. Des Weiteren wurden 7,9\% der Gene eine Funktion in der Proteastase und 6,4\% in metabolischen Prozessen zugeordnet. 12,7\% der Gene kodierten f{\"u}r zelloberfl{\"a}chenassoziierte Proteine. 11,9\% der Gene hatten anderen Funktionen, w{\"a}hrend 20\% der Gene keine putative Funktion zugeordnet werden konnte. Etwa 40\% der identifizierten Genprodukte waren bisher nicht in Proteomstudien nachgewiesen worden. In weiterf{\"u}hrenden Analysen wurden 34 Gene aus jeder ontologischen Gruppe ausgew{\"a}hlt und deren Expressionsver{\"a}nderung per quantitativer real time RT-PCR im Detail untersucht. F{\"u}r 29 Gene konnte dabei eine Transkriptexpression in Gametozyten nachgewiesen werden. Zudem wiesen 20 Gene eine erh{\"o}hte Expression in Gametozyten im Vergleich asexuellen Stadien auf. Insgesamt zeigten 8 Gene besonders hohe Transkriptlevel in aktivierten Gametozyten, was auf eine Funktion dieser Proteine w{\"a}hrend der {\"U}bertragung des Parasiten auf die M{\"u}cke hindeutet und diese somit potentielle Angriffspunkte f{\"u}r transmissionsblockierende Strategien darstellen k{\"o}nnten. Im letzten Teil dieser Arbeit stand die Untersuchung verschiedener antimikrobieller Substanzen in Bezug auf ihre transmissionsblockierenden Eigenschaften im Vordergrund. Die Substanzen waren entweder direkt aus der H{\"a}molymphe verschiedener Insekten isoliert oder rekombinant in transgenem Tabak exprimiert worden. Dabei wurden die rekombinanten Peptide so ausgew{\"a}hlt, dass sie entweder gegen die Mitteldarmstadien des Parasiten wirken oder m{\"u}ckenspezifische Rezeptoren blockieren, die der Parasit f{\"u}r seine weitere Entwicklung ben{\"o}tigt. Dabei konnte gezeigt werden, dass das antimikrobielle Molek{\"u}l Harmonin, ein Abwehrmolek{\"u}l aus der H{\"a}molymphe des asiatischen Marienk{\"a}fers Harmonia axyridis, antiplasmodiale als auch transmissions-blockierende Eigenschaften besitzt. Harmonin stellt daher eine potentielle Leitstruktur f{\"u}r die Entwicklung neuer Malariawirkstoffe dar}, subject = {Malariam{\"u}cke}, language = {en} } @phdthesis{Dunkel2013, author = {Dunkel, Nico}, title = {Regulation of virulence-associated traits of the human fungal pathogen Candida albicans by nitrogen availability}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-83076}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Nitrogen-regulated pathogenesis describes the expression of virulence attributes as direct response to the quantity and quality of an available nitrogen source. As consequence of nitrogen availability, the opportunistic human fungal pathogen Candida albicans changes its morphology and secretes aspartic proteases [SAPs], both well characterized virulence attributes. C. albicans, contrarily to its normally non-pathogenic relative Saccharomyces cerevisiae, is able to utilize proteins, which are considered as abundant and important nitrogen source within the human host. To assimilate complex proteinaceous matter, extracellular proteolysis is followed by uptake of the degradation products through dedicated peptide transporters (di-/tripeptide transporters [PTRs] and oligopeptide transporters [OPTs]). The expression of both traits is transcriptionally controlled by Stp1 - the global regulator of protein utilization - in C. albicans. The aim of the present study was to elucidate the regulation of virulence attributes of the pathogenic fungus C. albicans by nitrogen availability in more detail. Within a genome wide binding profile of Stp1, during growth with proteins, more than 600 Stp1 target genes were identified, thereby confirming its role in the usage of proteins, but also other nitrogenous compounds as nitrogen source. Moreover, the revealed targets suggest an involvement of Stp1 in the general adaption to nutrient availability as well as in the environmental stress response. With the focus on protein utilization and nitrogen-regulated pathogenesis, the regulation of the major secreted aspartic protease Sap2 - additionally one of the prime examples of allelic heterogeneity in C. albicans - was investigated in detail. Thereby, the heterogezygous SAP2 promoter helped to identify an unintended genomic alteration as the true cause of a growth defect of a C. albicans mutant. Additionally, the promoter region, which was responsible for the differential activation of the SAP2 alleles, was delimited. Furthermore, general Sap2 induction was demonstrated to be mediated by distinct cis-acting elements that are required for a high or a low activity of SAP2 expression. For the utilization of proteins as nitrogen source it is also crucial to take up the peptides that are produced by extracellular proteolysis. Therefore, the function and importance of specific peptide transporters was investigated in C. albicans mutants, unable to use peptides as nitrogen source (opt1Δ/Δ opt2Δ/Δ opt3Δ/Δ opt4Δ/Δ opt5Δ/Δ ptr2Δ/Δ ptr22Δ/Δ septuple null mutants). The overexpression of individual transporters in these mutants revealed differential substrate specificities and expanded the specificity of the OPTs to dipeptides, a completely new facet of these transporters. The peptide-uptake deficient mutants were further used to elucidate, whether indeed proteins and peptides are an important in vivo nitrogen source for C. albicans. It was found that during competitive colonization of the mouse intestine these mutants exhibited wild-type fitness, indicating that neither proteins nor peptides are primary nitrogen sources required to efficiently support growth of C. albicans in the mouse gut. Adequate availability of the preferred nitrogen source ammonium represses the utilization of proteins and other alternative nitrogen sources, but also the expression of virulence attributes, like Sap secretion and nitrogen-starvation induced filamentation. In order to discriminate, whether ammonium availability is externally sensed or determined inside the cell by C. albicans, the response to exterior ammonium concentrations of ammonium-uptake deficient mutants (mep1Δ/Δ mep2Δ/Δ null mutants) was investigated. This study showed that presence of an otherwise suppressing ammonium concentration did not inhibit Sap2 proteases secretion and arginine-induced filamentation in these mutants. Conclusively, ammonium availability is primarily determined inside the cell in order to control the expression of virulence traits. In sum, the present work contributes to the current understanding of how C. albicans regulates expression of virulence-associated traits in response to the presence of available nitrogen sources - especially proteins and peptides - in order to adapt its lifestyle within a human host.}, subject = {Candida albicans}, language = {en} } @article{SchulteWestermannVogel2012, author = {Schulte, Leon N. and Westermann, Alexander J. and Vogel, J{\"o}rg}, title = {Differential activation and functional specialization of miR-146 and miR-155 in innate immune sensing}, edition = {Advance Access}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-76365}, year = {2012}, abstract = {Many microRNAs (miRNAs) are co-regulated during the same physiological process but the underlying cellular logic is often little understood. The conserved, immunomodulatory miRNAs miR-146 and miR-155, for instance, are co-induced in many cell types in response to microbial lipopolysaccharide (LPS) to feedback-repress LPS signalling through Toll-like receptor TLR4. Here, we report that these seemingly co-induced regulatory RNAs dramatically differ in their induction behaviour under various stimuli strengths and act non-redundantly through functional specialization; although miR-146 expression saturates at sub-inflammatory doses of LPS that do not trigger the messengers of inflammation markers, miR-155 remains tightly associated with the pro-inflammatory transcriptional programmes. Consequently, we found that both miRNAs control distinct mRNA target profiles; although miR-146 targets the messengers of LPS signal transduction components and thus downregulates cellular LPS sensitivity, miR-155 targets the mRNAs of genes pervasively involved in pro-inflammatory transcriptional programmes. Thus, miR-155 acts as a broad limiter of pro-inflammatory gene expression once the miR-146 dependent barrier to LPS triggered inflammation has been breached. Importantly, we also report alternative miR-155 activation by the sensing of bacterial peptidoglycan through cytoplasmic NOD-like receptor, NOD2. We predict that dosedependent responses to environmental stimuli may involve functional specialization of seemingly coinduced miRNAs in other cellular circuitries as well.}, subject = {Medizin}, language = {en} } @article{PernitzschSharma2012, author = {Pernitzsch, Sandy R. and Sharma, Cynthia M.}, title = {Transcriptome Complexity and Riboregulation in the Human Pathogen Helicobacter pylori}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75096}, year = {2012}, subject = {Medizin}, language = {en} } @phdthesis{Masic2012, author = {Masic, Anita}, title = {Signaling via Interleukin-4 Receptor alpha chain during dendritic cell-mediated vaccination is required to induce protective immunity against Leishmania major in susceptible BALB/c mice}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75508}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Cutaneous leishmaniasis is endemic in tropical and subtropical regions of the world. Effective vaccination strategies are urgently needed because of the emergence of drug-resistant parasites and severe side effects of chemotherapy. The research group of Heidrun Moll previously established a DC-based vaccination strategy to induce complete and long-lasting immunity to experimental leishmaniasis using LmAg-loaded and CpG ODN-activated DC as a vaccine carrier. Prevention of tissue damages at the site of L. major inoculation can be achieved if the BALB/c mice were systemically given LmAg-loaded BMDC that had been exposed to CpG ODN. The interest in further exploring the role of IL-4 aroused as previous studies allowed establishing that IL-4 was involved in the redirection of the immune response towards a type 1 profile. Thus, wt BALB/c mice or DC-specific CD11ccreIL-4Rα-/lox BALB/c mice were given either wt or IL-4Rα-deficient LmAg-loaded BMDC exposed or not to CpG ODN prior to inoculation of 2 x 105 stationary phase L. major promastigotes into the BALB/c footpad. The results provide evidence that IL4/IL-4Rα-mediated signaling in the vaccinating DC is required to prevent tissue damages at the site of L. major inoculation, as properly conditioned wt DC but not IL-4Rα-deficient DC were able to confer resistance. Furthermore, uncontrolled L. major population size expansion was observed in the footpad and the footpad draining LN in CD11ccreIL-4Rα-/lox mice immunized with CpG ODN-exposed LmAg-loaded IL-4Rα-deficient DC, indicating the influence of IL-4R-mediated signaling in host DC to control parasite replication. In addition, no footpad damage was observed in BALB/c mice that were systemically immunized with LmAg-loaded wt DC doubly exposed to CpG ODN and recombinant IL-4. Discussing these findings allow the assumption that triggering the IL4/IL4Rα signaling pathway could be a precondition when designing vaccines aimed to prevent damaging processes in tissues hosting intracellular microorganisms.}, subject = {Leishmania major}, language = {en} } @phdthesis{Schnitzer2012, author = {Schnitzer, Johannes K.}, title = {Mechanism of dendritic cell-based vaccination against Leishmania major}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-74865}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Die Impfung mittels Antigen-beladener dendritischer Zellen [DZ] ist mittlerweile eine gut etablierte Technik, die dann zum Einsatz kommt, wenn Standard-Impftechniken versagen, vor Krankheiten zu sch{\"u}tzen beziehungsweise diese zu heilen. Die Effizienz dieser Technik konnte bereits f{\"u}r diverse Infektionskrankheiten und Krebserkrankungen in experimentellen Tiermodellen sowie am Menschen gezeigt werden. Hierbei ist die M{\"o}glichkeit zur wohldefinierten Manipulation und Antigenbeladung der DZ ein großer Vorteil gegen{\"u}ber den konventionellen Ans{\"a}tzen. Jedoch ist vor allem bei der Anwendung im klinischen Bereich die Pr{\"a}paration, Herstellung und Manipulation dieser autologen DZ mit einem erheblichen technischen, zeitlichen sowie finanziellen Aufwand verbunden. Hinsichtlich einer Pr{\"a}ventivimpfung gegen eine pandemische Infektionskrankheit, die in haupts{\"a}chlich unterentwickelten L{\"a}ndern vorkommt, wird dieser Aufwand sicherlich ein Hindernis darstellen. Daher muss f{\"u}r solche F{\"a}lle ein maßgeschneiderter Impfstoff entwickelt werden, der sich am Vorbild des effektiven DZ-basierten Impfstoffs orientiert. F{\"u}r die Impfung gegen die Leishmania Parasiten besteht so ein DZ-basierter Impfstoff bereits. Dessen Wirkung, eine T-Zell Antwort vom Typ Th1 zu induzieren, wurde bereits in mehreren Ver{\"o}ffentlichungen demonstriert. Zus{\"a}tzlich hat aber eine unserer Studien gezeigt, dass das typische Th1-bezogene Zytokin IL-12 zur Differenzierung naiver T-Zellen nicht von den injizierten DZ bereitgestellt werden muss, sondern von der geimpften Maus. Dies gab erste Hinweise auf eine st{\"a}rkere Beteiligung des Wirts-Immunsystems als zuvor angenommen. Daher sollte hier vertieft der Mechanismus dieser DZ-basierten Impfung untersucht werden, wobei modifizierte Impfstoff-Ans{\"a}tze zum Einsatz kommen sollten. Dabei wurden die Fragen nach der vom Impfstoff transportierten Information und dem Empf{\"a}nger dieser Information ber{\"u}cksichtigt. Das aktuelle Paradigma zur DZ-basierten Impfung besagt, dass transferierte DZ im direkten Kontakt mittels dreier Signale T-Zellen stimulieren und aktivieren. Daf{\"u}r m{\"u}ssen diese DZ mit dem entsprechenden Antigen beladen und aktiviert worden sein um das Antigen-Peptide mittels MHC Molek{\"u}l im Kontext der Co-Stimulation pr{\"a}sentieren zu k{\"o}nnen. Jedoch zeigt diese Studie hier, dass weder eine Aktivierung der DZ noch die Pr{\"a}sentation des Antigens mittels passender MHC Molek{\"u}le notwendig ist f{\"u}r die Induktion einer protektiven Immunantwort gegen Leishmania Parasiten. Aufgeschlossene, mit Antigen beladene DZ m{\"u}ssen nicht vor dem Transfer mit CpG ODN aktiviert worden sein, um entsprechende Immunit{\"a}t zu verleihen. Ebenso hat der MHC Typ in diesem Falle auch keinen Einfluss auf die Effektivit{\"a}t des Impfstoffs. Da im Weiteren aufgeschlossene mit Leishmania-Antigen beladene Makrophagen nach Impfung die gleiche Wirkung erzielen, wie vorangegangene DZ-basierte Impfstoffe, k{\"o}nnen keine DZ spezifischen Mechanismen Schl{\"u}sselkomponenten der Induktion einer protektiven Immunit{\"a}t sein. Dar{\"u}ber hinaus konnte gezeigt werden, dass die DZ der geimpften M{\"a}use, eine maßgebliche Rolle bei der Verarbeitung transferierter Signale spielen. Suspensionen aufgeschlossener DZ stellen eine Kombination aus freigesetzten l{\"o}slichen Molek{\"u}len sowie Membranvesikeln dar, die sich nach dem Aufschluss gebildet haben. Nach Auftrennung dieser beiden Fraktionen konnte gezeigt werden, dass ausschließlich die Membran-Fraktion nach Verimpfung eine geeignete Immunantwort zum Schutz vor Leishmania Parasiten induzieren kann. Als Vorteil dieser Aufreinigung erweist sich zudem die stabile Lagerm{\"o}glichkeit bei -80°C. Somit ist klar gezeigt, dass die Immunit{\"a}t-verleihende Einheit dieser Impfstoffvarianten in der Membran-Fraktion liegt. Verfolgt man die Induktion Th1-zugeh{\"o}riger Zytokine in in vivo Experimenten so ergibt sich im Falle der Gesamtsuspension aufgeschlossener, mit Leishmania-Antigen beladener DZ ein klares Bild. Diese Suspension erzeugt das volle Spektrum der DZ-basierten Impfung gegen Leishmania Parasiten. Es kann sowohl Produktion von IL-12 und IL-2 als auch eine antigenspezifische T-Zell Proliferation nach Stimulation von Splenozyten mit der entsprechenden Suspension verzeichnet werden. Außerdem produzieren Splenozyten von entsprechend geimpften M{\"a}usen nach Stimulation mit Leishmania-Antigen erhebliche Mengen des entscheidenden Zytokins IFNγ. Obwohl jedoch die Verimpfung aufgereinigter Membranvesikel dieses Ansatzes im Tierversuch zu biologisch sowie statistisch signifikanten Ergebnissen f{\"u}hrt, lassen sich die entsprechend Th1-bezogenen Zytokine im in vivo Ansatz nur in geringen Maße nachweisen. Ob dies jedoch f{\"u}r einen in vivo unbemerkten Aktivit{\"a}tsverlust des Vakzins oder f{\"u}r andere lymphatische Organe als Ort der T-Zell Instruktion spricht, ist noch unbekannt und muss noch gekl{\"a}rt werden.}, subject = {Leishmania major}, language = {en} } @article{HessStritzkerHaertletal.2011, author = {Hess, Michael and Stritzker, Jochen and H{\"a}rtl, Barbara and Sturm, Julia and Gentschev, Ivaylo and Szalay, Aladar}, title = {Bacterial glucuronidase as general marker for oncolytic virotherapy or other biological therapies}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69163}, year = {2011}, abstract = {Background: Oncolytic viral tumor therapy is an emerging field in the fight against cancer with rising numbers of clinical trials and the first clinically approved product (Adenovirus for the treatment of Head and Neck Cancer in China) in this field. Yet, until recently no general (bio)marker or reporter gene was described that could be used to evaluate successful tumor colonization and/or transgene expression in other biological therapies. Methods: Here, a bacterial glucuronidase (GusA) encoded by biological therapeutics (e.g. oncolytic viruses) was used as reporter system. Results: Using fluorogenic probes that were specifically activated by glucuronidase we could show 1) preferential activation in tumors, 2) rena l excretion of the activated fluorescent compounds and 3) reproducible detection of GusA in the serum of oncolytic vaccinia virus treated, tumor bearing mice in several tumor models. Time course studies revealed that reliable differentiation between tumor bearing and healthy mice can be done as early as 9 days post injection of the virus. Regarding the sensitivity of the newly developed assay system, we could show that a single infected tumor cell could be reliably detected in this assay. Conclusion: GusA therefore has the potential to be used as a general marker in the preclinical and clinical evaluation of (novel) biological therapies as well as being useful for the detection of rare cells such as circulating tumor cells}, subject = {Virologie}, language = {en} } @article{KuehnPradel2010, author = {Kuehn, Andrea and Pradel, Gabriele}, title = {The Coming-Out of Malaria Gametocytes [Review Article]}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68196}, year = {2010}, abstract = {The tropical disease malaria, which results in more than one million deaths annually, is caused by protozoan parasites of the genus Plasmodium and transmitted by blood-feeding Anopheline mosquitoes. Parasite transition from the human host to the mosquito vector is mediated by gametocytes, sexual stages that are formed in human erythrocytes, which therefore play a crucial part in the spread of the tropical disease. The uptake by the blood-feeding mosquito triggers important molecular and cellular changes in the gametocytes, thus mediating the rapid adjustment of the parasite from the warm-blooded host to the insect host and subsequently initiating reproduction. The contact with midgut factors triggers gametocyte activation and results in their egress from the enveloping erythrocyte, which then leads to gamete formation and fertilization. This review summarizes recent findings on the role of gametocytes during transmission to themosquito and particularly focuses on the molecular mechanisms underlying gametocyte activation and emergence from the host erythrocyte during gametogenesis.}, subject = {Malaria}, language = {en} } @article{HackerRdestWintermeyeretal.1991, author = {Hacker, J{\"o}rg and Rdest, Ursula and Wintermeyer, E. and Ludwig, B.}, title = {Legiolysin, a New Hemolysin from L. pneumophila}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73070}, year = {1991}, abstract = {Legionella pneumophila generares exotoxins, cytolysins, proteases oc hemolysins that darnage host cells llke erythrocytes or rissue cu lrure cells. The gene for a new L. pneumophila hemolysin withour a proteolytic activiry was idemified, cloned in E. coli and sequenced. The gene producr was analysed by SDS-Polyacrylamide-gel-electrophoresis.}, subject = {H{\"a}molysin}, language = {en} } @article{HackerSchrettenbrunnerSchroeteretal.1986, author = {Hacker, J{\"o}rg and Schrettenbrunner, A. and Schr{\"o}ter, G. and Schmidt, G. and D{\"u}vel, H. and Goebel, W.}, title = {Characterization of Escherichia coli wild-type strains by means of agglutination with antisera raised against cloned P-, S- and MS-fimbriae antigens, hemagglutination, serotyping and hemolysin-production}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72992}, year = {1986}, abstract = {E. coli stcains isolated from patients with urinary tcact infecrions (UTn very often possess mannose"sensitive (MS) and mannose-resistant (MR) adherence facmrs (fimbriae). According to their receptor specificity the mannose-resistant adhesins can be divided inm several types, P, S, M and X. We have cloned rhe determinants of rhree groups of UTI E. coli adhesins, MS, p and S, and prepared specific aorisera against the fimbriae antigens. 189 hernagglutination (HA+) -positive stcains, 96 fecal isolates and 93 strains isoJated from UTI . have been tesred with rhese specific antisera and further characterized by receptor specific : HA, HA parteras and further of rhe "common 0 serogroups" 01, 02, 04, 06, 07, 08, 018, ' 025, 075, most prevalenr in UTI, and hemolysin production. · 68 (73 \%) of the UTI srrains a.nd 50 (52\%) of the fecal isolates showed P-receptor specificiry; 16 (17\%) of the uropathogenic bacteria and 33 (34\%) of the fecal strains exhibited S, M or X-fimbriae antigens. 24\% of rhe P-hemagglutinating (P+) strains reacted wirb P (F8)-specific antiserum. In contrast, more than three quaner of the s+-srrains were agglutinated by S-specific antiserum. HA-pattern VJ and 018 amigen were found to be associared with P-fimbriae strains, wbereas HA-pattern V and VII and the 0 anrigens 02 (M-type), 06 and 018 (5-type) occurred most frequently in p- -strains. A high percentage of P-fimbriated strains showed mannose-sensitive hemagglurination and hemolysin production.}, subject = {Escherichia coli}, language = {en} } @article{HackerGadebergOrskov1989, author = {Hacker, J{\"o}rg and Gadeberg, Ole V. and Orskov, Ida}, title = {Role of alpha-Hemolysin for the in vitro Phagocytosis and intracellular killing of Escherichia coli}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73019}, year = {1989}, abstract = {The_role of a-hemolysin for the elimination of Eschericbia coli by phagocyres in vitro was investigated using sets of isogenic strains which included wild-type a -hemolyric srrains, derived strains with a reduced production of a-hemolysin and derived nonhemolytic strains. Phagocyrosis and intracellular killing of the bacteria by human blood granulocytes or monocytes were measured using growth inhibition rechniques. a-hemolytic strains were phagocytosed and killed ro a Jesser extent than isogenic strains with a reduced production of o:hemoJysin and isogenic nonhemolytic strains. The results obrained with granulocyres were similar to rhose obtained with monocyres although the elimination of bacteria by monocytes was less than that by granulocytes. These resulcs strongJy suggest that production of ahemolysin is a means by which E. coli counteracrs the activity of phagocytes by injuring these cells with the toxin.}, subject = {Escherichia coli}, language = {en} } @article{HackerUlmerFasskeetal.1987, author = {Hacker, J{\"o}rg and Ulmer, E. and Fasske, E. and Schmidt, G.}, title = {Isolation and characterization of coliphage Omega18A specific for Escherichia coli O18ac strains}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73001}, year = {1987}, abstract = {The bactedophage Q18A, specific for Escherichia coli 018ac srrains, was isolated frorn sewage. The results of host range and conjugation experiments showed that the sensitivity of bacteria to the phage is associated with rhe presence of 018ac antigens. With sorne of rhe 018 strains rhe phage Q18A produces clear Iysis on bacterial lawns only when applied at a high multiplicity and moreover the phage does not multiply. With rhe help of the phage Ql8A, E. coli 0 18ac strains could be divided inro rwo serologically clistinct subgroups called 018A and 018A1• E. coli strains belanging to the sugroup 0 ISAare sensitive to phage Q t8A wheteas bacteria of subgroup A1 are resistanr.}, subject = {Escherichia coli}, language = {en} } @phdthesis{Simon2012, author = {Simon, Nina Monica}, title = {Molecular interactions of the malaria parasite Plasmodium falciparum during the sexual reproduction in the mosquito midgut}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72403}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {The sexual phase of Plasmodium falciparum begins with the differentiation of intraerythrocytic sexual stages, termed gametocytes, in the human host. Mature gametocytes circulate in the peripheral blood and are taken up by the mosquito during the blood meal. These stages are essential for the spread of the malaria disease and form gametes in the mosquito midgut within minutes. A highly conserved family of six secreted proteins has been identified in Plasmodium falciparum. They comprise multiple adhesive domains and are termed PfCCp1 through PfCCp5, and PfFNPA. It was revealed in this work that PfCCp multi-domain adhesion proteins form protein complexes in gametocytes and on the surface of newly emerged macrogametes by adhesion domain-mediated binding. Co-Immunoprecipitation assays with activated gametocyte lysates show interactions between PfCCp proteins and indicate surface association via Pfs230 and Pfs25. Pfs230 is connected with the plasma membrane of the parasite by its interaction partner Pfs48/45. This protein is linked to the plasma membrane by a GPI anchor and presumably retains the multi-protein complex on the surface of newly emerged macrogametes in the mosquito midgut. A WD40 domain containing protein was identified to be part of this protein complex. It might serve as platform for the assembly of the multi protein complex or mediate the interplay among proteins, as suggested from known functions of the WD40 domain repeats. During egress from the host erythrocyte, the emerging gametes become vulnerable to factors of the human complement, which is taken up with the blood meal. In this thesis it was found that the complement system is active for about one hour post feeding. Macrogametes defend against complement-mediated lysis by co-opting the human complement regulators Factor H and FHL-1 from the blood-meal. These serum proteins bind via its SCR domains 5-7 to the surface of macrogametes. Once bound, they trigger complement inactivation of the alternative pathway, which prevents induction of complement lysis on the surface of the malaria parasite. Antibodies against Factor H are able to impair the sexual development in vitro and are able to block transmission to the mosquito. Interaction studies on endogenous proteins and immobilized recombinant proteins revealed the PfGAP50 protein as binding partner of Factor H and FHL-1. This protein was hitherto described as a glideosome-associated protein in invasive parasite stages, but has not yet been characterized in gametes. First localization studies indicate a relocation of PfGAP50 from the inner membrane complex to the surface of macrogametes. Malaria still persists as one of the deadliest infectious diseases worldwide. Investigations on the essential transmissive stages, gametocytes and gametes of Plasmodium falciparum, stood in the background of research for a long time. This work deciphered details on protein interactions on the surface of the malaria parasite and provides first information about coactions between the parasite and the human complement in the mosquito midgut.}, subject = {Plasmodium falciparum}, language = {en} } @phdthesis{Angermeier2011, author = {Angermeier, Hilde Gabriele}, title = {Molecular and ecological investigations of Caribbean sponge diseases}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-56855}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {W{\"a}hrend gewinnbringende Assoziationen von Schw{\"a}mmen mit Mikroorganismen in den letzten Jahren viel Aufmerksamkeit erhalten haben, wurde weit weniger in die Interaktion von Schw{\"a}mmen mit m{\"o}glicherweise pathogenen Mikroben investiert. Somit war es das Ziel dieser Studie zwei ausgew{\"a}hlte Karibische Schwammkrankheiten namens „Sponge Orange Band" und „Sponge White Patch" mittels {\"o}kologischer und molekularer Methoden zu untersuchen. Die Sponge Orange Band (SOB) Erkrankung bef{\"a}llt den bedeutenden karibischen Fass-Schwamm Xestospongia muta, der zu den bakterienhaltigen (HMA) Schw{\"a}mmen gez{\"a}hlt wird, w{\"a}hrend die Sponge White Patch (SWP) Erkrankung den h{\"a}ufig vorkommenden Seil-Schwamm Amphimedon compressa betrifft, der zu den bakterienarmen (LMA) Schw{\"a}mmen geh{\"o}rt. F{\"u}r beide Karibischen Schwammkrankheiten konnte ich einen Krankheitsverlauf beschreiben, der mit massiver Gewebszerst{\"o}rung und dem Verlust charakteristischer mikrobieller Signaturen einhergeht. Obwohl ich zeigen konnte, dass zus{\"a}tzliche Bakterienarten die gebleichten Schwammbereiche kolonisieren, lieferten meine Infektionsversuche in beiden F{\"a}llen keinen Beweis f{\"u}r die Beteiligung eines mikrobiellen Pathogens als Krankheitserreger. Somit liegen die eigentlichen Ausl{\"o}ser der Erkrankungen Sponge Orange Band als auch Sponge White Patch noch immer im Dunkeln.}, subject = {Meeresschw{\"a}mme}, language = {en} } @phdthesis{Seo2012, author = {Seo, Ean Jeong}, title = {Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72005}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Der probiotische Escherichia coli Stamm Nissle 1917 (EcN) ist eines der wenigen Probiotika, die als aktive Komponente eines Medikaments in mehreren L{\"a}ndern zugelassen sind. Am besten ist die Wirksamkeit des EcN f{\"u}r die Remissionserhaltung von an Colitis Ulcerosa leidenden Patienten dokumentiert. Diese F{\"a}higkeit ist vermutlich darauf zur{\"u}ckzuf{\"u}hren, dass EcN in der Lage ist die Produktion des humanen beta-Defensins 2 (HBD2) mittels seiner Flagelle zu Induzieren. In dieser Studie wurden rekombinante EcN St{\"a}mme konstruiert, die ein Defensin zu produzieren verm{\"o}gen. Zu diesem Zweck wurden Kodon-optimierte Defensingene in Expressionsplasmidvektoren kloniert, die entweder die Proform mit der Signalsequenz oder die reife Defensinform des humanen -Defensins 5 (HD5) oder des humanen -Defensins 2 (HBD2) unter der Kontrolle des T7-Promotors kodieren. Die Synthese dieser Defensine wurde mittels Western-Blot nach der Induktion der Expression und der Lyse der rekombinanten EcN St{\"a}mme demonstriert. Das rekombinante reife HBD2 mit einem N-terminalen His-Tag konnte mittels Ni-S{\"a}ulen-Chromatographie aufgereinigt werden. Das so gewonnene HBD2 zeigte antimikrobielle Aktivit{\"a}t gegen E. coli, Salmonella enterica Serovar Typhimurium und Listeria monocytogenes. In einem zweiten Ansatz wurde der Teil des HBD2-Gens mit dem yebF-Gen fusioniert, der das reife HBD2 kodiert. Das resultierende Fusionsprotein YebFMHBD2 wurde von dem entsprechenden EcN Stamm nach Induktion der Expression sekretiert. Die Pr{\"a}senz von YebFMHBD2 im Medium war nicht das Ergebnis von Zellyse wie Western-Blots spezifisch f{\"u}r die -Galaktosidase und das Maltose-Bindeprotein mit dem Kultur{\"u}berstand zeigten. Dieser Kultur{\"u}berstand inhibierte das Wachstum von E. coli, Salmonella enterica Serovar Typhimurium und Listeria monocytogenes nach Dialyse und Aufkonzentration sowohl in Agardiffusionsassays als auch in Fl{\"u}ssigcokultur. Damit konnte gezeigt werden, dass EcN ein f{\"u}r die Produktion von bestimmten humanen Defensinen geeignetes Probiotikum darstellt. EcN ist bei der Behandlung von Morbus Crohn Patienten nicht aktiv. Dies ist vermutlich in der genetisch bedingten Unf{\"a}higkeit zur ausreichenden Defensinproduktion solcher Individuen begr{\"u}ndet. Als ein erster Schritt in der Entwicklung von alternativen Ans{\"a}tzen zur Behandlung Morbus Crohn Patienten wurden in dieser Arbeit EcN St{\"a}mme konstruiert, die in der Lage sind HD5 oder HBD2 zu produzieren.}, subject = {Escherichia coli}, language = {en} } @phdthesis{Aminake2012, author = {Aminake, Makoah Nigel}, title = {Towards malaria combination therapy: Characterization of hybrid molecules for HIV/malaria combination therapy and of thiostrepton as a proteasome-targeting antibiotic with a dual mode of action}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71841}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Malaria and HIV are among the most important global health problems of our time and together are responsible for approximately 3 million deaths annually. These two diseases overlap in many regions of the world including sub-Saharan Africa, Southeast Asia and South America, leading to a higher risk of co-infection. In this study, we generated and characterized hybrid molecules to target P. falciparum and HIV simultaneously for a potential HIV/malaria combination therapy. Hybrid molecules were synthesized by covalent fusion between azidothymidine (AZT) and dihydroartemisinin (DHA), tetraoxane or chloroquine (CQ); and a small library was generated and tested for antiviral and antimalarial activity. Our data suggest that dihyate is the most potent molecule in vitro, with antiplasmodial activity comparable to that of DHA (IC50 = 26 nM, SI > 3000), a moderate activity against HIV (IC50 = 2.9 µM; SI > 35) and safe to HeLa cells at concentrations used in the assay (CC50 > 100 µM). Pharmacokinetic studies further revealed that dihyate is metabolically unstable and is cleaved following an O-dealkylation once in contact with cytochrome P450 enzymes. The later further explains the uneffectiveness of dihyate against the CQ-sensitive P. berghei N strain in mice when administered by oral route at 20 mg/kg. Here, we report on a first approach to develop antimalarial/anti-HIV hybrid molecules and future optimization efforts will aim at producing second generation hybrid molecules to improve activity against HIV as well as compound bioavailability. With the emergence of resistant parasites against all the counterpart drugs of artemisinin derivatives used in artemisinin based combination therapies (ACTs), the introduction of antibiotics in the treatment of malaria has renewed interest on the identification of antibiotics with potent antimalarial properties. In this study we also investigated the antiplasmodial potential of thiostrepton and derivatives, synthesized using combinations of tail truncation, oxidation, and addition of lipophilic thiols to the terminal dehydroamino acid. We showed that derivatives SS231 and SS234 exhibit a better antiplasmodial activity (IC50 = 1 µM SI > 59 and SI > 77 respectively) than thiostrepton (IC50 = 8.95 µM, SI = 1.7). The antiplasmodial activity of these derivatives was observed at concentrations which are not hemolytic and non-toxic to human cell lines. Thiostrepton and derivatives appeared to exhibit transmission blocking properties when administered at their IC50 or IC90 concentrations and our data also showed that they attenuate proteasome activity of Plasmodium, which resulted in an accumulation of ubiquitinated proteins after incubation with their IC80 concentrations. Our results indicate that the parasite's proteasome could be an attractive target for therapeutic intervention. In this regard, thiostrepton derivatives are promising candidates by dually acting on two independent targets, the proteasome and the apicoplast, with the capacity to eliminate both intraerythrocytic asexual and transmission stages of the parasite. To further support our findings, we evaluated the activity of a new class of antimalarial and proteasome inhibitors namely peptidyl sulfonyl fluorides on gametocyte maturation and analogues AJ34 and AJ38 were able to completely suppress gametocytogenesis at IC50 concentrations (0.23 µM and 0.17 µM respectively) suggesting a strong transmission blocking potential. The proteasome, a major proteolytic complex, responsible for the degradation and re-cycling of non-functional proteins has been studied only indirectly in P. falciparum. In addition, an apparent proteasome-like protein with similarity to bacterial ClpQ/hslV threonine-peptidases was predicted in the parasite. Antibodies were generated against the proteasome subunits alpha type 5 (α5-SU), beta type 5 (β5-SU) and pfhslV in mice and we showed that the proteasome is expressed in both sexual and asexual blood stages of P. falciparum, where they localize in the nucleus and in the cytoplasm. However, expression of PfhslV was only observed in trophozoites and shizonts. The trafficking of the studied proteasome subunits was further investigated by generating parasites expressing GFP tagged proteins. The expression of α5-SU-GFP in transgenic parasite appeared to localize abundantly in the cytoplasm of all blood stages, and no additional information was obtained from this parasite line. In conclusion, our data highlight two new tools towards combination therapy. Hybrid molecules represent promising tools for the cure of co-infected individuals, while very potent antibiotics with a wide scope of activities could be useful in ACTs by eliminating resistant parasites and limiting transmission of both, resistances and disease.}, subject = {Malaria}, language = {en} } @article{SasseSchilligDierolfetal.2011, author = {Sasse, Christoph and Schillig, Rebecca and Dierolf, Franziska and Weyler, Michael and Schneider, Sabrina and Mogavero, Selene and Rogers, David P. and Morschh{\"a}user, Joachim}, title = {The Transcription Factor Ndt80 Does Not Contribute to Mrr1-, Tac1-, and Upc2-Mediated Fluconazole Resistance in Candida albicans}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69201}, year = {2011}, abstract = {The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis, by the overexpression of genes encoding multidrug efflux pumps or ergosterol biosynthesis enzymes. Zinc cluster transcription factors play a central role in the transcriptional regulation of drug resistance. Mrr1 regulates the expression of the major facilitator MDR1, Tac1 controls the expression of the ABC transporters CDR1 and CDR2, and Upc2 regulates ergosterol biosynthesis (ERG) genes. Gain-of-function mutations in these transcription factors result in constitutive overexpression of their target genes and are responsible for fluconazole resistance in many clinical C. albicans isolates. The transcription factor Ndt80 contributes to the drug-induced upregulation of CDR1 and ERG genes and also binds to the MDR1 and CDR2 promoters, suggesting that it is an important component of all major transcriptional mechanisms of fluconazole resistance. However, we found that Ndt80 is not required for the induction of MDR1 and CDR2 expression by inducing chemicals. CDR2 was even partially derepressed in ndt80D mutants, indicating that Ndt80 is a repressor of CDR2 expression. Hyperactive forms of Mrr1, Tac1, and Upc2 promoted overexpression of MDR1, CDR1/CDR2, and ERG11, respectively, with the same efficiency in the presence and absence of Ndt80. Mrr1- and Tac1-mediated fluconazole resistance was even slightly enhanced in ndt80D mutants compared to wild-type cells. These results demonstrate that Ndt80 is dispensable for the constitutive overexpression of Mrr1, Tac1, and Upc2 target genes and the increased fluconazole resistance of strains that have acquired activating mutations in these transcription factors.}, subject = {Candida albicans}, language = {en} } @article{EulalioFroehlichManoetal.2011, author = {Eulalio, Ana and Fr{\"o}hlich, Kathrin S. and Mano, Miguel and Giacca, Mauro and Vogel, J{\"o}rg}, title = {A Candidate Approach Implicates the Secreted Salmonella Effector Protein SpvB in P-Body Disassembly}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68928}, year = {2011}, abstract = {P-bodies are dynamic aggregates of RNA and proteins involved in several post-transcriptional regulation processes. Pbodies have been shown to play important roles in regulating viral infection, whereas their interplay with bacterial pathogens, specifically intracellular bacteria that extensively manipulate host cell pathways, remains unknown. Here, we report that Salmonella infection induces P-body disassembly in a cell type-specific manner, and independently of previously characterized pathways such as inhibition of host cell RNA synthesis or microRNA-mediated gene silencing. We show that the Salmonella-induced P-body disassembly depends on the activation of the SPI-2 encoded type 3 secretion system, and that the secreted effector protein SpvB plays a major role in this process. P-body disruption is also induced by the related pathogen, Shigella flexneri, arguing that this might be a new mechanism by which intracellular bacterial pathogens subvert host cell function.}, subject = {Salmonella}, language = {en} } @article{WeibelRaabYuetal.2011, author = {Weibel, Stephanie and Raab, Viktoria and Yu, Yong A. and Worschech, Andrea and Wang, Ena and Marincola, Francesco M. and Szalay, Aladar A.}, title = {Viral-mediated oncolysis is the most critical factor in the late-phase of the tumor regression process upon vaccinia virus infection}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68691}, year = {2011}, abstract = {Background: In principle, the elimination of malignancies by oncolytic virotherapy could proceed by different mechanisms - e.g. tumor cell specific oncolysis, destruction of the tumor vasculature or an anti-tumoral immunological response. In this study, we analyzed the contribution of these factors to elucidate the responsible mechanism for regression of human breast tumor xenografts upon colonization with an attenuated vaccinia virus (VACV). Methods: Breast tumor xenografts were analyzed 6 weeks post VACV infection (p.i.; regression phase) by immunohistochemistry and mouse-specific expression arrays. Viral-mediated oncolysis was determined by tumor growth analysis combined with microscopic studies of intratumoral virus distribution. The tumor vasculature was morphologically characterized by diameter and density measurements and vessel functionality was analyzed by lectin perfusion and extravasation studies. Immunological aspects of viral-mediated tumor regression were studied in either immune-deficient mouse strains (T-, B-, NK-cell-deficient) or upon cyclophosphamide-induced immunosuppression (MHCII+-cell depletion) in nude mice. Results: Late stage VACV-infected breast tumors showed extensive necrosis, which was highly specific to cancer cells. The tumor vasculature in infected tumor areas remained functional and the endothelial cells were not infected. However, viral colonization triggers hyperpermeability and dilatation of the tumor vessels, which resembled the activated endothelium in wounded tissue. Moreover, we demonstrated an increased expression of genes involved in leukocyte-endothelial cell interaction in VACV-infected tumors, which orchestrate perivascular inflammatory cell infiltration. The immunohistochemical analysis of infected tumors displayed intense infiltration of MHCII-positive cells and colocalization of tumor vessels with MHCII+/CD31+ vascular leukocytes. However, GI-101A tumor growth analysis upon VACV-infection in either immunosuppressed nude mice (MHCII+-cell depleted) or in immune-deficient mouse strains (T-, B-, NK-cell-deficient) revealed that neither MHCII-positive immune cells nor T-, B-, or NK cells contributed significantly to VACV-mediated tumor regression. In contrast, tumors of immunosuppressed mice showed enhanced viral spreading and tumor necrosis. Conclusions: Taken together, these results indicate that VACV-mediated oncolysis is the primary mechanism of tumor shrinkage in the late regression phase. Neither the destruction of the tumor vasculature nor the massive VACV-mediated intratumoral inflammation was a prerequisite for tumor regression. We propose that approaches to enhance viral replication and spread within the tumor microenvironment should improve therapeutical outcome.}, subject = {Virusinfektion}, language = {en} } @article{ZdziarskiBrzuszkiewiczWulltetal.2010, author = {Zdziarski, Jaroslaw and Brzuszkiewicz, Elzbieta and Wullt, Bjorn and Liesegang, Heiko and Biran, Dvora and Voigt, Birgit and Gronberg-Hernandez, Jenny and Ragnarsdottir, Bryndis and Hecker, Michael and Ron, Eliora Z. and Daniel, Rolf and Gottschalk, Gerhard and Hacker, Joerg and Svanborg, Catharina and Dobrindt, Ulrich}, title = {Host Imprints on Bacterial Genomes-Rapid, Divergent Evolution in Individual Patients}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68594}, year = {2010}, abstract = {Bacteria lose or gain genetic material and through selection, new variants become fixed in the population. Here we provide the first, genome-wide example of a single bacterial strain's evolution in different deliberately colonized patients and the surprising insight that hosts appear to personalize their microflora. By first obtaining the complete genome sequence of the prototype asymptomatic bacteriuria strain E. coli 83972 and then resequencing its descendants after therapeutic bladder colonization of different patients, we identified 34 mutations, which affected metabolic and virulence-related genes. Further transcriptome and proteome analysis proved that these genome changes altered bacterial gene expression resulting in unique adaptation patterns in each patient. Our results provide evidence that, in addition to stochastic events, adaptive bacterial evolution is driven by individual host environments. Ongoing loss of gene function supports the hypothesis that evolution towards commensalism rather than virulence is favored during asymptomatic bladder colonization.}, subject = {Proteomanalyse}, language = {en} } @article{Moll1993, author = {Moll, Heidrun}, title = {Epidermal Langerhans cells are critical for immunoregulation of cutaneous leishmaniasis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-61323}, year = {1993}, abstract = {In leishmaniasis, macrophages are known to play a central role as modulators of the specific immune activity. In this article, Heidrun Moll presents evidence for the critical involvement of another component of the skin immune system, the epidermal Langerhans cell. She proposes that Langerhans cells take up parasites in the skin and transport them to the draining lymph node for presentation to T cells and initiation of the specific immune response.}, subject = {Biologie}, language = {en} } @article{MollMuellerGillitzeretal.1991, author = {Moll, Heidrun and M{\"u}ller, Christoph and Gillitzer, Reinhard and Fuchs, Harald and R{\"o}llinghoff, Martin and Simon, Markus M. and Kramer, Michael D.}, title = {Expression of T-cell-associated serine proteinase-1 during murine Leishmania major infection correlates with susceptibility to disease}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-61311}, year = {1991}, abstract = {The expression of T-cell-associated serine proteinase 1 (MTSP-1) in vivo during Leishmania major infection was analyzed in genetically resistant C57BL/6 mice and in genetically susceptible BALB/c mice. Using a monoclonal antibody as well as an RNA probe specific for MTSP-1 to stain tissue sections, we found T cells expressing MTSP-1 in skin lesions and spleens of mice of both strains. In skin lesions, MTSP-1-positive T cells could be detected as early as 3 days after infection. Most importantly, the frequency of T cells expressing MTSP-1 was significantly higher in susceptible BALB/c mice than in resistant C57BL/6 mice. These findings suggest that MTSP-1 is associated with disease-promoting T cells and that it may be an effector molecule involved in the pathogenesis of cutaneous leishmaniasis.}, subject = {Biologie}, language = {en} } @article{MollRoellinghoff1990, author = {Moll, Heidrun and R{\"o}llinghoff, Martin}, title = {Resistance to murine cutaneous leishmaniasis is mediated by T\(_H\)1 cells, but disease-promoting CD4\(^+\) cells are different from T\(_H\)2 cells}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-61305}, year = {1990}, abstract = {No abstract available}, subject = {Biologie}, language = {en} } @article{MollBinoederBogdanetal.1990, author = {Moll, Heidrun and Bin{\"o}der, Kerstin and Bogdan, Christian and Solbach, Werner and R{\"o}llinghoff, Martin}, title = {Production of tumour necrosis factor during murine cutaneous leishmaniasis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-61291}, year = {1990}, abstract = {We have assessed the role of tumour necrosis factor-a (TNF) during cutaneous leishmaniasis and demonstrated that significant levels of TNF were released by spleen cells from infected mice after in cirro restimulation with Leishmania major promastigotes. Spleen cells from both genetically resistant and genetically susceptible mice were equally capable of producing TNF. After challenge with bacterial endotoxin, TNF activity could also be demonstrated in the serum of L. mujor-infected mice and the titres correlated with the course of cutaneous disease in susceptible and resistant mice. TNF did not exert a direct leishmanicidal effect in uitro. Furthermore, our study indicated that macrophages are the source of L. major-induced TNF activity and that its elicitation is dependent on the presence of T cells. These findings suggest that TNF acts in concert with other cytokines produced during L. major infection and that its role depends on the composition of T cell subsets and cytokines present.}, subject = {Immunologie}, language = {en} } @article{HackerOttHof1993, author = {Hacker, J{\"o}rg and Ott, M. and Hof, H.}, title = {Effects of low, subinhibitory concentrations of antibiotics on expression of a virulence gene cluster of pathogenic E. coli by using a wild-type gene fusion}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59874}, year = {1993}, abstract = {No abstract available}, subject = {Infektionsbiologie}, language = {en} } @article{ZinglerBlumFalkenhagenetal.1993, author = {Zingler, G. and Blum, G. and Falkenhagen, U. and Orskov, I. and Orskov, F. and Hacker, J{\"o}rg and Ott, M}, title = {Clonal differentiation of uropathogenic E. coli isolates of serotype O6:K5 by fimbrial antigen typing and DNA long-range mapping techniques}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59865}, year = {1993}, abstract = {Escherichia coli isolates of serotype 06: K5 are the most common causative agents of cystitis and pyelonephritis in adults. To answer the question, as to whether strains of this particular serotype represent one special clonal group, out of a collection of 34 serotype 06: K5 isolates [Zingler et al. ( 1990) Zentralbl. Bakteriol Mikrobiol Hyg [A] 274:372-381] 15 strains were selected andanalyzed in detail. The flagellar (H) antigen and the outer membrane protein (OMP) pattern were determined. Furtherserum resistance properties and the genetic presence and expression of other virulence factors, including hemolysin, aerobactin, P fimbriae, S/F1C fimbriae and type 1 fimbriae was evaluated. In~laddition the Xbalmacrorestriction pattern of ten representative isolates was elaborated and the fimbrial (F) antigentype ofthe P fimbriae was determined, to obtain the complete 0: K: H: F pattern. These analyses could clearly show that the 06: K5 isolates do not represent one clonal group. The Xbal-macrorestriction profiles were heterogeneaus and marked differences in the hybridization patterns, using virulenceassociated gene probes in Southern hybridization of long-range-separated genomic DNA, were observed among the strains. However, some of strains showed similarities in the genomic profiles, arguing for clonal groupings among the 06: K5 isolates. lnterstingly the strains grouped tagether exhibited the same fimbrial F typethat many indicate a coincidence of this phenotypic trait with clonality.}, subject = {Infektionsbiologie}, language = {en} } @article{HackerKestlerHoschuetzkyetal.1993, author = {Hacker, J{\"o}rg and Kestler, H. and Hosch{\"u}tzky, H. and Jann, K. and Lottspeich, F. and Korhonen, T. K.}, title = {Cloning and characterization of the S fimbrial adhesin (SfaII) complex of an Escherichia coli O18:K1 meningitis isolate}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59853}, year = {1993}, abstract = {S fimbrial adbesins (Sfa), which are able to recognize sialic acid-containing receptors on eukaryotic cells, are produced by Escherichia coli strains causing urinary tract infections or newbom meningitis. We recently described tbe cloning and molecular cbaracterization of a determinant, termed sftJI, from the chromosome of an E. coli urinary tract infection strain. Herewe present data conceming a S fimbria-specific gene duster, designated sfall, of an E. coli newbom meningitis strain. Like tbe Sfal complex, Sfall consists of tbe major subunit protein SfaA (16 kDa) and the minor subunit proteins SfaG (17 kDa), SfaS (15 kDa), and SfaH (29 kDa). The genes encoding tbe subunit proteins of Sfall were identified and sequenced. Their protein sequences were calculated from the DNA sequences and compared with tbose of the Sfal complex subunits. Altbough the sequences ofthe two major SfaA subunits ditf'ered markedly, tbe sequences ofthe minor subunits sbowed only a few amino acid exchanges (SfaG, SfaH) or were completely identical (SfaS). The introduction of a site-specific mutation into the gene sfaSII and subsequent analysis of an SfaS-negative clone indicated that sfaSII codes for the sialic acid-specific adhesin of tbe meninigitis isolate. These data were confirmed by tbe isolation and characterization of tbe SfaSII protein and the determination of its N-terminal amino acid sequence. The identity between the sialic acid-specific adhesins of Sfal and Sfall revealed that difl'erences between the two Sfa complexes with respect to tbeir capacities to agglutinate erythrocytes must result from sequence alterations of subunit proteins other tban SfaS.}, subject = {Infektionsbiologie}, language = {en} } @article{MorschhaeuserUhlinHacker1993, author = {Morschh{\"a}user, J. and Uhlin, B. E. and Hacker, J{\"o}rg}, title = {Transcriptional analysis and regulation of the sfa determinant coding for S fimbria of pathogenic E. coli strains}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59844}, year = {1993}, abstract = {The sfa determinant codes for S fimbrial adhesins which constitute adherence factors of pathogenic Escherichia coli strains. Wehave recently shown that the sfa determinant is transcribed from three pr{\"o}moters, pA, pB, and pC. In comparison with the promoters pB and pC, promoter pA, which is located in front of the structural gene sfaA, showed very weak activity. Herewe have determined the exact positions ofthe mRNA start points by primer extension studies. We have also shown that mRNAs of 500, 700 and 1400 bases can be detected using oligonucleotide probes specific for the genes sfaB, sfaC and sfaA. SfaB and SfaC arepositive regulators infiuencing fimbriation and the production of the S-specific adhesin which is encoded by the gene sfaS Iocated in the distal half of the determinant. In addition, it is demonstrated that SfaB and SfaC interfere with the regulatory effect of the histone-like protein H-NS, encoded by a locus termed drdX or osmZ. In a drdx+ strain the regulators are necessary for transcription of the sfa determinant. In contrast, sfa expression is activator-independent in a drdx- strain. In this latter genetic background, a substantial fraction of the sfa transcripts is initiated from promoter pA. On the basis of these data we discuss a model for the regulation of this adhesin-specific determinant.}, subject = {Infektionsbiologie}, language = {en} } @article{SchrotenSteinigPlogmannetal.1992, author = {Schroten, H. and Steinig, M. and Plogmann, R. and Hanisch, F. G. and Hacker, J{\"o}rg and Herzig, P. and Wahn, V}, title = {S-fimbriae mediated adhesin of Escherichia coli to human buccal epithelial cells is age independent}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59830}, year = {1992}, abstract = {S-fimbriated Escherichia coli, which cause sepsis and meningitis in the newbom, bind to sialic acid-containing glycoprotein structures on the surface of human buccal epithelial cells. The dependence of · this binding on host age was examined. S-fimbriated · E. coli adhered in comparable numbers to cells in newborns, infants, children and adults (23.0 ± 8.6; 23.1 ± 11.5; 24.7 ± 7.9; 28.9 ± 8.8). Thus, the increased susceptibility of neonates to infections caused by S-fimbriated E. coli cannot be explained by enhanced · adhesion to epithelial cells}, subject = {Infektionsbiologie}, language = {en} } @article{OttBenderLuecketal.1992, author = {Ott, M. and Bender, L. and L{\"u}ck, P. C. and Meyer, P. and Hacker, J{\"o}rg}, title = {Distribution of Legionellae in a hospital water system: prevalence of immunologically and genetically related Legionella pneumophila serogroup 6 isolates}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59827}, year = {1992}, abstract = {A hospital warm water system was monitored for the prcsence and distribution of lcgionellac. Subtyping of ten scletled Legionella pneumophiltl isolates. originating from four different sites in the system by using serogroup spccific antisera in an indircct immunofluorcscence tcst, rcvcalcd that nine of the tcn isolatcs belonged to scrogroup 6, while the remaining one was serogroup I 0. Two monoclonal antibodics (mAbs) spccific for a subgroup of serogroup 6 strains were further used for characterization. None of the strains reactcd with these mAbs. Genome analysis by elaborating Not I profiles using the pulscd field gel electrophoresis (PFGE) technique revealed that nearly all serogroup 6 isolates dcrived from different sites, including a new building connected hy a ring pipe. wcrc identical according to restriction fragment pattems. The patterns were distinguishable from those of the two L. pnewnophi/a serogroup 6 rcfcrencc strains, and ftom that of thc L. pneumophila scrogroup 10 isolate. These data arguc for a relatively homogeneaus L. pneunwpltila serogroup 6 population in the entire watcr system.}, subject = {Infektionsbiologie}, language = {en} } @article{LinhardtZiebuhrMeyeretal.1992, author = {Linhardt, F. and Ziebuhr, W. and Meyer, P. and Witte, W. and Hacker, J{\"o}rg}, title = {Pulsed-field gel electrophoresis of genomic restriction fragments as a tool for the epidemiological analysis of Staphylococcus aureus and coagulase-negative Staphylococci}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59811}, year = {1992}, abstract = {Thirtccn StttJ1hylococcus dw·eus and s: <'pid<'l'· midis strains ohtaincd from nnsc and hand nf twn cmployccs and onc paticnt uf a mcdical ward as weil as two S. hemol.\"licus strains wcrc analyscd according to thcir rcstrktion fmgmcnt lcngth pattcrns ( RFLP) hy pulscd-ficld gcl clcctrophorcsis (PFGE) using thc rcslriction cnzymcs SmaJ and s.. .· tll. Spccics idcntification nf thc isolatcs was pcrformcd hy a systcm which includcs :!O hiochcmical rc"ctions. Furthcrmorc. thc antillintic resistancc pattcrns of thc stmins wcrc dctcrmincd. Whilc scvcral isolatcs cxhihitcd idcnticaf antihiotic susccptihilitics and hiochcmical prnfilcs. diffcrences in thc RFLP wcrc ohtaincd. ln thrcc cascs, S. epiderm{\"u}lis strains colonizing thc skin showcd an idcntical rcstriction profilc as isollltcs from thc mucous mcmhrancs of thc samc pcrson. Wc C(mcludcd that thc analysis of staphylococcal strains hy PFGE is an important cpidcmiolngical tnnl with high discrimination power.}, subject = {Infektionsbiologie}, language = {en} } @article{SchrotenLethenHanischetal.1992, author = {Schroten, H. and Lethen, A. and Hanisch, F., G. and Plogmann, R. and Hacker, J{\"o}rg and Nobis-Bosch, R. and Wahn, V.}, title = {Inhibition of adhesion of S-fimbriated Escherichia coli to epithelial cells by meconium feces of breast fed and formula fed newborns - mucins are the major inhibitor component}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59804}, year = {1992}, abstract = {We investigated the ability of meconium, feces from human milk-fed (HMF) newborns, and feces from formula-fed (FF) newborns to inhibit adhesion of S-fimbriated E. coli to human buccal epithelial cells. S-fimbriae are a common property of E.·coli strains causing sepsis and meningitis in neonates. Meconium had the highest content of neuraminic acid and the strongest inhibitory effect on bacterial adhesion. HMF also exerted high inhibitory activity while FF was markedly less active: To achieve inhibitory effects comparable to HMF a sixfold amount of FF was required. Glycoproteins from excretions were separated by gel chromatography. Fractions obtained were analyzed for adhesion-inhibiting activity. In all excretions analyzed, the mucin-containing fraction could be identified as the major inhibitory component. Inhibition was probably mediated by specific interaction of this fraction with S-fimbriae, as shown by binding of isolated fimbriae on Western blots after electrophoretic separation of glycoproteins. In conclusion, our data support the view that the mucin-containing fraction from meconium and human milk exerts antibacterial functions by preventing adhesin-mediated binding of pathogenic bacteria to mucosal epithelia. Key Words: S-fimbriated E. coli-Inhibition of adhesion-Meconium- Feces of human milk-fed newborns-Feces of formula-fed newborns-Mucins.}, subject = {Infektionsbiologie}, language = {en} } @article{ZinglerOttBlumetal.1992, author = {Zingler, G. and Ott, M. and Blum, G. and Falkenhagen, U. and Naumann, G. and Sokolowska-K{\"o}hler, W. and Hacker, J{\"o}rg}, title = {Clonal analysis of Escherichia coli serotype O6 strains from urinary tract infections}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59786}, year = {1992}, abstract = {A total of 36 Escherichia coli urinary tract isolates (UTI) of serotype 06, with different combinations of capsule ( K) and flagellin ( H) antigens, were analysed according to the outer membrane pattern (OMP), serum resistance properties, mannose-resistant hemagglutination using various types of erythrocytes, and also for the genetic presence and the expression of Pfimbriae. S fimbriae/F1 C fimbriae, Type 1 fimbriae, aerobactin and hemolysin. Twenty selected strains were further analysed by pulsed field gel electrophoresis (PFGE), elaborating genomic profilas by Xba I cleavage and subsequent Southern hybridization to virulence-associated DNA probes. lt could be shown that 06 UTI isolates represent a highly heterogeneaus group of strains according to the occurrence and combination of these traits. Relatedness an the genetic and the phenotypic Ievei was found for some of the strains exhibiting the same 0: K: H: F serotype. DNA Iang-range mapping further indicated some interesting features, according to the copy number and the genomic linkage of virulence genes.}, subject = {Infektionsbiologie}, language = {en} } @article{SchrotenHanischPlogmannetal.1992, author = {Schroten, H. and Hanisch, F. G. and Plogmann, R. and Hacker, J{\"o}rg and Uhlenbruck, G. and Wahn, V.}, title = {Inhibition of Adhesion of S-fimbriated Escherichia coli to buccal epithelial cells by human milk fat globule membrane components: a novel aspect of protective function of mucins in the non-immunoglobulin fraction}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59793}, year = {1992}, abstract = {We investigated the presence of factors in human milkthat inhibit Invasion of pathogenic bacteria. The efl'ect of human milk fat globule membrane (HMFGM) components on adhesion of cloned S-fimbriated Escherichia coli to human buccal epithelial cells was analyzed. S fimbriae are a common feature of E. coli strains causing sepsis and meningitis in newborns and are bound to epithelia via sialyl-(a-2-3)galactoside structures. Human milk fat globules (HMFG) could be agglutinated by the above-mentioned bacteria. Agglutination could be inhibited by fetuin, human glycophorin, and a 1-acid glycoprotein. In addition, pretreatment of HMFG with Jlibrio cholerae neuraminidase markedly reduced bacterium-induced agglutinations, indicating the involvement of neuraminic acid-containing glycoproteins. In contrast, Iipid droplets of infant formula or artificiallipid emulsions (Intralipid) could not be agglutinated. HMFG were present in stools of breast-fed neonates as shown by indirect immunofluorescence staining with a monoclonal antibody directed against carbohydrate residues present on HMFGM. These HMFG could be agglutinated by bacteria. HMFG inhibited E. coli adhesion to buccal epithelial cells. To further characterize relevant E. coli binding structures, HMFGM components w~re separated by gel chromatography. The mucin fraction showed the most pronounced inhibitory efrect on adhesion of S-fimbriated E. coli to human buccal epithelial cells. Our data soggest that HMFG inhibit bacterial adhesion in the entire intestine and thereby may provide protection against bacterial infection.}, subject = {Infektionsbiologie}, language = {en} } @article{FischerBangLudwigetal.1992, author = {Fischer, G. and Bang, H. and Ludwig, B. and Mann, K. H. and Hacker, J{\"o}rg}, title = {Mip protein of Legionella pneumophila exhibits peptidyl-prolyl cis-trans-isomerase (PPIase) activity}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59778}, year = {1992}, abstract = {Legfonells pneumoph/la is an intracellular paraslte which ts able to survtve and multipJy in human monocytes and alveolar macrophages. The Mtp (macrophage lnfectiv1ty potentlator) protein has been shown to be an essential virulente factor. A search of translated nuclelt .acld data ba.ses has shown that the Mip proteJn from strain Wadsworth possesses reglons homologaus to those found in the FK.506-bindfng proteins (FKBPs) of several different eukaryotlc organisms. FKBPs are abte to bind to the fmmunosuppressant macrollde FK506 and possess peptidyf .. prolyl cisltrans Isomerase (PPiase) activlty. The gene coding for the Mlp proteln was cloned from the ehromo. some of L. pneumophila straln Philadelph·a I and sequenced. II was synthesl\%ed in Escherichla coll ·K- 12 and alter purlfication it exhibited PPiase activity catalyslng the slow clsltrans lsomerization of prolyl peptlde bonds. ln ollgopeptides. Mip ls inhibi~ted by FK506 and fully reslstant to cyclosporln A, as was also found for the recently characterlzed FKBP-type PPiases of eukaryotes. However, the N-terminal extenslon of Mip and/or the substltutrons of the vari· ab1e amlno acrds ln the C-termlnal FKBP core Iead to variatlons,. when compared with eukaryotlc FKBPs, Jn substrate specfflclty wlth the Oligopeptide substrates of' type Suc-Aia-Xaa-Pro-Phe·4·nitroanUide. Never· theless, the Legionella Mip factor represents a bacte· rial gene product whtch shares some characteristics normally found in eukaryotic proteins. ln view of the activity of PPiases in protein-folding reactlonsf such prokaryotic FKBP analogues may represent a new class of bacterial. pathogenicity factors.}, subject = {Infektionsbiologie}, language = {en} } @article{OttBenderChirinosetal.1991, author = {Ott, M. and Bender, L. and Chirinos, E. and Ehret, W. and Hacker, J{\"o}rg}, title = {Phenotype versus genotype of the 19 kd peptido-glycan associated protein of Legionella (PplA) among Legionellae and other gram-negative bacteria}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59768}, year = {1991}, abstract = {The protein PpiA (19 kD) cloned from a genomic library of Legionella pneumophila, Philadelphia 1, represents a peptido-glycan associated outer membrane protein in recombinant E. coli K-12 and L. pneumophila. lt exhibits distinct sequence homology to Iipoproteins of Haemophilus influenzae and E. coli. A ppiA specific DNA probe generated by PCR was used in Southern hybridizations of chromosomal DNA of Legionella strains and other Gram-negative pathogens. Under conditions of high stringency, hybridization could only be observed in L. pneumophila isolates, but alt other Legionella strains tested displayed hybridization under lower stringency. No signals appeared after hybridization of chromosomal DNA from a variety of other bacteria. Using anti-PpiA monospecific polyclonal antibodies in Western blots, it was demonstrated that PpiA related proteins of nearly the same size are found in all L. pneumophila isolates and in a variety of, but not alt, the Legionella species analysed here.}, subject = {Infektionsbiologie}, language = {en} } @article{OttMessnerHeesemannetal.1991, author = {Ott, M. and Messner, P. and Heesemann, J. and Marre, R. and Hacker, J{\"o}rg}, title = {Temperature dependent expression of flagella in Legionella}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59755}, year = {1991}, abstract = {Legionel/a pneumophila, the causative agent of Legionnaires' disease, was analysed by electron microscopy for production of surface structures. Crystalline surface (S-) layers and fimbriae were not detected, but monotrichous flagellation was seen. Polyclonal antibodies specific for the 47 kDa ftagellin subunit of L. pneumophila Philadelphia I were used in Western blots to confirm the presence of flagella subunits in various L. pneumophila strains tested, but the antiserumalso reacted with flagellin subunits of L. micdlulei, L. hackelia (serogroup (SG) l and SG21 and L./ongbetichae (SG2). Flagellation of Legionellae was shown to be temperature regulated. When the growth temperature of virulent and avirulent variants of strain L. pneumophila Philadelphia I was shifted from 30 oc to either 37 or 41 oc, a decrease in the percentage offtagellated bacteria within the populationwas observed.}, subject = {Infektionsbiologie}, language = {en} } @article{BenderOttDebesetal.1991, author = {Bender, L. and Ott, M. and Debes, A. and Rdest, U. and Heesemann, J. and Hacker, J{\"o}rg}, title = {Distribution, expression and long range mapping of legiolysin gene (lly) specific DNA sequences in Legionellae}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59744}, year = {1991}, abstract = {The legiolysin gene (lly) cloned from Legionella pneumophila Philadelphia 1 confers the phenotypes of hemolysis and browning of the culture medium. An internal Uy-specific DNA probe was used in Southern hybridizations for the detection of Uy-specific DNA in the genomes of legioneUae and other gram-negative pathogenic bacteria. Under conditi9ns of high stringency, tlie Uy DNA probe specifically reacted with DNA fragments fr9m L. pneumophi{\"u}z isolates; by reducing stringency, hybridization was also observed for all other Legionella strains tested. No hybridization occurred with DNAs isolated from bact~ria of other genera. The Uy genewas mapped by pulsed-field gel electrophoresis to the respective genomic Notl fragments of Legionelltz isolates. By using antilegiolysin monospecific polyclonal antibodies in Western blots (immunoblots), Lly proteins could be detected only in L. pneumophila isolates.}, subject = {Infektionsbiologie}, language = {en} } @article{OttBenderBlumetal.1991, author = {Ott, M. and Bender, L. and Blum, G. and Schmittroth, M. and Achtmann, M. and Tsch{\"a}pe, H. and Hacker, J{\"o}rg}, title = {Virulence patterns and long range mapping of extraintestinal Escherichia coli K1, K5 and K100 isolates: Use of pulse field gel electrophoresis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59738}, year = {1991}, abstract = {A total of 127 extraintestinal Escherichia coli strains of the capsule serotypes Kl, KS, and KlOO from human and animal sources were analyzed for DNA sequences specific for the genes for various adhesins (P fimbriae fpap] and P-related sequences fprs], S fimbriae [s/a)/FlC fimbriae [foc], and type I fimbriae lfim]), aerobactin (aer), and hemolysin (hly). The expression of corresponding virulence factors was also tested. Twenty-four selected strains were analyzed by long-range DNA mapping to evaluate their genetic relationships. DNA sequences for the adhesins were often found in strains not expressing them, while strains with hemolysin and aerobactin genes usually did express them. Different isolates of the same serotype orten expressed different virulence patterns. The use of virulence-associated gene probes for Southern hybridization with genomic DNA fragments separated by pulsed-field gel electrophoresis revealed that a highly heterogeneous restriction fragment length and hybridization pattern existed even within strains of the same serotype. Long-range DNA mapping is therefore useful for the evaluation of genetic relatedness among individual isolates and facilitates the performance of .precise molecular epidemiology.}, subject = {Infektionsbiologie}, language = {en} } @article{LudwigSchmidMarreetal.1991, author = {Ludwig, B. and Schmid, A. and Marre, R. and Hacker, J{\"o}rg}, title = {Cloning, genetic analysis and nucleotide sequence of a determinant coding for a 19 kd peptidoglycan-associated protein (Ppl) of Legionella pneumophila}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59721}, year = {1991}, abstract = {A genomic library of Legionello pneumophihz, the causative agent of Legionnaires disease in humans, was constructed in Escherichill coli K-12, and the recombinant clones were screened by immuno-colony blots with im antiserum raised against heat-killed L. pneumophilo. Twenty-three clones coding for a LegioneUa-specific protein of 19 kDa were isolated. The 19-kDa protein, which represents an outer membrane protein, was found tobe associated with the peptidoglycan layer bothin L. pneumophilo andin the recombinant E. coli clones. This was shown by electrophoresis and Western immunoblot analysis of bacterial cell membrane fractions witb a monospecific polyclonal 19-kDa protein-specific antiserum. Tbe protein was termed peptidoglycan-associated protein of L. pneumophilo (Ppl). The corresponding genetic determinant, ppl, was subcloned on a 1.8-kb Clol fragment. DNA sequence studies revealed that two open reading frames, pplA and pplB, coding for putative proteins of 18~9 and 16.8 kDa, respectively, were located on the Clol fragment. Exonuclease 111 digestion studies confirmed tbat pplA is the gene coding for the peptidoglycan.;.associated 19-kDa protein of L. pneumophilo. The amino acid sequence of PpiA exhibits a high degree of homology to the sequences of the Pal Iipoproteins of E. coli K-12 and liaemophilus injluenvze.}, subject = {Infektionsbiologie}, language = {en} } @article{BlumOttCrossetal.1991, author = {Blum, G. and Ott, M. and Cross, A. and Hacker, J{\"o}rg}, title = {Virulence determinants of Escherichia coli O6 extraintestinal isolates analysed by Southern hybridizations and DNA long range mapping techniques}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59717}, year = {1991}, abstract = {A total of 16 Escherichia coli 06 strains isolated from cases of extraintestinal infections were analysed for the genetic presence and phenotypic expression of fimbrial adhesins ( P, S/FIC, type I), aerobactin and hemolysin. ln addition restriction fragment length polymorphisms (RFLPs) of Xbal-cleaved genomic DNA of seven selected strains, separated by orthogonal field alternation gel electrophoresis {OFAGE) were determined and virulence-associated DNA probes were used for Southern hybridization studies of the Xbal-cleaved genomic DNAs. The virulence characteristics and hybridization patterns obtained differed between the various isolates. ln three isolates hemolysin genes and P fimbrial determinants were located on the same Xbal fragments. Furthermore, multiple copies of FIC determinants (foc) could be detected in two strains. Our data show that the new technique of pulse field electrophoresis tagether with Southern hybridization represents a powerful tool for the genetic analysis of pathogenic bacteria.}, subject = {Infektionsbiologie}, language = {en} } @article{WintermeyerRdestLudwigetal.1991, author = {Wintermeyer, E. and Rdest, U. and Ludwig, B. and Debes, A. and Hacker, J{\"o}rg}, title = {Characterization of legiolysin (lly); responsible for hemolytic activity, colour production and fluorescence of Legionella pneumophila}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59706}, year = {1991}, abstract = {No abstract available}, subject = {Infektionsbiologie}, language = {en} } @article{OttHacker1991, author = {Ott, M. and Hacker, J{\"o}rg}, title = {Analysis of the variability of S fimbriae expression in an Escherichia coli pathogen.}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59695}, year = {1991}, abstract = {The uropathogenic Escherichia coli wiJd..:type strain 536 produces S-fimbriae, P-related fimbriae and type I fimbriae. Using immuno-colony dot and ELISA techniques, variants were detected showing an increased degree of S-fimbrial production. It was demonstrated by itrtmunofluorescence microscopy that in noimal (wild-type) and hyperS- fimbriated E. coli populaiions non-fimbriated cells also · exist, and that the percentage of Sfinibrlated and non-fimbriated bacteria was roughly identica1 in either population. Hyper-Sfimbriated variants could be stably maintained. The transition from wild-type to hyper-S-fimbriation, which occurs spontaneously, is markedly higher than vice versa. Southern blot analysis of the S fimbrial adhesin (sfa) determinants of normal and hyper-fimbriated strains revealed no marked difference in the gene structure.}, subject = {Infektionsbiologie}, language = {en} } @article{HackerOttLudwigetal.1991, author = {Hacker, J{\"o}rg and Ott, M. and Ludwig, B. and Rdest, U.}, title = {Intracellular survival and expression of virulence determinants of Legionella pneumophila}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59681}, year = {1991}, abstract = {Legionella pneumophila, the causative agent of Legionnaires' disease is able to live and multiply within macrophages as weil as within protozoan organisms. Legionella strains inhibit phagosome-lysosome fusion and phagosome acidification. By using two different cell culture systems, one derived from human macrophages and the other from human.embryo lung fibro:blastic cells, it is demonstrated that Legionella strains lose their virulence following cultivation in the laboratory. In order to study the mechanisms involved in intracellular survival of Legionella a genomic library of strain Legionella pneumophila Philadelphia I was established in Escherichia coli K-12. By cosmid cloning technique we were able to clone five putative virulence factors, two of which exhibit hemolytic activities and three of which represent membrane-associated proteins of 19, 26 and 60 kilodalton. One of the hemolytic proteins, termed legiolysin, represents a new toxin which specifically lyses human erythrocytes. The other hemolysin exhibits proteolytic properties in addition and is cytolytic for Vero and CHO cells. Further sturlies will be necessary to determine the exact role of the cloned proteins in the pathogenesis of Legionella. Zusammenfassung: Intrazellul{\"a}res {\"U}berleben}, subject = {Infektionsbiologie}, language = {en} } @article{OttBenderMarreetal.1991, author = {Ott, M. and Bender, L. and Marre, R. and Hacker, J{\"o}rg}, title = {Pulsed field electrophoresis of genomic restriction fragments for the detection of nosocomial Legionella pneumophila in hospital water supplies}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59672}, year = {1991}, abstract = {Ten Legionella pneumophUa strains isolated from dift'erent sources were analyzed according to their restriction fragment patterils obtained by cle~vage of gen.omic DNA With Notl and Sftl and separation by pulsed field electrophoresis. Three L. pneumophila isolate~ from a nosocomial outbreak in L{\"u}~k (Germany) and three other L. prreumophilll stralns independently isolated from a water tap located in the care unit where tbe patients were bospitalized 'xhibited identical restricti9n fragment profiles. Therefore, we concluded that these environment81 spee~ens were the source of the Legionnatres dlsease. Anotber two isolates from patients and two strains from the environment, all unrelated to the outJlreak described, sbowed different cleavage patterns.}, subject = {Infektionsbiologie}, language = {en} } @article{SchmollMorschhaeuserOttetal.1990, author = {Schmoll, T. and Morschh{\"a}user, J. and Ott, M. and Ludwig, B. and Van Die, I. and Hacker, J{\"o}rg}, title = {Complete genetic organization and functional aspects of the Escherichia coli S fimbrial adhesin determinant: nucleotide sequence of the genes sfaB, C, D, E, F.}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59661}, year = {1990}, abstract = {The S fimbrial adhesin (sfa) determinant of E. co/i comprises nine genes situated on a stretch of 7.9 kilobases (kb) DNA. Here the nucleotide sequence of the genes sfa B and sfaC situated proximal to the main structural gene sfaA is described. Sfa-LacZ fusions show that the two genes are transcribed in opposite directions. The isolation of mutants in the proximal region of the sfa gene cluster, the construction of sfa-phoA gene fusions and subsequent transcomplementation sturlies indicated that the genes sfaB and sfaC play a role in regulation of the sfa determinant. ln addition the nucleotide sequence of the genes sfa D, sfa E and sfa F situated between the genes sfaA and sfaG responsible for S subunit proteins, were determined. lt is suggested that these genes are involved in transport and assembly of fimbrial subunits. Thus the entire genetic organization of the sfa determinant is presented and compared with the gene clusters coding for P fimbriae (pap), F1 C fimbriae (foc) and type I fimbriae ( fim). The evolutionary relationship of fimbrial adhesin determinants is discussed.}, subject = {Infektionsbiologie}, language = {en} } @article{BenderOttMarreetal.1990, author = {Bender, L. and Ott, M. and Marre, R. and Hacker, J{\"o}rg}, title = {Genome analysis of Legionella spp. by orthogonal field alternation gel electrophoresis (OFAGE)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59657}, year = {1990}, abstract = {Various Legionella isolates from different sources and origins were analysed by orthogonal field alternation gel electrophoresis of Not I cleaved genomic DNA. The genome of L pneumophila Philadelphia I, the original isolate of the epidemics in 1976, exhibits only five Not I fragments. Two virulent derivatives. derived from L pneumophila Philadelphia I. which were obtained by prolonged passage on artificial cuhure media, did not differ from their isogenic virulent strain according the Not I fragment pattern. By summing the lengths of the Notl fragments, the genome size of L. pneumophila Philadelphia I was calculated as approximately 3.9 Mb. Environmental L pneumophila strains exhibited different Not I pattems, as did Legionella strains not belongi'ng to the species pneumophila. The usefulness of DNA long range mapping of Legionella ssp. with Notl for epidemiology and evaluation of their evolutionary rela· tionships is discussed.}, subject = {Infektionsbiologie}, language = {en} } @article{MarreKreftHacker1990, author = {Marre, R. and Kreft, B. and Hacker, J{\"o}rg}, title = {Genetically engineered S and F1C fimbriae differ in their contribution to adherence of Escherichia coli to cultured renal tubular cells}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59644}, year = {1990}, abstract = {Escherichia coU K-12 strains producing S-fimbrial adhesins, FlC fimbriae, and mutagenized fimbriae were tested in a binding assay with a renal tubular cell line. S-fimbrial adhesins and FlC fimbriae mediated bindlog to tubular cells. The SfaA, SfaG, and SfaS subunits of S fimbriae contributed to attachment. Site-specific mutations in the sfaS gene reduced binding. The Inhibitionprofile of FlC fimbriae resembled that of S fimbriae.}, subject = {Infektionsbiologie}, language = {en} } @article{VenturSchefferHackeretal.1990, author = {Ventur, Y. and Scheffer, J. and Hacker, J{\"o}rg and K{\"o}nig, W.}, title = {Effects of adhesins from mannose-resistant Escherichia coli on mediator release from human lymphocytes, monocytes and basophils and from polymorphonuclear granulo-cytes}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59636}, year = {1990}, abstract = {We investigated the roJe of Escherichia coU expressing mannose-resistant hemagglutination and adhesins with regard to the induction of leukotrienes from a suspension of human lymphocytes, monocytes, and basophils (LMBs) compared with human polymorphonuclear granulocytes (PMNs). Genetically cloned E. coli strains expressing various types of mannose-resistant hemagglutination (MRH+) were phagocytosed to a higher degree by monocytes than the nonadherent E. coli strain. The various strains dUfered in their capacity to induce a chemiluminescence response, which showed the same pattern for LMBs and PMNs. Stimulation of LMBs with bacteria alone, unlike granulocytes, did not activate the cells for the release of leukotrienes. However, preincubation of LMBs with bacteria decreased subsequent leukotriene formation when the cells were stimulated with calcium ionophore. The inhibitory eft'ect was dependent on the concentration of bacteria used for preincubation as weil as on the preincubation temperature. The various bacterial strains dift'ered in inhibitory potency for mediator release. Preincubation of LMBs with zymosan, opsonized zymosan, the bacterfal peptide FMLP, and peptidoglycan bad no inhibitory eft'ect or even increased subsequent IeukotrieDe formation. Opsonized bacteria were far less inhibitory than nonopsonized bacteria. In contrast to human LMBs, preincubation of human PMNs with mannose-resistant bacteria led to increased leukotriene 84 generation and reduced w-oxidation of leukotriene 84 • Our data soggest that phagocytes (neutrophils, monocytes) respond in a different way for leukotriene formation after Interaction with mannose-resistant E. coli.}, subject = {Infektionsbiologie}, language = {en} } @article{SchmollOttOugedaetal.1990, author = {Schmoll, T. and Ott, M. and Ougeda, B. and Hacker, J{\"o}rg}, title = {Use of a wild-type gene fusion to determine the influence of environmental conditions on expression of the S fimbrial adhesin in an Escherichia coli pathogen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59625}, year = {1990}, abstract = {S fimbrial adhesins (Sfa) enable pathogenic Escherichia coli strains to bind to sialic acid-containing eucaryotic receptor molecules. In order to determine the inftuence of culture conditions on the expression of the sfa determinant in a wild-type strain, we fused the gene lacZ, coding for the enzyme ß-galactosidase, to the sfaA gene, responsible for the major protein subunit of S fimbriae. By using a plasmid which carries an R6K origin, the sfaA-Iac hybrid construct was site-specifically integrated into the chromosome of the uropathogenic E. coli strain S36WT. The expression of lacZ, which was under the control of the sfa wild-type promoters, was now equivalent to the sfa expression of strain S36WT. With the help of this particular wild-type construct, it was demonstrated that the sfa determinant is better expressed on solid media than in liquid broth. The growth rate bad a strong inftuence on Sfa expression under aerobic but not under anaerobic conditions. Production of Sfa was further regulated by catabolite repression, osmolarity, and temperature.}, subject = {Infektionsbiologie}, language = {en} } @article{MorschhaeuserHoschuetzkyJannetal.1990, author = {Morschh{\"a}user, J. and Hosch{\"u}tzky, H. and Jann, K. and Hacker, J{\"o}rg}, title = {Functional analysis of the Sialic acid-binding adhesin SfaS of pathogenic Escherichia coli by site-specific mutagenesis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59613}, year = {1990}, abstract = {The gene coding for the sialic acid-specific adhesin SfaS produced by the S fimbrial adhesin (sfa) determinant of Escherichia coli has been modified by oligonucleotide-directed, site-specific mutagenesis. Lysine 116, arginine 118, and Iysine 122 were replaced by threonine, serine, and threonine, respectively. The mutagenized gene dusters were able to produce S fimbrial adhesin complexes consisting of the S-specific subunit proteins including the adhesin SfaS. The mutant clones were further characterized by hemagglutination and by enzyme-linked immunoassay tests with antifimbria- and anti-adhesin-specific monoclonal antibodies, one of which is able to block S-specific binding (Moch et al., Proc. Natl. Acad. Sei. USA 84:3462-3466, 1987). The lysine-122 mutantclone was indistinguishable from the wild-type clone in these assays. Replacement of Iysine 116 and ai'ginine 118, however, abolished hemagglutination and resulted in clones which showed a weak (Iysine 116) or a negative (arginine 118) reaction with the antiadhesin-specific antibody Al. We therefore suggest that Iysine 116 and arginine 118 have an inßuence on binding of SfaS to the sialic acid residue of the receptor molecule. Substitution of arginine 118 by serine also had a negative efl"ect on the amount of SfaS adhesin proteins isolated from the S fimbrial adhesin complex.}, subject = {Infektionsbiologie}, language = {en} } @article{HackerBenderOttetal.1990, author = {Hacker, J{\"o}rg and Bender, L. and Ott, M. and Wingeder, J. and Lund, B. and Marre, R. and Goebel, W.}, title = {Deletions of chromosomal regions coding for fimbriae and hemolysins occur in vivo and in vitro in various extraintestinal Escherichia coli isolates}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59608}, year = {1990}, abstract = {No abstract available}, subject = {Infektionsbiologie}, language = {en} } @article{RiegmannKustersVanVeggeletal.1990, author = {Riegmann, N. and Kusters, R. and Van Veggel, H. and Bergmans, H. and Van Bergen en Henegouwen, P. and Hacker, J{\"o}rg and Van Die, I.}, title = {F1C fimbriae of an uropathogenic Escherichia coli: Genetic and functional organization of the foc gene cluster and identification of minor subunits}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59597}, year = {1990}, abstract = {Tbe genetic organization of tbe foc gene duster bas been studied; six genes involved in tbe biogenesis of Fl C fimbriae were identifi.ed.focA encodes tbe major fimbrial subunit, focC encodes a product tbat is indispensable for fimbria formation,focG andjocH encode minor ftmbrial subunits, andfocl encodes a protein wbicb sbows similarities to the subunit protein FocA. Apart from tbe FocA major subunits, purified FlC fimbriae contain at least two minor subunits, FocG and FocH. Minor proteins of similar size were observed in purified S fimbriae. Remarkably, some mutations in tbe foc gene duster result in an altered 6mbrial morpbology, i.e., rigid stubs or long, curly ftmbriae.}, subject = {Infektionsbiologie}, language = {en} } @article{SchmollHoschuetzkyMorschhaeuseretal.1989, author = {Schmoll, T. and Hosch{\"u}tzky, H. and Morschh{\"a}user, J. and Lottspeich, F. and Jann, K. and Hacker, J{\"o}rg}, title = {Analysis of genes coding for the Sialic acid-binding adhesin and two other minor fimbrial subunits of the S-fimbrial adhesin determinant of Escherichia coli}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59585}, year = {1989}, abstract = {The S flmbrial adhesln (Sfa) enables Esch richla colito attach to slalfc acld-containing receptor molecules of eukaryotJc cells. As prevlously reported, the genetlc determinant coding for the Sfa of an E. co/1 06 strain was cloned, the gene codlng for the major fimbrfal subunit was ldentlfled and sequenced and th.e S speclflc adhesin was detected. Here we present evidence that ln addltlon to the major subunit proteln SfaA three other minor subunit proteins, SfaG (17 kD), SfaS (14kD) and SfaH (31 kD) can be isolated from the S..speclfic flmbrial adhesln complex. The genes coding for these minor subunits were ldenblied, mutagenlzed separately and sequenced. Using haemagglutlnatton tests. electron-microscopy and quantitative ELISA assays with monoclonal anti-SfaA and anti-SfaS antlbodles the functlons of the minor subunlts were determined. lt was determlned that SfaS ls ldentlcal to the S-specific adhesln; whlch also plays a role ln deterrninatlon of the degree of fimbri· ation ofthe cell. The mlnor subunit SfaH also had some Jnfluence on the Ievei of fimbrlation of the cell. while StaG ls necessary for full expression of S·specific binding. lt was further shown that the amino-terminal proteln sequence of the isolated SfaS profein was identJcal to the proteln sequence calculated from the DNA sequence of the sfaS gene locus.}, subject = {Infektionsbiologie}, language = {en} } @article{MarreHackerBraun1989, author = {Marre, R. and Hacker, J{\"o}rg and Braun, V.}, title = {The cell-bound hemolysin of Serratia marcescens contributes to uropathogenicity}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59576}, year = {1989}, abstract = {No abstract available}, subject = {Infektionsbiologie}, language = {en} }