TY - JOUR A1 - Siegel, T. Nicolai A1 - Hon, Chung-Chau A1 - Zhang, Qinfeng A1 - Lopez-Rubio, Jose-Juan A1 - Scheidig-Benatar, Christine A1 - Martins, Rafeal M. A1 - Sismeiro, Odile A1 - Coppée, Jean-Yves T1 - Strand-specific RNA-Seq reveals widespread and developmentally regulated transcription of natural antisense transcripts in Plasmodium falciparum JF - BMC Genomics N2 - 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. KW - directional RNA-Seq KW - ncRNA KW - natural antisense transcripts KW - 3′ UTR KW - polyadenylation sites KW - genes KW - antisense RNA KW - plasmodium falciparum Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119892 VL - 15 ER - TY - JOUR A1 - Adelfinger, Marion A1 - Gentschev, Ivaylo A1 - de Guibert, Julio Grimm A1 - Weibel, Stephanie A1 - Langbein-Laugwitz, Johanna A1 - Härtl, Barbara A1 - Escobar, Hugo Murua A1 - Nolte, Ingo A1 - Chen, Nanhai G. A1 - Aguilar, Richard J. A1 - Yu, Yong A. A1 - Zhang, Qian A1 - Frentzen, Alexa A1 - Szalay, Aladar A. T1 - Evaluation of a New Recombinant Oncolytic Vaccinia Virus Strain GLV-5b451 for Feline Mammary Carcinoma Therapy JF - PLoS ONE N2 - 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. KW - antibodies KW - cancer treatment KW - carcinomas KW - vaccinia virus KW - oncolytic viruses KW - viral replication KW - cell cultures KW - enzyme-linked immunoassays Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119387 VL - 9 IS - 8 ER - TY - JOUR A1 - Li, Lei A1 - Wong, Hin-chung A1 - Nong, Wenyan A1 - Cheung, Man Kit A1 - Law, Patrick Tik Wan A1 - Kam, Kai Man A1 - Kwan, Hoi Shan T1 - Comparative genomic analysis of clinical and environmental strains provides insight into the pathogenicity and evolution of Vibrio parahaemolyticus JF - BMC Genomics N2 - 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. " KW - comparative genomics KW - clinical KW - environment KW - vibrio parahaemolyticus Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-118080 SN - 1471-2164 VL - 15 IS - 1135 ER - TY - JOUR A1 - Reynolds, David A1 - Cliffe, Laura A1 - Förstner, Konrad U. A1 - Hon, Chung-Chau A1 - Siegel, T. Nicolai A1 - Sabatini, Robert T1 - Regulation of transcription termination by glucosylated hydroxymethyluracil, base J, in Leishmania major and Trypanosoma brucei JF - Nucleic Acids Research N2 - 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. KW - RNA-polymerase-II KW - variant surface glycoprotein KW - SWI2/SNF2-like protein KW - messenger RNA KW - polycistronic transcription KW - DNA glycosation KW - hela cells KW - gene expression KW - genome KW - 5-bromodeoxyuridine Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117863 VL - 42 IS - 15 ER - TY - JOUR A1 - Jun, Kyong-Hwa A1 - Gholami, Spedideh A1 - Song, Tae-Jin A1 - Au, Joyce A1 - Haddad, Dana A1 - Carson, Joshua A1 - Chen, Chun-Hao A1 - Mojica, Kelly A1 - Zanzonico, Pat A1 - Chen, Nanhai G. A1 - Zhang, Qian A1 - Szalay, Aladar A1 - Fong, Yuman T1 - A novel oncolytic viral therapy and imaging technique for gastric cancer using a genetically engineered vaccinia virus carrying the human sodium iodide symporter JF - Journal of Experimental & Clinical Cancer Research N2 - 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. KW - oncolytic viral therapy KW - GLV-1 h153 KW - gastric cancer KW - human sodium iodide symporter (hNIS) KW - radioiodine therapy KW - gene therapy KW - expression KW - replication KW - stomach KW - tumors KW - surgery Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117716 SN - 1756-9966 VL - 33 IS - 2 ER - TY - JOUR A1 - Bielaszewska, Martina A1 - Schiller, Roswitha A1 - Lammers, Lydia A1 - Bauwens, Andreas A1 - Fruth, Angelika A1 - Middendorf, Barbara A1 - Schmidt, M. Alexander A1 - Tarr, Phillip I. A1 - Dobrindt, Ulrich A1 - Karch, Helge A1 - Mellmann, Alexander T1 - Heteropathogenic virulence and phylogeny reveal phased pathogenic metamorphosis in Escherichia coli O2:H6 JF - EMBO Molecular Medicine N2 - 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. KW - phased metamorphosis KW - phylogeny KW - heteropathogenicity KW - Shiga toxin-producing Escherichia coli KW - hemolytic-uremic syndrome KW - urinary-tract-infection KW - cytolethal distending toxin KW - shiga toxin KW - Crohns-disease KW - outbreak KW - genes KW - island KW - strains KW - parallel evolution KW - uropathogenic Escherichia coli Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117254 SN - 1757-4684 VL - 6 IS - 3 ER - TY - JOUR A1 - Nguyen, Tu N. A1 - Müller, Laura S. M. A1 - Park, Sung Hee A1 - Siegel, T. Nicolai A1 - Günzl, Arthur T1 - Promoter occupancy of the basal class I transcription factor A differs strongly between active and silent VSG expression sites in Trypanosoma brucei JF - Nucleic Acid Research N2 - 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. KW - RNA-polymerase-I KW - blood-stream forms KW - acrican trypanosomes KW - gene expression KW - antigenic variation KW - ribosomal RNA KW - plasmodium falciparum KW - virulence genes KW - subunit KW - complex Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117232 SN - 1362-4962 VL - 42 IS - 5 ER - TY - JOUR A1 - Bischler, Thorsten A1 - Kopf, Matthias A1 - Voss, Bjoern T1 - Transcript mapping based on dRNA-seq data JF - BMC Bioinformatics N2 - 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. KW - transcriptional start site KW - dynamic programming KW - RNA-seq KW - differential KW - segmentation KW - transcriptional uni KW - transcriptome KW - reveals KW - model Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-116663 SN - 1471-2105 VL - 15 IS - 122 ER - TY - JOUR A1 - Wagner, Ines A1 - Volkmer, Michael A1 - Sharan, Malvika A1 - Villaveces, Jose M. A1 - Oswald, Felix A1 - Surendranath, Vineeth A1 - Habermann, Bianca H. T1 - morFeus: a web-based program to detect remotely conserved orthologs using symmetrical best hits and orthology network scoring JF - BMC Bioinformatics N2 - 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. KW - reciprocal best hit KW - finder using symmetrical best hits KW - sequences KW - annotation KW - identification KW - database KW - genomes KW - proteins KW - homologs KW - hidden markov-models KW - phylogenetic trees KW - PSI-blast KW - eigenvector centrality KW - meta-analysis based orthology KW - orthology KW - remote sequence conservation KW - alignment clustering KW - orthology network Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115590 VL - 15 IS - 263 ER - TY - JOUR A1 - Talman, Arthur M. A1 - Prieto, Judith H. A1 - Marques, Sara A1 - Ubaida-Mohien, Ceereena A1 - Lawniczak, Mara A1 - Wass, Mark N. A1 - Xu, Tao A1 - Frank, Roland A1 - Ecker, Andrea A1 - Stanway, Rebecca S. A1 - Krishna, Sanjeev A1 - Sternberg, Michael J. E. A1 - Christophides, Georges K. A1 - Graham, David R. A1 - Dinglasan, Rhoel R. A1 - Yates, John R., III A1 - Sinden, Robert E. T1 - Proteomic analysis of the Plasmodium male gamete reveals the key role for glycolysis in flagellar motility JF - Malaria Journal N2 - 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. KW - glycolysis KW - gamete KW - energy metabolism KW - tandem mass-spectra KW - YoelII-Nigeriensis KW - haemoproteus-columbae KW - chlamydomonas flagella KW - life cycle KW - microtubule motor KW - hexose transporter KW - membrane-protein topology KW - malaria parasite KW - subcellular localization KW - flagellum KW - plasmodium Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115572 N1 - Additional files are available here: http://www.malariajournal.com/content/13/1/315/additional VL - 13 IS - 315 ER - TY - JOUR A1 - Remes, Bernhard A1 - Berghoff, Bork A. A1 - Förstner, Konrad U. A1 - Klug, Gabriele T1 - Role of oxygen and the OxyR protein in the response to iron limitation in Rhodobacter sphaeroides JF - BMC Genomics N2 - 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. KW - oxidative stress KW - Rhodobacter sphaeroides KW - RNAseq KW - OxyR KW - iron limitation KW - transcriptomics KW - dependent gene-expression KW - hydrogen-peroxide KW - escherichia coli Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115357 SN - 1471-2164 VL - 15 IS - 794 ER - TY - JOUR A1 - Lindgreen, Stinus A1 - Umu, Sinan Uğur A1 - Lai, Alicia Sook-Wei A1 - Eldai, Hisham A1 - Liu, Wenting A1 - McGimpsey, Stephanie A1 - Wheeler, Nicole E. A1 - Biggs, Patrick J. A1 - Thomson, Nick R. A1 - Barquist, Lars A1 - Poole, Anthony M. A1 - Gardner, Paul P. T1 - Robust Identification of Noncoding RNA from Transcriptomes Requires Phylogenetically-Informed Sampling JF - PLOS Computational Biology N2 - 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. KW - protein families database KW - small nucleolar RNAs KW - bacterial genomes KW - comparative genomics KW - dark-matter KW - homology search KW - archaea KW - sequence KW - alignment KW - insights Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115259 VL - 10 IS - 10 ER - TY - JOUR A1 - Rico, Sergio A1 - Yepes, Ana A1 - Rodriguez, Hector A1 - Santamaria, Jorge A1 - Antoraz, Sergio A1 - Krause, Eva M. A1 - Diaz, Margarita A1 - Santamaria, Ramon I. T1 - 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 JF - PLOS ONE N2 - 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. KW - signal-transduction systems KW - biosynthetic gene-cluster KW - escherichia coli KW - response regulator KW - oviedomycin KW - expression KW - organization KW - integration KW - bacteria KW - sequence Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115151 SN - 1932-6203 VL - 9 IS - 10 ER - TY - JOUR A1 - Möller, Philip A1 - Overlöper, Aaron A1 - Förstner, Konrad U. A1 - Wen, Tuan-Nan A1 - Sharma, Cynthia M. A1 - Lai, Erh-Min A1 - Narberhaus, Franz T1 - Profound Impact of Hfq on Nutrient Acquisition, Metabolism and Motility in the Plant Pathogen Agrobacterium tumefaciens JF - PLOS ONE N2 - 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. KW - regulatory small RNAs KW - messenger-RNA KW - protein HFQ KW - bacillus subtilis KW - RNA CHAPERONE HFQ KW - flagellar basal body KW - escherichia coli KW - stress resistance KW - transport systems KW - Erwinia amylovora Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114874 VL - 9 IS - 10 ER - TY - JOUR A1 - Zukher, Inna A1 - Novikova, Maria A1 - Tikhonov, Anton A1 - Nesterchuk, Mikhail V. A1 - Osterman, Ilya A. A1 - Djordjevic, Marko A1 - Sergiev, Petr V. A1 - Sharma, Cynthia M. A1 - Severinov, Konstantin T1 - Ribosome-controlled transcription termination is essential for the production of antibiotic microcin C JF - Nucleic Acids Research N2 - 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. KW - escherichia coli KW - messenger-RNA decay KW - translation KW - expression KW - synthetase KW - enterobacteria KW - inhibitors KW - maturation KW - target KW - stability Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114839 SN - 0305-1048 VL - 42 IS - 19 ER - TY - JOUR A1 - Dimastrogiovanni, Daniela A1 - Fröhlich, Kathrin S. A1 - Bandyra, Katarzyna J. A1 - Bruce, Heather A. A1 - Hohensee, Susann A1 - Vogel, Jörg A1 - Luisi, Ben F. T1 - Recognition of the small regulatory RNA RydC by the bacterial Hfq protein JF - eLife N2 - 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. KW - Hfq KW - small RNA KW - natively unstructured protein KW - protein-RNA recognition KW - gene regulation KW - Escherichia coli-Hfq KW - SM-like protein KW - messenger-RNA KW - chaperone Hfq KW - target recognition KW - noncoding RNAs KW - interaction surfaces KW - crystal-structures KW - soluble-RNAs KW - C-Terminus Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114191 SN - 2050-084X VL - 3 IS - e05375 ER - TY - JOUR A1 - Glaser, Jan A1 - Schultheis, Martina A1 - Hazra, Sudipta A1 - Hazra, Banazri A1 - Moll, Heidrun A1 - Schurigt, Uta A1 - Holzgrabe, Ulrike T1 - Antileishmanial Lead Structures from Nature: Analysis of Structure-Activity Relationships of a Compound Library Derived from Caffeic Acid Bornyl Ester N2 - 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 KW - Valeriana wallichii KW - leishmaniasis KW - caffeic acid bornyl ester KW - structure-activity relationship Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112835 ER - TY - JOUR A1 - Gorski, Stanislaw A. A1 - Vogel, Jörg A1 - Saliba, Antoine-Emmanuel A1 - Westermann, Alexander J. T1 - Single-cell RNA-seq: advances and future challenges N2 - 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. KW - RNS Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-110993 ER - TY - JOUR A1 - Hofmann, Elisabeth A1 - Weibel, Stephanie A1 - Szalay, Aladar A. T1 - Combination treatment with oncolytic Vaccinia virus and cyclophosphamide results in synergistic antitumor effects in human lung adenocarcinoma bearing mice N2 - 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. KW - Vaccinia virus KW - Chemotherapy KW - Combination therapy KW - Cyclophosphamide KW - Lung cancer Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-110168 ER - TY - THES A1 - Schiller, Roswitha Dorothee T1 - Studien zu Virulenzeigenschaften typischer und atypischer uropathogener Escherichia coli T1 - Studies on virulence properties of typical and atypical uropathogenic Escherichia coli N2 - Die Forschungsergebnisse der letzten Jahre liefern immer mehr Hinweise darauf, dass eine klare Unterscheidung von Fitness- und Virulenzfaktoren in vielen Fällen, insbesondere bei extraintestinal pathogenen Escherichia coli, nicht möglich ist. So lässt sich auch bei Harnwegsinfektionen verursachenden E. coli den bakteriellen und teils stammspezifischen Faktoren oftmals nicht eindeutig eine typische Virulenz- oder Fitness-assoziierte Funktion zuordnen. Zudem werden in neueren Studien immer häufiger atypische uropathogene Isolate von E. coli beschrieben, die in ihrem „Virulenzrepertoire“ deutlich von typischen uropathogenen E. coli (UPEC) abweichen, da sie keine klassischen UPEC-Virulenzfaktoren aufweisen. In dieser Arbeit wurden daher Virulenzeigenschaften typischer als auch atypischer UPEC untersucht. Der Effekt eines bestimmten bakteriellen Faktors auf den Wirtsorganismus wird teilweise indirekt durch sekundäre Modifikation bedingt. Dies offenbart sich beispielsweise am Autotransporterprotein AIDA-I, dessen Konformation durch posttranslationale Glykosylierung stabilisiert wird, wodurch es seine Funktionalität als Adhäsin erhält. Da bisherige Studien zum AIDA-I homologen Autotransporterprotein Antigen 43 (Ag43) auf der Analyse von künstlich glykosyliertem Protein basieren, lag ein Schwerpunkt dieser Arbeit auf der Untersuchung der natürlichen Glykosylierung von Ag43 in UPEC Stamm 536. Es zeigte sich, dass beide Ag43-Varianten von E. coli Stamm 536 natürlicherweise glykosyliert vorliegen, der Grad der Glykosylierung jedoch wesentlich geringer ausfällt als bei natürlich glykosyliertem AIDA-I. Inwieweit die natürliche Glykosylierung von Ag43 zu dessen Funktionalität beiträgt, kann erst durch die Identifizierung der für die Ag43-Glykosylierung verantwortlichen Glykosyltransferase geklärt werden. Die in silico-Analyse des Genoms von UPEC Stamm 536 für potentielle Glykosyltransferasen von Ag43 lieferte neun Kandidatengene. Die Gene wurde teils im Wildtyp-Hintergrund, teils im rfaH-negativen Hintergrund von E. coli Stamm 536 deletiert und die Mutanten im Anschluss phänotypisch charakterisiert. Die Deletion der Kandidatengene waaF, waaG und waaQ, die für Glykosyltransferasen des LPS-Biosynthesesystems kodieren, führte zu den deutlichsten Unterschieden in Bezug auf Motilität, Curli/Zellulose-Produktion, Hämolyseaktivität und Expression von Typ 1 Fimbrien. Der Einfluss des „knock-out“ der Kandidatengene auf die Glykosylierung von Ag43 muss in weiterführenden Studien untersucht werden. Zur Charakterisierung des uropathogenen Virulenzpotentials verschiedener E. coli Stämme in vivo hat sich in den letzten Jahren das murine Modell der aufsteigenden Harnwegsinfektion etabliert. Mit Hilfe dieses Modells wurden in der vorliegenden Arbeit sowohl spezifische Deletionsmutanten prototypischer UPEC als auch atypische E. coli Harnwegsisolate bezüglich ihrer Urovirulenz getestet und verglichen. Bei der Untersuchung der klassischen UPEC lag der Fokus auf der möglichen Urovirulenzmodulation durch die folgenden spezifischen Faktoren: dem Autotransporterprotein Ag43, dem „Response regulator“ UvrY, dem Polyketid Colibactin sowie dem Exopolysaccharid poly-β-1,6-N-Acetylglucosamin (PGA). Für Ag43 war bei der Etablierung einer Harnwegsinfektion keine eindeutige Funktion feststellbar. Es ist jedoch denkbar, dass Ag43 zur Langzeitpersistenz im Harnwegstrakt beitragen kann, was in weiteren Studien belegt werden sollte. Die Expression von UvrY in der natürlichen uvrY-Deletionsmutante UPEC Stamm 536 ließ keine Erhöhung des Urovirulenzpotentials im Mausmodell erkennen. In diesem Zusammenhang konnte allerdings gezeigt werden, dass die Expression des Genotoxins Colibactin in UPEC Stamm 536 dessen Virulenz signifikant herabsetzte. Die Untersuchungen zur Relevanz des Exopolysaccharids PGA belegen deutlich, dass PGA für die Langzeitpersistenz von E. coli im murinen Harnwegstrakt benötigt wird. Für die initiale Kolonisierung scheint PGA hingegen keine Bedeutung zu haben. Für atypische UPEC Isolate, die Charakteristika von STEC und EAEC zeigen und sich in ihrem Virulenzmuster deutlich von prototypischen UPEC unterscheiden, ließ sich im murinen Modell der aufsteigenden Harnwegsinfektion, verglichen mit dem UPEC Modellorganismus 536, ein ähnliches, teils sogar erhöhtes uropathogenes Virulenzpotential nachweisen. Die Ergebnisse der Arbeit untermauern somit die heutige Vorstellung bezüglich der Entwicklung und Etablierung einer Harnwegsinfektion, dass verschiedene E. coli Stämme unterschiedliche (Kontroll-) Mechanismen entwickelt haben, um erfolgreich den Harnwegstrakt kolonisieren und eine Infektion auslösen zu können. Zudem weisen sie darauf hin, dass diese Fähigkeit nicht auf Isolate typischer phylogenetischer UPEC Entwicklungslinien beschränkt und auf das Vorhandensein charakteristischer UPEC Virulenzfaktoren angewiesen ist. N2 - Research findings over the last years indicate that in many cases, including extraintestinal pathogenic Escherichia coli, a clear distinction between fitness and virulence factors is not possible. Accordingly, the classical distinction of often strain-specific virulence- and fitness-related traits of uropathogenic E. coli (UPEC) can often not be made. Furthermore, recent studies describe atypical UPEC isolates. These isolates remarkably differ in their “virulence repertoire” compared to typical UPEC, because they lack classical UPEC-related virulence factors. Therefore, the aim of the present study was the investigation of virulence properties of typical as well as atypical UPEC strains. The effect of a certain bacterial factor upon the host organism is in part indirectly influenced by secondary modifications. For instance, the conformation of the autotransporter protein AIDA-I is stabilized by posttranslational glycosylation which in turn confers its functionality as an adhesin. Prior studies on the AIDA-I homologous autotransporter protein antigen 43 (Ag43) are based on the analysis of the artificially glycosylated protein. Thus, a key aspect of the current work was to elucidate the naturally occurring glycosylation of Ag43 in UPEC strain 536. For both Ag43 variants of E. coli 536 natural glycosylation was detected. However, Ag43 was less glycosylated than naturally glycosylated AIDA-I. The future identification of the glycosyltransferase responsible for natural glycosylation of Ag43 will help to determine the impact of this posttranslational modification on the functionality of Ag43. In silico analysis of the UPEC strain 536 genome regarding potential glycosyltransferases of Ag43 revealed nine candidate genes. Corresponding deletion mutants of the identified genes were constructed in part in the wild type strain background and in part in the rfaH-negative background of UPEC 536. The most prominent differences concerning motility, curli/cellulose production, hemolytic activity and expression of type 1 fimbriae were observed upon deletion of the genes waaF, waaG or waaQ coding for glycosyltransferases of the LPS biosynthesis pathway. The impact of the deleted candidate genes on the glycosylation of Ag43 has to be further investigated. In recent years the murine model of ascending urinary tract infection was established to characterize the uropathogenic potential of E. coli strains in vivo. By means of this model the uropathogenic potential of different specific “knock-out” mutants of prototypic UPEC strains as well as of atypical E. coli urinary tract isolates was tested and compared. The analysis of the impact of specific factors on the uropathogenic potential of classical UPEC strains focused on the autotransporter protein Ag43, the response regulator UvrY, the genotoxin colibactin, and the exopolysaccharide poly-β-1,6-N-acetylglucosamine (PGA). Ag43 did not exhibit a distinct function during the establishment of urinary tract infection in mice. However, it is conceivable that Ag43 can contribute to long-term persistence in the urinary tract, which should be covered in further studies. Expression of UvrY in the natural uvrY-negative UPEC strain 536 did not increase the uropathogenic potential. However, expression of the genotoxin colibactin significantly reduced the urovirulence of UPEC strain 536. The exopolysaccharide PGA was shown to contribute to long-term persistence of UPEC in the murine model of urinary tract infection. For the initial colonization of the urinary tract, PGA seems to be dispensable. The atypical UPEC isolates investigated in this study display typical characteristics of STEC and EAEC and differ significantly in their virulence gene content compared to prototypic UPEC strains. Nevertheless, in the murine model of ascending UTI many atypical UPEC isolates exhibited a comparable and sometimes even increased uropathogenic potential relative to UPEC model strain 536. The results of this work support the current idea regarding the development and establishment of a urinary tract infection that different E. coli strains have evolved diverse (control-) mechanisms to successfully colonize the urinary tract and provoke an infection. In addition, the findings point out that the ability to cause a urinary tract infection is not limited to phylogenetic lineages of classical UPEC isolates and the presence of characteristic UPEC virulence traits. KW - Escherichia coli KW - Harnwegsinfektion KW - Glykosylierung KW - UPEC KW - Autotransporterprotein KW - Glykosyltransferase KW - murines Modell der aufsteigenden Harnwegsinfektion Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-103907 ER - TY - CHAP A1 - Förstner, Konrad A1 - Hagedorn, Gregor A1 - Koltzenburg, Claudia A1 - Kubke, Fabiana A1 - Mietchen, Daniel T1 - Collaborative platforms for streamlining workflows in Open Science T2 - Proceedings of the 6th Open Knowledge Conference N2 - 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. KW - Open Science KW - Virtual Research Environment KW - collaboratories KW - workflow platform KW - automation Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-101678 ER - TY - JOUR A1 - Mietchen, Daniel A1 - Hagedorn, Gregor A1 - Förstner, Konrad U. A1 - Kubke, M Fabiana A1 - Koltzenburg, Claudia A1 - Hahnel, Mark J. A1 - Penev, Lyubomir T1 - Wikis in scholarly publishing N2 - 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. KW - Elektronisches Publizieren KW - wikis KW - scientific publishing KW - scholarly publishing KW - reputation KW - version control Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-87770 ER - TY - JOUR A1 - Schroten, Horst A1 - Wolske, Anja A1 - Plogmann, Ricarda A1 - Hanisch, Franz-Georg A1 - Hacker, Jörg A1 - Uhlenbrück, Gerhard A1 - Wahn, Volker T1 - Binding of cloned S-fimbriated E. coli to human buccal epithelial cells-different inhibition of binding by neonatal saliva and adult saliva. N2 - 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. N2 - bestätigt werden. Wurde als Inhibitor Speichel eingesetzt, so ergab sich für den Speichel Neugeborener eine 4-Sfach stärkere Inhibition als für Erwachsenenspeichel Parallel dazu ergab die Untersuchung der Speichelproben für Neugeborene einen 4-Sfachen höheren. Die Adhäsion clonierter, Fluoresceinisothiocyanat (FITC)-markierter, S-Fimbrien tragender E. coli an menschliche Mundschleimhautzellen wurde untersucht. Die fluoreszenzmikroskopische Auswertung ergab, daß 75-95% der Schleimhautzellen Bakterien gebunden hatten. Die Spezifität der Bindung der Bakterien an Sialoglykoproteine konnte durch Inhibitionsexperimente mit Fetuin, saurem arGlykoprotein und N-acetyl-Neuraminsäure Gehalt an Gesamt-N-acetyl-Neuraminsäure. In Westerohlot Analysen konnten nur in Proben nativen Speichels Neugeborener mit Wheat Germ Agglutinin (WGA) reagierende Sialoglykoproteinbanden mit Molekülmassen > 200 kD identifiziert werden, die aufgrund ihres Molekulargewichtes und Färbeverhaltens der Klasse der Mucine zuzuordnen sind. Speichelmucine können einen wichtigen Abwehrmechanismus gegen Infektionen in einer Periode der kindlichen Entwicklung darstellen, in der das sekretorische IgA-System noch nicht voll entwickelt ist. KW - Escherichia coli KW - Speichel KW - Neugeborenes KW - Erwachsener KW - Adhäsion Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86291 ER - TY - JOUR A1 - Morschhäuser, Joachim A1 - Vetter, Viktoria A1 - Korhonen, Timo A1 - Uhlin, Bernt Eric A1 - Hacker, Jörg T1 - Regulation and binding properties of S fimbriae cloned from E. coli strains causing urinary tract infection and meningitis N2 - 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. KW - Escherichia coli KW - Harnwegsinfekt KW - Hirnhautentzündung Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86140 ER - TY - JOUR A1 - Tschäpe, Helmut A1 - Bender, Larisa A1 - Ott, Manfred A1 - Wittig, Walter A1 - Hacker, Jörg T1 - Restriction fragments length polymorphism and virulence pattern of the veterinary pathogen Escherichia coli O139:K82:H1 N2 - 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. KW - Escherichia coli Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86131 ER - TY - JOUR A1 - Hacker, Jörg A1 - Ott, Manfred A1 - Wintermeyer, Eva A1 - Ludwig, Birgit A1 - Fischer, Gunter T1 - Analysis of virulence factors of Legionella pneumophila. N2 - 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. KW - Legionella pneumophila Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-70620 ER - TY - JOUR A1 - Hacker, Jörg A1 - Ott, Manfred A1 - Blum, Gabriele A1 - Marre, Reinhard A1 - Heesemann, Jürgen A1 - Tschäpe, Helmut A1 - Goebel, Werner T1 - Genetics of Escherichia coli uropathogenicity: Analysis of the O6:K15:H31 isolate 536 N2 - 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. KW - Escherichia coli KW - Genetik Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-71578 ER - TY - JOUR A1 - Parkkinen, Jaakko A1 - Hacker, Jörg A1 - Korhonen, Timo K. T1 - Enhancement of tissue plasminogen activator-catalyzed plasminogen activation by Escherichia coli S fimbriae associated with neonatal septicaemia and meningitis. N2 - 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. KW - Escherichia coli Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-71566 ER - TY - JOUR A1 - Morschhäuser, Joachim A1 - Ramírez-Zavala, Bernardo A1 - Weyler, Michael A1 - Gildor, Tsvia A1 - Schmauch, Christian A1 - Kornitzer, Daniel A1 - Arkowitz, Robert T1 - Activation of the Cph1-Dependent MAP Kinase Signaling Pathway Induces White-Opaque Switching in Candida albicans JF - PLoS Pathogens N2 - 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. KW - biofilms KW - candida albicans KW - library screening KW - pheromones KW - protein expressions KW - protein kinase signaling cascade KW - regulator genes KW - transcription factors Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-97281 ER - TY - JOUR A1 - Sharma, Cynthia M. A1 - Dugar, Gaurav A1 - Herbig, Alexander A1 - Förstner, Konrad U. A1 - Heidrich, Nadja A1 - Reinhardt, Richard A1 - Nieselt, Kay T1 - High-Resolution Transcriptome Maps Reveal Strain-Specific Regulatory Features of Multiple Campylobacter jejuni Isolates JF - PLoS Genetics N2 - 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. KW - bacterial genomics KW - CRISPRs KW - genome annotation KW - campylobacter KW - genomic libraries KW - genomic library construction KW - sequence motif analysis KW - transcriptome analysis Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96610 ER - TY - JOUR A1 - Szalay, Aladar A A1 - Weibel, Stephanie A1 - Hofmann, Elisabeth A1 - Basse-Luesebrink, Thomas Christian A1 - Donat, Ulrike A1 - Seubert, Carolin A1 - Adelfinger, Marion A1 - Gnamlin, Prisca A1 - Kober, Christina A1 - Frentzen, Alexa A1 - Gentschev, Ivaylo A1 - Jakob, Peter Michael T1 - Treatment of malignant effusion by oncolytic virotherapy in an experimental subcutaneous xenograft model of lung cancer JF - Journal of Translational Medicine N2 - 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. KW - Oncolytic virotherapy KW - Malignant effusion KW - Lung cancer KW - VEGF KW - Lungenkrebs KW - Vascular endothelial Growth Factor Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96016 UR - http://www.translational-medicine.com/content/11/1/106 ER - TY - THES A1 - Fröhlich, Kathrin T1 - Assigning functions to Hfq-dependent small RNAs in the model pathogen Salmonella Typhimurium T1 - Funktionelle Charakterisierung Hfq-abhängiger kleiner RNAs im Modellpathogen Salmonella Typhimurium N2 - 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. N2 - Die bakterielle Genexpression wird unter anderem maßgeblich von nicht-kodierenden RNAs bestimmt. Kleine regulatorische RNAs (sRNAs) sind eine bezüglich Größe und Struktur heterogene Gruppe von Riboregulatoren, die ihre in cis oder in trans-kodierten Zielgene mittels direkter Basenpaarungen kontrollieren. Während der Großteil der sRNAs reprimierend wirkt, konnte für einige RNAs gezeigt werden, dass sie die Expression ihres Zieltranskripts verstärken. Ein wichtiger Kofaktor für die regulatorische Funktion der sRNAs ist das RNA-Chaperon Hfq, welches sowohl die Umfaltung intramolekularer Sekundärstrukturen ermöglicht, als auch die Ausbildung von Basenpaarungen zwischen komplementären RNA-Sequenzen steuert. Zusätzlich schützt Hfq nicht-gepaarte RNAs vor dem Abbau durch Ribonukleasen, und trägt damit zur Stabilität der Moleküle bei. Durch Ko-Immunopräzipitation mit Hfq sowie in weiteren experimentellen als auch bioinformatischen Studien konnten im letzten Jahrzehnt in diversen Enterobakterien, wie z.B. auch Escherichia coli und Salmonellae, mehr als 150 verschiedene sRNAs bestimmt werden. Von so genannten "core sRNAs" (Kern-sRNAs) wird aufgrund ihres hohen Grades an Konservierung in unterschiedlichen Spezies angenommen, dass sie zentrale Funktionen erfüllen. Etwa 25 core sRNAs agieren unter verschiedenen Umweltbedingungen als Regulatoren. Ihre exakte biologische Rolle, sowie ihre Funktionsweise sind jedoch größtenteils noch unbekannt. In der vorliegenden Arbeit wurden die beiden konservierten, Hfq-abhängigen sRNAs, SdsR und RydC, im Modellpathogen Salmonella Typhimurium charakterisiert. SdsR war als eine der abundantesten sRNAs der stationären Phase in E. coli beschrieben worden. Durch Auswertung der Konservierungsmuster der sdsR Promotorsequenz sowie klassische genetische Analyse konnte SdsR als erste sRNA unter direkter Kontrolle des alternativen σ Faktors σS bestimmt werden. In Salmonella führt die Überexpression von SdsR zur Reprimierung des Membranporins OmpD, und die Bindestelle von SdsR auf dem ompD Transkript wurde mittels einer auf 3'-RACE basierenden Methode ermittelt. Obwohl die Expression von ompD auf post-transkriptionaler Ebene von drei weiteren sRNAs kontrolliert wird, konnte eine spezische Regulation des Porins durch SdsR während Aminosäure-Hungerung gezeigt werden. Auch RydC, die zweite in dieser Studie analysierte sRNA, wurde zunächst in E. coli beschrieben und ist aber auch in weiteren γ-Proteobakterien konserviert. Interessanterweise enthält die Sekundärstruktur dieser Hfq-abhängigen sRNA einen Pseudoknoten, während das 5'-Ende ungepaart ist. Die Zielgene von RydC wurden mittels einer Transkriptomanalyse bestimmt, in der die Änderung der Häufigkeitsverteilung aller mRNAs nach kurzzeitiger Überexpression der sRNA auf Microarrays untersucht wurde. RydC bewirkte die spezifische Aktivierung des längeren von insgesamt zwei Versionen der cfa mRNA, die für eine Cyclopropan-fettsäuresynthase kodiert, ein Enzym das zur Modifikation von Phospholipiden dient. Eine Basenpaarung über das freie 5'-Ende der sRNA RydC führt zur Aktivierung der cfa-Expression, und maskiert eine Erkennungssequenz der Endoribonuklease, RNase E, innerhalb des Transkripts. KW - Small RNA KW - Genexpression KW - Hfq KW - Small RNA KW - Hfq KW - Salmonella KW - Salmonella typhimurium Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-85488 ER - TY - THES A1 - Ngwa, Che Julius T1 - The mosquito midgut-specific stages of the malaria parasite as targets for transmission blocking interventions T1 - Die Moskitomitteldarmstadien des Malariaparasiten als Ziele für übertragungsblockierende Eingriffe N2 - Die Tropenkrankheit Malaria, wird durch eine Infektion mit einzelligen Parasiten der Gattung Plasmodium verursacht und durch den Stich der weiblichen Anopheles-Mücke von Mensch zu Mensch verbreitet. Dabei kann eine erfolgreiche Übertragung des Parasiten auf den Menschen nur dann stattfinden, wenn der Parasit seine sexuelle Entwicklungsphase im Mitteldarm der Mücke erfolgreich durchläuft. Ziel dieser Arbeit war es daher, die Wechselwirkungen des Malariaparasiten im Mitteldarm der Mücke in Hinblick auf die Identifizierung möglicher neuer transmissionsblockierender Strategien zu untersuchen. Der Zweck von transmissionsblockierende Strategien ist es, der Verbreitung der Malaria durch die Mücke entgegenzuwirken, indem die Entwicklung des Parasiten in der Mücke 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 Mücke. Dabei sollte der mögliche Einfluss des Parasiten auf die Darmflora untersucht werden und weiterführend die potentielle Verwendung von Darmbakterien als Vehikel für die Herstellung paratransgener Mücken erforscht werden. Vergleichende16S-rRNA- und DGGE-Analysen an der Darmflora des asiatischen Malariavektors Anopheles stephensi zeigten eine deutliche Reduktion der mikrobiellen Diversität während der Entwicklung vom Ei zur adulten Mücke. Zudem konnte das gram-negative Bakterium Elizabethkingia meningoseptica, das sich stadien- und generationsübergreifend verbreitet, als dominante Darmspezies bei im Labor aufgezogenen weiblichen und männlichen An. stephensi festgestellt werden. Die Dominanz von E. meningoseptica wurde zudem nicht durch die Aufnahme von infiziertem Blut oder einer veränderten Nahrung beeinflusst. Für die Studien wurde sowohl der humanpathogene Parasit P. falciparum als auch der Nagermalariaerreger P. berghei verwendet. Weiterführende Versuche zeigten, dass Extrakte von E. meningoseptica antibakterielle, antifungale und antiplasmodiale Aktivitäten aufwiesen, die ein möglicher Grund für 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-Mücken dar. Ein zweites Ziel dieser Arbeit war es, mögliche Unterschiede in der Genexpression von P. falciparum darzustellen, die in den ersten 30 Minuten nach dessen Übertragung auf die Mücke erfolgen. Dies hatte zum einen zum Zweck, die durch den Wirtswechsel hervorgerufenen Genregulationen besser zu verstehen, und bot zum anderen die Möglichkeit, neue Proteine zu identifizieren, die als potentielle transmissionsblockierende Ziele genutzt werden können. Mittels supression substractive hybridization (SSH) konnten insgesamt 126 Gene identifiziert werden, deren Expression sich während der Gametogenese verändert. 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 für zelloberflächenassoziierte Proteine. 11,9% der Gene hatten anderen Funktionen, während 20% der Gene keine putative Funktion zugeordnet werden konnte. Etwa 40% der identifizierten Genprodukte waren bisher nicht in Proteomstudien nachgewiesen worden. In weiterführenden Analysen wurden 34 Gene aus jeder ontologischen Gruppe ausgewählt und deren Expressionsveränderung per quantitativer real time RT-PCR im Detail untersucht. Für 29 Gene konnte dabei eine Transkriptexpression in Gametozyten nachgewiesen werden. Zudem wiesen 20 Gene eine erhöhte Expression in Gametozyten im Vergleich asexuellen Stadien auf. Insgesamt zeigten 8 Gene besonders hohe Transkriptlevel in aktivierten Gametozyten, was auf eine Funktion dieser Proteine während der Übertragung des Parasiten auf die Mücke hindeutet und diese somit potentielle Angriffspunkte für transmissionsblockierende Strategien darstellen könnten. 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 Hämolymphe verschiedener Insekten isoliert oder rekombinant in transgenem Tabak exprimiert worden. Dabei wurden die rekombinanten Peptide so ausgewählt, dass sie entweder gegen die Mitteldarmstadien des Parasiten wirken oder mückenspezifische Rezeptoren blockieren, die der Parasit für seine weitere Entwicklung benötigt. Dabei konnte gezeigt werden, dass das antimikrobielle Molekül Harmonin, ein Abwehrmolekül aus der Hämolymphe des asiatischen Marienkäfers Harmonia axyridis, antiplasmodiale als auch transmissions-blockierende Eigenschaften besitzt. Harmonin stellt daher eine potentielle Leitstruktur für die Entwicklung neuer Malariawirkstoffe dar N2 - Malaria is a vector-borne disease caused by the protozoan parasite of the genus Plasmodium and it is transmitted from human to human by female Anopheles mosquitoes during a blood meal. For malaria transmission to occur, the malaria parasite must undergo a crucial developmental sexual phase inside the mosquito midgut. In this study, we sought to investigate the interplay of the malaria parasite in the mosquito midgut with regard to the identification of novel types of transmission blocking intervention strategies. These strategies are aimed at reducing the spread of malaria by blocking the development of the mosquito midgut-specific stages of Plasmodium. We focused on three aspects. The first aspect was to investigate the interplay between mosquito midgut bacteria and malaria parasites in order to determine the potential influence of malaria parasites on the composition of the mosquito gut microbiota and also determine midgut bacteria which could be exploited as vehicles for the generation of paratransgenic Anopheles mosquitoes. We analyzed the microbial diversity of gut bacteria of the Asian malaria vector Anopheles stephensi during development and under different feeding regimes, including feeds on malaria parasite-infected blood, using the human pathogenic P. falciparum as well as the rodent malaria model P. berghei. 16S rRNA and DGGE analyses demonstrated a reduction in the microbial diversity during mosquito development from egg to adult and identified the gram-negative bacterium Elizabethkingia meningoseptica as the dominant species in the midgut of laboratory-reared male and female mosquitoes. E. meningoseptica is transmitted between generations and its predominance in the mosquito midgut was not altered by diet, when the gut microbiota was compared between sugar-fed and blood-fed female mosquitoes. Furthermore, feeds on blood infected with malaria parasites did not impact the presence of E. men-ingoseptica in the gut. Interestingly, extracts from E. meningoseptica exhibited antibacterial, antifungal and antiplasmodial activities, which may account for its dominance in the midgut of the malaria vector. Isolates of E. meningoseptica were cultivable, making the bacterium a potential candidate vehicle for the generation of paratransgenic Anopheles mosquitoes. The second aspect of this thesis was to determine transcriptome changes that occur during the first half hour following transmission of P. falciparum to the mosquito vector in order to better understand gene regulation mechanisms important for the change of hosts and determine novel proteins which could be exploited in malaria transmission blocking interventions. We initially used suppression subtractive hybridization (SSH) to compare mRNA levels of P. falciparum gametocytes before and 30 min fol-lowing activation. We identified a total of 126 genes for which transcript expression changed during gametogenesis. Among these, 17.5% had putative functions in signaling, 14.3% were assigned to cell cycle and gene expression, 8.7% were linked to the cytoskeleton or motor complex, 7.9% were involved in proteostasis and 6.4% in metabolism, 12.7% were genes encoding for cell surface associated proteins, 11.9% were assigned to other functions, and 20.6% represented genes of unknown function. For 40% of the identified genes there has as yet not been any protein evidence. We further selected a subset of 34 genes from all the above ontology groups and analyzed the transcript changes during gametogenesis in detail by quantitative realtime RT-PCR. Of these, 29 genes were expressed in gametocytes, and for 20 genes transcript expres-sion in gametocytes was increased compared to asexual blood stage parasites. Transcript levels of eight genes were particularly high in activated gametocytes, pointing at functions downstream of gametocyte transmission to the mosquito which could be exploited in malaria transmission blocking strategies. The last aspect of this thesis was to determine the transmission blocking effect of a range of antimicrobial molecules as transmission blocking agents. The molecules were either isolated from insect hemolymph or recombinantly expressed in tobacco and designed to act either directly on the mosquito midgut stages or cover receptors on mosquito tissues like the midgut epithelium which the parasite would need for transit. We were able to show an antiplasmodial and transmission blocking effect of the anti-microbial molecule harmonine, a defense compound isolated from the hemolymph of the Asian ladybug Harmonia axyridis. Harmonine thus represents a potential lead structure for the development of novel antimalarials. KW - Malariamücke KW - Anopheles stephensi KW - Mitteldarm KW - Malaria KW - Mosquito KW - midgut KW - transmission KW - Malaria KW - Krankheitsübertragung KW - Mücke KW - Mitteldarm Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-83594 ER - TY - THES A1 - Dunkel, Nico T1 - Regulation of virulence-associated traits of the human fungal pathogen Candida albicans by nitrogen availability T1 - Regulation Virulenz-assoziierter Faktoren im humanpathogenen Pilz Candida albicans durch Stickstoffverfügbarkeit N2 - 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. N2 - Stickstoffregulierte Pathogenität bezeichnet die Kontrolle von Virulenz-assoziierten Eigenschaften als direkte Folge der verfügbaren Quantität und Qualität einer Stickstoffquelle. Im Zusammenhang mit der Stickstoffverfügbarkeit verändert der opportunistisch krankheitserregende Pilz Candida albicans seine Morphologie und sekretiert Aspartat-Proteasen [SAPs], beides gut charakterisierte Virulenzattribute. Im Gegensatz zu seinem normalerweise apathogenen Verwandten Saccharomyces cerevisiae ist C. albicans in der Lage Proteine zu verwerten, welche als sehr häufige und wichtige Stickstoffquelle im menschlichen Wirt angesehen werden. Zur Nutzung von Proteinen sekretiert C. albicans Aspartat-Proteasen für den außerzellulären Verdau der Proteine und exprimiert Peptidtransporter (Di- /Tripeptidtransporter [PTRs] und Oligopeptidtransporter [OPTs]) um die Abbauprodukte aufzunehmen. Beide Eigenschaften werden transkriptionell von Stp1 - dem globalen Regulator zur Verwertung von Proteinen - kontrolliert. Ziel der vorliegenden Arbeit war es, die Regulation von Virulenzattributen im pathogenen Pilz C. albicans durch die Verfügbarkeit von Stickstoff genauer zu untersuchen. Innerhalb einer genomweiten Bindestudie von Stp1 wurden mehr als 600 Stp1-Zielgene während des Wachstums mit Proteinen identifiziert. Dadurch bestätigte sich die Funktion von Stp1 in der Proteinverwertung und wurde zudem auch auf die allgemeine Verwertung von Stickstoffquellen erweitert. Des Weiteren deuten die aufgedeckten Zielgene an, dass Stp1 womöglich in der Adaption an die generelle Nährstoffverfügbarkeit sowie in der Antwort auf Stresssignale beteiligt ist. Mit dem Fokus auf die Proteinverwertung und stickstoffregulierter Pathogenität wurde die Regulation der wichtigsten sekretierten Protease Sap2 - welche außerdem ein Paradebeispiel für allelische Heterogenität ist - im Detail untersucht. Dabei half der heterogene SAP2-Promoter bei der Identifizierung einer unbeabsichtigten genomischen Veränderung als wahren Grund eines Wachstumsdefektes einer C. albicans Mutante. Zusätzlich wurde der Promotorbereich eingegrenzt, welcher für die unterschiedliche Aktivierung der beiden SAP2 Allele verantwortlich ist. Weiterhin wurden verschiedene cis-aktive Elemente identifiziert, die entweder für eine hohe oder eine niedrige SAP2 Expression benötigt werden. Die Aufnahme von Peptiden, die durch den außerzellulären Verdau entstehen, ist für die Verwertung von Proteinen ebenso wichtig. Deshalb wurde die Funktion und Bedeutung der spezifischen Peptidtransporter anhand von C. albicans Mutanten untersucht, welche Peptide nicht aufnehmen können (opt1Δ/Δ opt2Δ/Δ opt3Δ/Δ opt4Δ/Δ opt5Δ/Δ ptr2Δ/Δ ptr22Δ/Δ Septuplemutanten). Die Überexpression von individuellen Transportern in diesen Septuplemutanten offenbarte unterschiedliche Substratspezifitäten und erweiterte die Spezifität für die OPTs auf Dipeptide, eine komplett neue Facette dieser Transporter. Des Weiteren ermöglichten die Septuplemutanten eine Aufklärung, ob Proteine und Peptide tatsächlich eine wichtige In Vivo Stickstoffquelle für C. albicans sind. Dieses Arbeit zeigte, dass während der kompetitiven Kolonisierung des Mäusedarms die Septuplemutanten wildtypische Fitness aufwiesen. Dies deutet daraufhin, dass weder Proteine noch Peptide eine wichtige Stickstoffquelle für ein effizientes Wachstum in diesem In Vivo Model sind. Die ausreichende Verfügbarkeit der bevorzugten Stickstoffquelle Ammonium unterdrückt die Verwertung von Proteinen und anderen alternativen Stickstoffquellen. Aber auch die Expression von Virulenzattributen, wie die Proteasesekretion und die stickstoffmangel-induzierte Filamentierung, wird durch Ammonium inhibiert. Um zu unterscheiden, ob C. albicans die Ammoniumverfügbarkeit außerzellulär oder in der Zelle bestimmt, wurde das Verhalten auf außerzelluläre Ammoniumkonzentrationen in Mutanten untersucht, welche Ammonium nicht aufnehmen können (mep1Δ/Δ mep2Δ/Δ Mutanten). Diese Arbeit zeigte, dass in diesen Mutanten eine ansonsten inhibierende Ammoniumkonzentration nicht in der Lage war, die Sekretion der Sap2-Protease oder die Arginin-induzierte Hyphenbildung zu unterdrücken. Folglich wird, um die Expression von Virulenzattributen zu regulieren, die Ammoniumverfügbarkeit vorrangig in der Zelle bestimmt. Zusammenfassend erweitert die vorliegende Arbeit das Verständnis zur Regulation der Expression von Virulenzattributen durch die Verfügbarkeit von Stickstoffquellen - insbesondere Proteine und Peptide - die eine Anpassung von C. albicans an ein Leben im menschlichen Wirt ermöglichen. KW - Candida albicans KW - Regulation KW - Stickstoff KW - Virulenz KW - Proteasen KW - Nitrogen KW - SAP2 KW - STP1 KW - peptide KW - transport KW - ammonium KW - protease KW - Proteine Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-83076 ER - TY - THES A1 - Schillig, Rebecca T1 - Funktionelle Analyse der Zink-Cluster-Transkriptionsfaktorfamilie von Candida albicans durch artifizielle Aktivierung T1 - Functional analysis of the zinc cluster transcription factor family of Candida albicans by artificial activation N2 - Der Hefepilz Candida albicans gehört zu den opportunistischen Infektionserregern. Er ist Teil der natürlichen Mikroflora der Schleimhäute des Gastrointestinal- und Urogenitaltraktes des Menschen. Bei Störungen des natürlichen Gleichgewichts dieser Flora kann es zu oberflächlichen Mykosen, z. B. der oropharyngealen Candidiasis (Mundsoor), kommen. Besonders immunsupprimierte Patienten, wie AIDS-Patienten, leiden häufig unter immer wiederkehrenden Infektionen, die mitunter auch zu schwerwiegenden Infektionsverläufen, bis hin zu lebensbedrohlichen systemischen Mykosen führen können. Zur Therapie solcher Erkrankungen werden oft Ergosterolbiosyntheseinhibitoren, wie Fluconazol, eingesetzt. Besonders bei wiederkehrenden Infektionen und wiederholender Therapie ist C. albicans in der Lage, gegen diese häufig verabreichten Antimykotika Resistenzen zu entwickeln. Hierbei spielen Zink-Cluster-Transkriptionsfaktoren eine zentrale Rolle. Zink-Cluster-Proteine gehören zu einer pilzspezifischen Familie von Transkriptionsfaktoren, die ein großes Spektrum an zellulären Prozessen regulieren. Die gut charakterisierten Regulatoren Upc2, Tac1 und Mrr1 gehören zu den Zink-Cluster-Transkriptionsfaktoren, die maßgeblich zur Resistenzentwicklung von C. albicans beitragen. Upc2 kontrolliert die Expression vieler Ergosterolbiosynthesegene, besonders die von ERG11, welches für die Zielstruktur des gängigen Antimykotikums Fluconazol kodiert. Tac1 und Mrr1 hingegen regulieren die Expression von Multidrug-Effluxpumpen, den ABC-Transportern CDR1 und CDR2 bzw. dem Major Facilitator MDR1. Gain-of-function-Mutationen in diesen Transkriptionsfaktoren resultieren in einer konstitutiven Überexpression ihrer Zielgene und sind verantwortlich für die Resistenz vieler klinischer Isolate. In dieser Arbeit wurde gezeigt, dass die Fusion von Mrr1 mit der Gal4-Aktivierungsdomäne von Saccharomyces cerevisiae zu einem konstitutiv aktiven Hybridtranskriptionsfaktor führte, der eine MDR1-Überexpression bewirkte und Fluconazolresistenz vermittelte. Dieses Hybridprotein vermittelte sogar eine höhere Resistenz als ein Mrr1 mit natürlich vorkommenden gain-of-function-Mutationen. Analoge Fusionen mit Tac1 und Upc2 resultierten ebenfalls in einer konstitutiven Aktivierung dieser Transkriptionsfaktoren, die einen starken Anstieg der Fluconazolresistenz zur Folge hatte. Daraus ergab sich die Schlussfolgerung, dass dies eine generelle Methode sein könnte, die Zink-Cluster-Transkriptionsfaktoren künstlich zu aktivieren und so ihre biologischen Funktionen zu offenbaren, ohne die genauen Bedingungen für ihre Aktivität zu kennen. Deshalb wurde auf der Basis dieser Strategie eine Bibliothek von C.-albicans-Stämmen konstruiert, in der alle 82 putativen Zink-Cluster-Transkriptionsfaktoren in dieser möglicherweise hyperaktiven Form exprimiert werden. Untersuchungen dieser Bibliothek offenbarten neue Transkriptionsfaktoren, die Fluconazolresistenz vermittelten, aber auch noch unbekannte Regulatoren der Morphogenese und andere Phänotypen konnten beobachtet werden. Um einen tieferen Einblick in die Funktionsweise zu bekommen, wurden die Transkriptionsprofile der vier Transkriptionsfaktoren ermittelt, die in ihrer hyperaktiven Form die höchste Fluconazolresistenz bewirkten. Dabei stellte sich heraus, dass die zwei künstlich aktivierten (*) Regulatoren ZCF34* und ZNC1* die Expression der wichtigsten Multidrug-Effluxpumpe CDR1 stark hochregulierten. Der Transkriptionsfaktor mit dem vorläufigen Namen ZCF34 konnte im Verlauf dieser Arbeit als ein wichtiger Regulator für die CDR1-Expression identifiziert werden. Er ist sowohl an der Aktivierung der Expression von CDR1 beteiligt als auch für die basale CDR1-Promotoraktivität notwendig. Aus diesem Grund wurde er in MRR2 (multidrug resistance regulator 2) umbenannt. Mit der Entdeckung eines neuen Regulators der wichtigsten Multidrug-Effluxpumpe von C. albicans wurde ein wichtiger Beitrag zum Verständnis der Regulation solcher Transporter geleistet. Die Überexpression dieser Pumpen ist einer der häufigsten Resistenzmechanismen in C. albicans. Auf diesem Wege kann Resistenz gegen strukturell völlig unterschiedliche Antimykotika bewirkt werden. Somit stellen sowohl diese Effluxpumpen, als auch deren Regulatoren mögliche Angriffsziele für die Entwicklung neuer oder Weiterentwicklung bereits vorhandener Antimykotika dar. N2 - The yeast Candida albicans is an oppotunistic fungal pathogen, usually a harmless colonizer of mucosal surfaces of the gastrointestinal und urogenital tract of healthy people. If the balance of this microflora is disturbed, it can cause superficial mycoses, like oropharyngeal candidiasis. Especially immunocompromised patients, like AIDS patients suffer from recurrent infections, occasionally causing life-threatening systemic infections. The antifungal agent fluconazole, which inhibits ergosterol biosynthesis, is frequently used to treat Candida-infections. Particularly during long term treatments of recurrent infections, C. albicans can develop resistance to the commonly used antifungal drugs. Zinc cluster transcription factors often play key roles in the development of such resistances. The zinc cluster proteins are a fungus-specific family of transcription factors that regulate a variety of cellular processes. The well characterized regulators Upc2, Tac1 und Mrr1 are among these zinc cluster transcription factors, being significantly involved in mediating drug resistance. Upc2 controls the expression of ergosterol biosynthesis genes, e. g. of ERG11, encoding the target enzyme of fluconazole. Tac1 and Mrr1 regulate the expression of multidrug efflux pumps, the ABC transporters CDR1 and CDR2 and the major facilitator MDR1, respectively. Gain-of-function mutations in these transcription factors result in constitutive overexpression of their target genes and are responsible for drug resistance in many clinical C. albicans strains. In this thesis it could be shown that fusion of the full-length Mrr1 with the Gal4 activation domain from Saccharomyces cerevisiae produced a constitutively active hybrid transcription factor that mediated MDR1 overexpression and increased drug resistance. The hybrid transcription factor exhibited even higher activity than Mrr1 with a naturally occurring gain-of-function mutation. Analogous fusions with Tac1 and Upc2 also resulted in constitutively activated transcription factors that conferred strongly increased drug resistance, suggesting that this might be a generally applicable approach for the artificial activation of zinc cluster transcription factors, which could reveal their biological function without prior knowledge about inducing conditions. Therfore a library of C. albicans strains expressing all 82 predicted zinc cluster transcription factors of this pathogen was constructed, by using this strategy, resulting in strains with potentially hyperactive regulators. Screening of this comprehensive set of strains revealed novel transcription factors mediating drug resistance, but also previously unknown regulators of morphogenesis and other phenotypes. To gain insight into their mechanism of action, transcriptional profiles were determined of the four transcription factors that produced the strongest increase in fluconazole resistance when expressed in a hyperactive form. This analysis revealed that two out of these four artificially activated (*) transcription factors, ZCF34* and ZNC1*, strongly upregulate the expression of the most important multidrug efflux pump CDR1, which could be verified by Northern hybridization. The transcription factor previously named ZCF34 could be identified as a new and important regulator of CDR1, being involved in the activation of CDR1 expression as well as in basal promoter activity of this pump. Therefore it was renamed MRR2 (multidrug resistance regulator 2). The identification of MRR2 as a new regulator of the most important multidrug efflux pump in C. albicans represents a major step forward in understanding the regulation of such transporters. The overexpression of these efflux pumps is one of the most common resistance mechanism in C. albicans, conferring resistance to many structurally and functionally unrelated toxic compounds. Therefore these transporters, as well as their regulators, provide potential tagets of new or further developed antifungal agents. KW - Candida albicans KW - Zink-Cluster-Transkriptionsfaktoren KW - Resistenzmechanismen KW - artifizielle Aktivierung KW - CDR1-Effluxpumpe KW - Fluconazol KW - Zink-Finger-Proteine KW - Resistenz KW - Antimykotikum Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-79608 ER - TY - JOUR A1 - Schulte, Leon N. A1 - Westermann, Alexander J. A1 - Vogel, Jörg T1 - Differential activation and functional specialization of miR-146 and miR-155 in innate immune sensing N2 - 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. KW - Medizin Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-76365 ET - Advance Access ER - TY - JOUR A1 - Pernitzsch, Sandy R. A1 - Sharma, Cynthia M. T1 - Transcriptome Complexity and Riboregulation in the Human Pathogen Helicobacter pylori KW - Medizin KW - RNA-seq KW - sRNA KW - Helicobacterpylori KW - post-transcriptionalregulation KW - transcriptomeanalysis Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-75096 ER - TY - THES A1 - Bergmann, Anna T1 - Untersuchungen zur Verwertung proteinhaltiger Substrate als mögliche Virulenzdeterminante des humanpathogenen Schimmelpilzes Aspergillus fumigatus T1 - Studies on utilisation of proteinaceous substrates as potential virulence determinant of the human pathogen Aspergillus fumigatus N2 - Die asexuellen Sporen von Aspergillus fumigatus sind ubiquitär verbreitete Luftkeime. Als Saprophyt ist dieser opportunistisch humanpathogene Pilz darauf spezialisiert, polymere Substanzen aus dem umgebenden Milieu zu zersetzen, um daraus die von ihm benötigten Nährstoffe zu generieren und aufzunehmen. Die Fähigkeit, verschiedene Stickstoff- und Kohlenstoffquellen zu verwerten, trägt dabei zu seiner Virulenz bei und hierbei scheint die extrazelluläre Proteolyse eine wichtige Rolle zu spielen. Sekretierte Proteasen, die das umgebende Gewebe während einer Infektion mit A. fumigatus erschließen, könnten somit zu dessen Pathogenität beitragen. Dementsprechend sollte im Rahmen dieser Arbeit die Bedeutung einer Regulation der extrazellulären proteolytischen Aktivität von A. fumigatus für dessen Virulenz untersucht werden. Dies geschah durch Untersuchungen eines konservierten Transkriptionsfaktors, PrtT. Dabei stellte sich heraus, dass PrtT die Expression der drei Hauptproteasen von A. fumigatus, Alp, Mep und Pep stark beeinflusst, in einem murinen Tiermodell der pulmonaren Aspergillose scheint dieser Regulator jedoch keine Rolle für die Pathogenität von A. fumigatus zu spielen. Um einen weiteren Aspekt des pilzlichen Aminosäurestoffwechsels zu beleuchten, wurde die Biosynthese der aromatischen Aminosäuren als mögliche Virulenzdeterminate untersucht. Für den Menschen sind diese Aminosäuren essentiell, weshalb dieser Syntheseweg ein mögliches Ziel für antimykotische Substanzen darstellen könnte. Es konnten mehrere für A. fumigatus essentielle Komponenten des Shikimatweges identifiziert werden, des Weiteren wurden Deletionsmutanten in den Genen aroC und trpA, die für die Chorismatmutase bzw. Anthranilatsynthase der Biosynthese von Phenylalanin und Tyrosin bzw. Tryptophan kodieren, erzeugt und phänotypisch charakterisiert. Deren Untersuchung in einem alternativen Tiermodell der Aspergillose zeigte eine deutlich attenuierte Virulenz. Diese Ergebnisse verdeutlichen, wie wichtig die Biosynthese der aromatischen Aminosäuren für das Wachstum von A. fumigatus ist, und dass ein Eingriff in diesen Syntheseweg eine lohnende Strategie zur Entwicklung neuer Antimykotika sein könnte. Die hier präsentierten Ergebnisse unterstreichen die für den Schimmelpilz A. fumigatus typische Redundanz bezüglich extrazellulärer proteolytischer Enzyme und dass diese nur bedingt hinsichtlich ihres Virulenzbeitrags untersucht werden können. Im Gegensatz hierzu lassen sich bestimmte Stoffwechselwege, die oftmals durch einzigartige Genprodukte katalysiert werden, unter Umständen besser als unspezifische aber vielversprechende Virulenzdeterminanten identifizieren. N2 - The air-borne spores of Aspergillus fumigatus are ubiquitously distributed. As a saprophyte, this fungus is well adapted to feed from the environment by degradation of polymeric substances and uptake of breakdown products. The nutritional versatility has to be regarded as virulence determinant in the development of pulmonary aspergillosis. Secreted proteolytic activities that degrade the surrounding lung tissue during infection may contribute to pathogenicity. Until now, knowledge on the regulation of the expression and secretion of proteases by A. fumigatus is scarce. Therefore, the role of extracellular proteolytic activity for pathogenicity of A. fumigatus was examined by characterisation of a global regulatory factor, PrtT, that acts on expression of secreted proteases. It could be shown that PrtT regulates the transcription of the major secreted proteases Alp, Mep and Pep. When tested in a leukopenic mouse model, the deletant strain is not attenuated in virulence, suggesting that the PrtT transcription factor - and accordingly extracellular proteolysis - supports virulence of this opportunistic pathogen only to a limited extent. To gain insight into the fungal biosynthesis pathway of amino acids, the aromatic amino acid biosynthesis was investigated concerning the aspect of a virulence determinant. In contrast to mammals, fungi are able for de novo synthesis of the aromatic amino acids. Therefore it might be a usable target for antifungal therapy since such pathway does not exist in humans. Some genes of the shikimate pathway could be shown to be essential for the survival of A. fumigatus. Virulence tests of strains with deletion of the genes aroC or trpA which encodes for the chorismate mutase and anthranilate synthase respectively, showed attenuated virulence of both strains. These results clarify the stringent necessity of the aromatic amino acid biosynthesis for the survival of A. fumigatus, concluding this biosynthesis pathway as a usable target for antimycotic substances. The results of this work emphasise the redundancy of extracellular proteolytic activities. In contrast specific pathways which are mostly catalysed by unique gene products may be identified as virulence determinant rather than unspecific factors. KW - Aspergillus fumigatus KW - Proteasen KW - Proteolyse KW - Posttranskriptionelle Regulation KW - Biosynthese der aromatischen Aminosäuren KW - Aspergillus fumigatus KW - Proteases KW - Proteolysis KW - posttranscriptional regulation KW - aromatic amino acid biosynthesis Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-67333 ER - TY - THES A1 - Masic, Anita T1 - 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 T1 - Die auf dendritischen Zellen basierende Immunisierungsstrategie gegen Leishmania major in BALB/c Mäusen ist abhängig von der Stimulation der Interleukin-4 Rezeptor alpha Kette N2 - 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. N2 - Die kutane Leishmaniose ist vor allem in den tropischen und subtropischen Regionen endemisch. Die Notwendigkeit der Erforschung und Etablierung einer Impfstoffstrategie basiert auf dem Auftreten von starken Nebenwirkungen während einer medikamentösen Behandlung, als auch auf die Entwicklung von Resistenzen des Parasiten gegenüber herkömmlichen Behandlungsmethoden. Die Arbeitsgruppe um Heidrun Moll etablierte eine auf dendritischen Zellen (DZ) basierende Immunisierungsstrategie, welche langlebige Immunität gegen experimentelle Leishmaniose vermittelt. Dabei dienen CpG ODN-stimulierte DZ als Adjuvans für L.-major–Antigene (LmAg). Die durch Infektion mit Leishmania-Parasiten hervorgerufene Gewebeschädigung kann in BALB/c-Mäusen verhindert werden, vorausgesetzt eine systemische Verabreichung von LmAg-beladenen und CpG ODN-aktivierten DZ erfolgte eine Woche vor der Infektion. Es konnte gezeigt werden, dass der Schutz durch die Induktion einer von Interleukin (IL)-12 und Interferon (IFN)-gamma dominierten T-Helfer (Th)1-Immunantwort herbeigeführt wurde und kranke Kontrollmäuse eine IL-4-dominierte Th2 Immunantwort aufwiesen. Mittlerweile zeigen zahlreiche Studien, dass IL-4 nicht ausschließlich eine krankheitsfördernde Funktion innehat, sondern auch die Fähigkeit zur Einleitung eine Typ-1-Immunantwort besitzt. Auf Grund dieser Studien wurde das Augenmerk auf die Rolle von IL-4 in der DZ-basierten Immunisierung gegen Leishmaniose in BALB/c Mäusen gelegt. In der vorliegenden Arbeit wurde die Notwendigkeit der Stimulation der IL-4 Rezeptor alpha (IL-4Rα) Kette auf DZ, während einer DZ-basierten Immunisierung gegen Leishmaniose in BALB/c Mäusen gezeigt. Um dies zu erreichen, wurden Wildtyp (wt)-BALB/c-Mäuse oder DZ-spezifische CD11ccreIL-4Rα-/lox BALB/c Mäuse entweder mit wt oder IL-4Rα-defizienten LmAg-beladenen DZ mit oder ohne Aktivierung durch CpG ODN, eine Woche vor der Infektion mit 2x105 L. major Promastigoten in den Hinterfuß, immunisiert. Die in dieser Doktorarbeit gezeigten Ergebnisse lassen den Schluss zu, dass die Stimulation der IL-4Rα-Kette auf den als Adjuvans eingesetzten DZ erforderlich ist, um eine Gewebsschädigung an der Infektionsstelle zu verhindern, da konditionierte wt DZ, nicht aber IL-4Rα-defiziente DZ in der Lage sind, Schutz gegen Leishmaniose zu vermitteln. Des Weiteren konnte eine unkontrollierte Ausdehnung von Leishmania-Parasiten im infizierten Fuß und in den angrenzenden Lymphknoten von CD11ccreIL-4Rα-/lox Mäusen beobachtet werden, welche mit CpG ODN-aktivierten und LmAg-beladenen IL-4Rα-defizienten DZ immunisiert wurden. Dieser Befund zeigt den Einfluss der Stimulation der IL-4Rα-Kette auf wirtsansässigen DZ im Hinblick auf die Eindämmung der Parasitenreplikation und Parasitenverbreitung. Zusätzliche Analysen in BALB/c-Mäusen, welche mit LmAg-beladenen, CpG ODN- und rekombinanten IL-4-stimulierten DZ immunisiert wurden, zeigten einen resistenten klinischen Verlauf der Infektion. Die hier gezeigten Ergebnisse lassen die Vermutung zu, dass die durch die IL-4/IL-4Rα-Kette ausgelösten Signale in den DZ eine Grundvoraussetzung für eine erfolgreiche Immunisierung sind und sollten deswegen unbedingt bei der Entwicklung eines Impfstoffes gegen die gewebsschädigenden Folgen einer Leishmaniose oder anderer durch intrazelluläre Mikroorganismen verursachten Infektionen berücksichtigt werden. KW - Leishmania major KW - Dendritische Zelle KW - Immunisierung KW - murine leishmaniasis KW - IL-4 Rezeptor alpha KW - Leishmania major KW - murine leishmaniasis KW - dendritic cells KW - IL-4 KW - IL-4 receptor alpha chain KW - vaccine Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-75508 ER - TY - THES A1 - Schnitzer, Johannes K. T1 - Mechanism of dendritic cell-based vaccination against Leishmania major T1 - Mechanismus der auf dendritischen Zellen beruhenden Impfung gegen Leishmania major N2 - 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 schützen beziehungsweise diese zu heilen. Die Effizienz dieser Technik konnte bereits für diverse Infektionskrankheiten und Krebserkrankungen in experimentellen Tiermodellen sowie am Menschen gezeigt werden. Hierbei ist die Möglichkeit zur wohldefinierten Manipulation und Antigenbeladung der DZ ein großer Vorteil gegenüber den konventionellen Ansätzen. Jedoch ist vor allem bei der Anwendung im klinischen Bereich die Präparation, Herstellung und Manipulation dieser autologen DZ mit einem erheblichen technischen, zeitlichen sowie finanziellen Aufwand verbunden. Hinsichtlich einer Präventivimpfung gegen eine pandemische Infektionskrankheit, die in hauptsächlich unterentwickelten Ländern vorkommt, wird dieser Aufwand sicherlich ein Hindernis darstellen. Daher muss für solche Fälle ein maßgeschneiderter Impfstoff entwickelt werden, der sich am Vorbild des effektiven DZ-basierten Impfstoffs orientiert. Für 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 Veröffentlichungen demonstriert. Zusätzlich 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 stärkere Beteiligung des Wirts-Immunsystems als zuvor angenommen. Daher sollte hier vertieft der Mechanismus dieser DZ-basierten Impfung untersucht werden, wobei modifizierte Impfstoff-Ansätze zum Einsatz kommen sollten. Dabei wurden die Fragen nach der vom Impfstoff transportierten Information und dem Empfänger dieser Information berücksichtigt. Das aktuelle Paradigma zur DZ-basierten Impfung besagt, dass transferierte DZ im direkten Kontakt mittels dreier Signale T-Zellen stimulieren und aktivieren. Dafür müssen diese DZ mit dem entsprechenden Antigen beladen und aktiviert worden sein um das Antigen-Peptide mittels MHC Molekül im Kontext der Co-Stimulation präsentieren zu können. Jedoch zeigt diese Studie hier, dass weder eine Aktivierung der DZ noch die Präsentation des Antigens mittels passender MHC Moleküle notwendig ist für die Induktion einer protektiven Immunantwort gegen Leishmania Parasiten. Aufgeschlossene, mit Antigen beladene DZ müssen nicht vor dem Transfer mit CpG ODN aktiviert worden sein, um entsprechende Immunität zu verleihen. Ebenso hat der MHC Typ in diesem Falle auch keinen Einfluss auf die Effektivität des Impfstoffs. Da im Weiteren aufgeschlossene mit Leishmania-Antigen beladene Makrophagen nach Impfung die gleiche Wirkung erzielen, wie vorangegangene DZ-basierte Impfstoffe, können keine DZ spezifischen Mechanismen Schlüsselkomponenten der Induktion einer protektiven Immunität sein. Darüber hinaus konnte gezeigt werden, dass die DZ der geimpften Mäuse, eine maßgebliche Rolle bei der Verarbeitung transferierter Signale spielen. Suspensionen aufgeschlossener DZ stellen eine Kombination aus freigesetzten löslichen Molekülen 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 Lagermöglichkeit bei -80°C. Somit ist klar gezeigt, dass die Immunität-verleihende Einheit dieser Impfstoffvarianten in der Membran-Fraktion liegt. Verfolgt man die Induktion Th1-zugehöriger 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 Mäusen 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 führt, lassen sich die entsprechend Th1-bezogenen Zytokine im in vivo Ansatz nur in geringen Maße nachweisen. Ob dies jedoch für einen in vivo unbemerkten Aktivitätsverlust des Vakzins oder für andere lymphatische Organe als Ort der T-Zell Instruktion spricht, ist noch unbekannt und muss noch geklärt werden. N2 - Dendritic cell-based vaccination is a well established technique for preventive and therapeutic instruction of the immune system where conservative vaccine formulations fail to cure or prevent diseases, respectively. Efficiency of this technique already was demonstrated in infectious diseases as well as for cancer in animal or human studies. Well controlled manipulation and antigen-loading of immature DC is most beneficial to this technique. But, time-consuming and cost-extensive procedures for preparation of DC precursors, expansion and stimulation of DC and inpatient administration are big disadvantages regarding vaccine development for pandemic infectious diseases that occur mainly in underdeveloped countries. Therefore vaccines are needed that are pathogen-tailored and able to induce equal immune responses as their DC-based vaccine models. For vaccination against Leishmania parasites such a DC-based vaccine is feasible and its efficacy to induce protective Th1-based immune responses was already demonstrated in several animal studies. But, one of our own studies indicated supportive activity of host cells exceeding the allocation of T cells to become activated by transferred DC. IL-12, an important cytokine for the induction of Th1-related immune responses, has to be produced by host cells. Therefore, the aim of this study was to investigate the mechanism of BMDC-based vaccination with regard to simplification of the vaccine formulation. Key questions that have been addressed are: Which cells process the information that is transferred by the injected DC and what are the key components of this information? Further more, it was looked at whether altered vaccine formulations are able to induce protective immunity and whether they share equal molecular mechanisms. The current paradigm of BMDC-based vaccination proposes direct interaction of transferred BMDC with host T cells. These BMDC have to be antigen-loaded for stimulation via antigen-peptide-MHC molecule-complexes and they have to be activated for proper co-stimulation of T cells. Here, this study demonstrates that neither activation for co-stimulation nor direct interaction with adequate MHC molecules is needed for the induction of protective immunity against infection with Leishmania-parasites. Disrupted antigen-loaded BMDC are able to induce protective immunity in BALB/c mice without pre-stimulation via CpG ODN. Beyond, if BMDC were used with a different MHC-background than recipient mice then the vaccine still would be efficient in terms of reduction of footpad swelling and parasite load in draining lymph nodes. Even more, DC-specific features are no key component that leads to protective immunity as vaccination with disrupted antigen-loaded MΦ shows equal properties than before mentioned vaccine formulations. Further more, it was found that host DC play a major role in transforming the incoming signal, received from transferred antigen-loaded DC, into Th1-related stimuli and Leishmania-antigen-specific T cell activation. Suspensions of disrupted antigen-loaded DC resemble a combination of laid off soluble molecules together with exosome-like vesicles that formed after disruption of membranes. Here it was shown that separation of the membranous and soluble fractions and subsequent transfer into BALB/c mice will lead to protection of these mice against infection with L. major promastigotes only if the membranous fraction is used as vaccine. More, this vaccine formulation takes advantage of easy storage at -80°C with no need of fresh production. This clearly demonstrates that the immunity-inducing principle of disrupted DC-based vaccination lies within the membrane enclosed fraction. On a molecular level, disrupted antigen-loaded DC induce Th1-related cytokines during vaccination and as response on pathogen encounter. In vivo assays revealed IL-12 production and antigen-specific T cell proliferation among splenocytes that were stimulated with disrupted antigen-loaded DC. Splenocytes of accordingly vaccinated mice produce tremendous amounts of IFNγ after stimulation with Leishmania parasites. In summary, disrupted antigen-loaded BMDC fulfil all characteristics of DC-based vaccination against Leishmania major. But, while purification of membranes of antigen-loaded DC and subsequent transfer to BALB/c mice leads to control of the disease in the animal model, only slight levels of Th1-related cytokines are seen in the in vivo assays. Whether this points towards a loss of vaccine activity on unseen levels or unknown sites where Th1-related immunity is induced by both, complete solution and purified membranes, still has to be determined. KW - Leishmania major KW - Immunsystem KW - Impfung KW - Dendritische Zelle KW - Makrophage KW - Interferon KW - Interleukin 12 KW - Leishmania KW - Immunologie KW - Dendritic cell Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-74865 ER - TY - THES A1 - Troge, Anja T1 - Studien am Flagellensystem des Escherichia coli Stammes Nissle 1917 (EcN) im Hinblick auf seine Funktion als Probiotikum T1 - Studies on the flagellar system of Escherichia coli Nissle 1917 (EcN) with regard to its function as a probiotic N2 - Escherichia coli Nissle 1917 (EcN) gehört zu den am besten untersuchten und charakterisierten probiotischen Bakterienstämmen. Seit Beginn des letzten Jahrhunderts wird er als Medikament eingesetzt, um verschiedene Darmerkrankungen wie z.B. Diarrhöe, entzündliche Darmerkrankungen und Verstopfung zu behandeln. Die Flagelle des EcN vermittelt Beweglichkeit und kann die Produktion von humanem β-Defensin 2 (hBD2) durch Epithelzellen induzieren. Somit ist dieses Organell direkt in die probiotische Funktion des EcN involviert. Es konnte gezeigt werden, dass die Flagellen anderer Bakterien, wie z.B. dem probiotischen Stamm Bacillus cereus CH oder den pathogenen Stämmen Pseudomonas aeruginosa und Clostridium difficile, die Adhäsion an intestinalen Mucus, welcher von Epithelzellen sekretiert wird, vermitteln. Allerdings blieb unklar, welcher Teil der Flagelle an welche Mucuskomponente bindet. Die Fähigkeit effizient an Wirtgewebe zu adhärieren wird als wichtiges Attribut eines probiotischen Stammes angesehen. Ex vivo Adhäsionsstudien mit Kryoschnitten humaner Darmbiopsien haben gezeigt, dass die Flagelle des EcN in die effiziente Adhäsion an humanes Darmgewebe involviert sein muss. Aus diesem Grund wurde in dieser Arbeit die Funktion der Flagelle des EcN als Adhäsin untersucht. Zunächst wurde die hyperflagellierte Variante EcN ATHF isoliert und durch verschiedene Experimente, z.B. Schwärmagartests und Elektronenmikroskopie, charakterisiert. Weitere ex vivo Adhäsionsstudien mit EcN ATHF zeigten eine höhere Adhäsionseffizienz dieser hyperflagellierten Variante und bestätigten damit die Rolle der Flagelle bei der effizienten Adhäsion von EcN an die Kryoschnitte der humanen Darmbiopsien. Interessanterweise fungierte die Flagelle in in vitro Studien mit den humanen Epithelzellen Caco-2 und T24 nicht als Adhäsin. Diese Unterschiede zwischen den in vitro und ex vivo Studien führten zu der Annahme, dass die Flagelle des EcN in vivo die Adhäsion an Mucus vermittelt, welcher von den Caco-2- und T24-Zellen nicht produziert wird, aber in den Kryoschnitten der Darmbiopsien nachgewiesen wurde. Diese Vermutung wurde durch in vitro Adhäsionsstudien mit der Mucin-produzierenden Epithelzelllinie LS174-T bestätigt, da die Flagellen für eine effektive Adhäsion an diese Zellen essentiell waren. Zudem reduzierte die Präinkubation flagellierter EcN-Stämme mit Mucin2 ihre Adhäsionseffizienz an Kryoschnitte humaner Darmbiopsien. Um die direkte Interaktion zwischen Flagellen des EcN Wildtyps und Mucus zu zeigen, wurde ein ELISA etabliert. Es konnte eine direkte konzentrationsabhängige Interaktion zwischen isolierten Flagellen des EcN Wildtyps und Mucin2, bzw. humanem Mucus (Kolon) beobachtet werden. Interessanterweise konnte keine Interaktion zwischen isolierten Flagellen des EcN Wildtyps und murinem Mucus (Duodenum, Ileum, Caecum, Colon) festgestellt werden. Dies weist darauf hin, dass die Mucuszusammensetzung zwischen verschiedenen Spezies variiert. Verschiedene Kohlenhydrate, welche bekannte Mucusbestandteile sind, wurden auf ihre Interaktion mit der Flagelle von EcN getestet und Gluconat wurde als ein Rezeptor identifiziert. Die Präinkubation isolierter Flagellen mit Gluconat reduzierte ihre Interaktion mit Mucin2, bzw. humanem Mucus signifikant. Zudem wurde die oberflächenexponierte Domäne D3 des Flagellins, der Hauptuntereinheit der Flagelle, als möglicher Interaktionspartner von Mucin2, bzw. humanem Mucus ausgeschlossen. Flagellen, die aus einer Domäne D3 Deletionsmutante isoliert wurden, zeigten sogar eine effizientere Bindung an Mucin2, bzw. humanen Mucus. Weiterhin konnte gezeigt werden, dass Änderungen des pH-Wertes signifikante Effekte auf die Interaktion zwischen Mucus und isolierten Flagellen hatten, vermutlich aufgrund von Konformationsänderungen. Zusammenfassend wurde in dieser Arbeit die Flagelle als neues und scheinbar wichtigstes Adhäsin in vivo für den probiotischen Stamm EcN identifiziert. Hierfür wurden sowohl eine hyperflagellierte Variante, eine ΔfliC Mutante, sowie der dazugehörige komplementierte Stamm verwendet. EcN ist zudem der erste probiotische Stamm für den eine direkte Bindung der Flagellen an humanen Mucus nachgewiesen werden konnte. Die Mucuskomponente Gluconat konnte dabei als wichtiger Rezeptor identifiziert werden. Da einige pathogene Bakterien ihre Flagelle zur Adhäsion an Wirtsgewebe nutzen, könnte dieses Organell EcN dazu befähigen, mit Pathogenen um die erfolgreiche Kolonisierung des Darms zu konkurrieren, was als wichtige Eigenschaft eines Probiotikums betrachtet wird. N2 - Escherichia coli Nissle 1917 (EcN) is one of the best studied and characterized probiotic bacterial strains. It is in use as a drug since the beginning of last century to treat various diseases and dysfunctions of the human intestinal tract, e.g. diarrhea, inflammatory bowel diseases and obstipation. The flagellum of EcN mediates motility and is able to induce human beta defensin 2 (hBD2) production by epithelial cells. Therefore, this organelle is directly involved in EcN’s probiotic function. It has been shown that the flagella of several other bacteria, including the probiotic strain Bacillus cereus CH or the pathogenic strains Pseudomonas aeruginosa and Clostridium difficile, mediate adhesion to intestinal mucus, which is secreted by epithelial cells. However it remained unclear which part of the flagella binds to which mucus component. The ability to adhere efficiently to host tissue is considered to be an important attribute for a probiotic strain. Ex vivo adhesion studies with cryosections of human gut biopsies have revealed, that the flagellum of EcN must be involved in efficient adhesion to human intestinal tissue. Thus, the function of EcN’s flagellum as an adhesin was investigated in this work. First, the hyperflagellated variant EcN ATHF was isolated and characterized by several experiments, e.g. motility tests and electron microscopy. Further ex vivo adhesion studies with EcN ATHF demonstrated a higher adhesion efficiency of this hyperflagellated variant confirming the role of the flagellum for adhesion of EcN to cryosections of human gut biopsies. Interestingly, EcN’s flagellum did not function as an adhesin in in vitro adhesion studies with the human epithelial cells Caco-2 and T24. These differences between the in vitro and ex vivo studies led to the assumption, that in vivo the flagellum of EcN mediates adhesion to mucus, which is not produced by Caco-2 and T24 cells, but was shown to be present in the cryosections of human gut biopsies. This was confirmed by in vitro adhesion studies with the mucin-producing epithelial cell line LS174-T, as flagella were essential for efficient adhesion to these cells. Furthermore, preincubation of flagellated EcN strains with mucin2 (porcine stomach) reduced their adhesion effiency to cryosections of human gut biopsies. To demonstrate the direct interaction between flagella from EcN wildtype and mucus, an ELISA was established. A direct concentration-dependent interaction between isolated flagella from EcN wildtype and mucin2 as well as human mucus (Colon) could be observed. In contrast, there was no direct interaction between isolated flagella from EcN wildtype and murine mucus (Duodenum, Ileum, Ceacum, Colon), indicating that mucus composition varies among different species. By testing different carbohydrates - known to be constituents of mucus - for their interaction with the flagellum of EcN, gluconate was identified as one receptor. Preincubation of isolated flagella with gluconate significantly reduced their interaction with mucin2 or human mucus. Additionally, the surface exposed domain D3 of flagellin, the major subunit of the flagellum, could be excluded to be responsible for the interaction with mucin2 or human mucus. Flagella, which were isolated from a domain D3 deficient mutant, bound even more efficient to mucin2 as well as to human mucus. Furthermore the change of pH had significant effects on the interaction between mucus and isolated flagella, probably due to conformational changes. In summary, this study identified the flagellum as a novel and apparently major adhesin in vivo of the probiotic EcN by employing a hyperflagellated variant, a ΔfliC mutant as well as the corresponding complemented strain. Additionally, EcN is so far the first probiotic strain, for which it has been shown, that its flagella directly bind to human mucus. Thereby the mucus component gluconate was identified as an important receptor. As some pathogens have been reported to use their flagella for adhesion to human host tissue, this organelle might enable EcN to compete with pathogens for successful colonization of the gut, which has been postulated to be a prerequisite for probiotics. KW - Escherichia coli KW - Probiotikum KW - Geißel KW - Adhäsion KW - Adhärenz KW - E.coli Nissle 1917 KW - Flagelle KW - adhesion KW - E. coli Nissle 1917 KW - flagellum Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-74201 ER - TY - JOUR A1 - Hess, Michael A1 - Stritzker, Jochen A1 - Härtl, Barbara A1 - Sturm, Julia A1 - Gentschev, Ivaylo A1 - Szalay, Aladar T1 - Bacterial glucuronidase as general marker for oncolytic virotherapy or other biological therapies N2 - 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 KW - Virologie KW - beta-glucuronidase KW - oncolytic virus KW - cancer KW - reporter KW - fluorescent probe Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-69163 ER - TY - JOUR A1 - Kuehn, Andrea A1 - Pradel, Gabriele T1 - The Coming-Out of Malaria Gametocytes [Review Article] N2 - 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. KW - Malaria Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68196 ER - TY - JOUR A1 - Hacker, Jörg A1 - Rdest, Ursula A1 - Wintermeyer, E. A1 - Ludwig, B. T1 - Legiolysin, a New Hemolysin from L. pneumophila N2 - 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. N2 - Legionella pneum.ophila bildet Exoroxine, Zytolysine, Proteasen oder Hämolysine, die Wirtszellen wie Erythrozyten oder Animalzellen schädigen. Das Gen für ein neues L. pneumophila Hämolysin ohne proteolytische Aktivität wurde identifiziert, in E. coli kloniert und sequenziert. Das Genprodukt wurde durch SDS-Gelelcktropborese analysiert. KW - Hämolysin KW - Legionella pneumophila Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-73070 ER - TY - JOUR A1 - Hacker, Jörg A1 - Schrettenbrunner, A. A1 - Schröter, G. A1 - Schmidt, G. A1 - Düvel, H. A1 - Goebel, W. T1 - 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 N2 - 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. N2 - E. co/i-Stämme, die von Patienten mit Urogenitaltraktinfektionen (UTI) isoliert werden, weisen oftmals Mannose-sensitive (MS) und Mannose-resistente (MR) Adhärenzfaktoren (Fimbrien) auf. Entsprechend ihrer Rezeptorspezifität können die MR-Adhäsine in verschiedene Gruppen, P, S, M und X unterteilt werden. Vor kurzem haben wir die Determinanten von drei Gruppen der UTI E. coli-Adhäsine, MS, P und S, kloniert und spezifische Antiseren gegen diese Fimbrienantigene hergestellt. 189 Hämagglutinations (HA +)-positive Stämme,. 96 Isolate aus Stuhlproben und 93 Stämme von Patienten mit UTl, wurden mit diesen Fimbrienantigen-spezifischen Antiseren getestet. Sie wurden weiterhin bezüglich ihrer rezeptorspezifischen HA, ihres HA-Musters, dem Vorkommen der 0-Serogruppen 01, 02, 04, 06, 07, 08, 018, 025, 075 ("common 0-serogroups"), die bei Harnwegsisolaten vorherrschen, und der Hämolysinbildung charakterisiert. 68 (73 %) der UTl-Stämme und 50 (52%) der fäkalen lsolate zeigten P-Rezeptorspezifität; 16 ( 17%) uropathogene Stämme und 33 (34%) Stämme aus Stuhlproben prägten S, M oder X-Fimbrienantigene aus. 24% der P-hämagglutinierenden (P+) Stämme reagierten mit P (F8)-spezifischem Antiserum. lm Gegensatz dazu reagierten mehr als drei Viertel der s+ -Stämme mit dem S-spezifischen Antiserum. Das HA-Muster Vl und 018- Antigen wurden meistenteils bei p+ -Stämmen gefunden; die HA-Muster V und Vll und die 0-Antigene 02 (M-Typ), 06 und 018 (S-Typ) wurden vorzugsweise bei p--Stämmen nachgewiesen. Ein hoher Prozentsatz von P-fimbrierten Stämmen zeigte Mannose-sensitive Hämagglutination und Hämolysinbildung. KW - Escherichia coli Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-72992 ER - TY - JOUR A1 - Hacker, Jörg A1 - Gadeberg, Ole V. A1 - Orskov, Ida T1 - Role of alpha-Hemolysin for the in vitro Phagocytosis and intracellular killing of Escherichia coli N2 - 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. N2 - Die Rolle von a-Hämolysin bei der in vitro·Eliminierung von Escherichia coli durch Phagozyten wurde unter Verwendung isogener Stämme einschließlich von a-hämolysierenden Wildstämmen, davon abstammenden Stämmen mit reduzierter a-Hämolysin-Bildung und davon abstammenden nicht hämolysierenden Stämmen untersuche. Phagozytose und intrazelluläre Abtötung der Bakterien durch Granulozyten oder Monozyten im menschlieben Blut wurden u.nter Verwendung von Wachstums-Hemmtechniken gemessen. o:-hämolysierende Stämme wurden in geringerem Maße als isogene Stämme mit einer geringeren Hämolysin-Bildung und isogene nicht hämolysierende Stämme pbago:z.ytiert und ;~bgetötet. Die mit Granulozyten erzielten Ergebnisse waren den bei Monozyten ähnlich, obwohl die Bakterienelimination durch Monozyten geringer war als durch Granulozyten. Diese Ergebnisse deuten stark darauf hin, daß die Bildung von a-Hämolysin ein Mine) ist, mit dem E. coli der Aktivität der Phagozyten durch Schädigung dieser Zellen mit dem Toxin entgegenwirkt. KW - Escherichia coli Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-73019 ER - TY - JOUR A1 - Hacker, Jörg A1 - Ulmer, E. A1 - Fasske, E. A1 - Schmidt, G. T1 - Isolation and characterization of coliphage Omega18A specific for Escherichia coli O18ac strains N2 - 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. N2 - Der Bakteriophage Q18A, der spezifisch Escherichia coli 018ac Bakterien lysierr, wurde aus Abwasser isoliert. Die Untersuchungen des Wirtsbereichs und Konjugationsversuche zeigten, daß die Sensitivität der Bakterien gegenüber dem Phagen mit dem Vorhandensein des 0 '18ac Antigens assoziiert ist. Bei eir1igen 0 18 Stämmen wird nur bei Anwendung hoher Phagenkonzentrationen eine klare Lysis auf dem Bakterienrasen erzeugt. Darüber hinaus läßt sich der Phage auf diesen Stämmen nicht vermehren. Mit Hilfe des Phagen Q l8A konnten E, wli 0 18ac Stämme in zwei serologische Subgruppen unteneilt werden, die als 0 lHA und 0 l8A 1 bezeichnet werden. E. coli Bakterien der Subgruppe 0 ISA sind gegenüber dem Phagen Ql8A sensitiv und diejenigen der Subgruppe 0 18A 1 sind resistent. KW - Escherichia coli Y1 - 1987 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-73001 ER - TY - THES A1 - Simon, Nina Monica T1 - Molecular interactions of the malaria parasite Plasmodium falciparum during the sexual reproduction in the mosquito midgut T1 - Molekulare Wechselwirkungen des Malariaparasiten Plasmodium falciparum während der sexuellen Fortpflanzung im Mitteldarm der Mücke N2 - 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. N2 - Die Sexualphase von Plasmodium falciparum beginnt mit der Ausbildung von intraerythrozytären Sexualstadien, sogenannten Gametozyten, im menschlichen Wirt. Reife Gametozyten zirkulieren im peripheren Blut und werden während der Blutmahlzeit von der Mücke aufgenommen. Dieses Parasitenstadium ist ausschlaggebend für die Verbreitung von Malaria und bildet im Mückendarm innerhalb von Minuten Gameten. In Plasmodium falciparum wurde eine hochkonservierte Familie bestehend aus sechs sekretierten Proteinen entdeckt. Diese bestehen aus verschiedenen Adhäsionsdomänen und werden PfCCp1 bis PfCCp5 und PfFNPA genannt. In dieser Arbeit wurde gezeigt, dass PfCCp Multiadhäsionsproteine Komplexe in Gametozyten und auf der Oberfläche von jungen Makrogameten mittels domänenvermittelter Bindungen bilden. Ko-Immunpräzipitationen mit Lysat aus aktivierten Gametozyten zeigten oberflächenvermittelte Interaktionen der PfCCp Proteine durch Pfs230 und Pfs25. Pfs230 ist mit seinen Interaktionspartner Pfs48/45 durch einen GPI-Anker mit der Plasmamembran des Parasiten verbunden. Der Multi-Proteinkomplex wird somit auf der Oberfläche von jungen weiblichen Gameten festgehalten. Zudem wurde in dem neu identifizierten Proteinkomplex ein Protein entschlüsselt welches WD40-Domänen aufweist. Bereits bekannte Funktionen von sich wiederholenden WD40-Domänen lassen vermuten, dass dieses Protein möglicher-weise als Plattform für den Zusammenbau des Proteinkomplexes dient oder das Wechselspiel zwischen Proteinen vermittelt. Während des Ausbruchs aus der Wirtszelle, dem Erythrozyten, werden Gameten angreifbar für Faktoren des humanen Komplements, welches mit der Blutmahlzeit in den Mückendarm aufgenommen wird. In dieser Arbeit wurde ermittelt, dass das Komplementsystem nach der Blutmahlzeit etwa eine Stunde lang im Mückendarm aktiv ist. Durch die Bindung der Regulatoren Faktor H und FHL-1 des menschlichen Komplementsystems aus der Blutmahlzeit, schützen sich Makrogameten gegen eine komplementvermittelte Lyse. Diese Serumproteine binden mittels ihrer SCR-Domänen 5-7 an die Oberfläche von Makrogameten und vermitteln damit die Inaktivierung des alternativen Komplementweges. Dadurch schützen sie sich vor der komplementinduzierten Lyse auf der Oberfläche des Parasiten. Antikörper gegen Faktor H vermindern die sexuelle Entwicklung in vitro und können die Weiterentwicklung des Erregers in der Mücke blockieren. Interaktionsstudien mit endogenen Proteinen und immoblilisierten rekombinanten Proteinen offenbarten PfGAP50 als Bindungspartner von Faktor H und FHL-1. PfGAP50 wurde bislang einem Motorkomplex zugeschrieben, welcher für die Parasitenbewegung von invasiven Stadien zuständig ist. Es wurde jedoch bis heute nicht in Gameten charakterisiert. Erste Lokalisationsstudien weisen auf eine Relokalisierung von PfGAP50 vom inneren Membrankomplex zur Oberfläche von Makrogameten hin. Malaria ist weiterhin eine der tödlichsten Infektionskrankheiten weltweit. Die Erforschung dieser für die Übertragung essentiellen Stadien, den Gametozyten und Gameten von Plasmodium falciparum, stand lange im Hintergrund der Forschung. Diese Arbeit entschlüsselt Details über Proteininteraktionen auf der Oberfläche des Malariaparasiten und beschreibt das Zusammenwirken des Parasiten mit dem menschlichen Komplementsystem im Darm der Mücke. KW - Plasmodium falciparum KW - Sexuelle Entwicklung KW - Malaria KW - Transmissionsblockierende Impfstoffe KW - Oberflächenproteine der Sexualstadien KW - Malaria KW - Gametozyt KW - humaner Faktor H KW - transmission blocking vaccine KW - sexual stage surface proteins KW - malaria KW - gametocyte KW - human factor H Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-72403 ER - TY - THES A1 - Angermeier, Hilde Gabriele T1 - Molecular and ecological investigations of Caribbean sponge diseases T1 - Molekulare und ökologische Untersuchungen zu Krankheiten Karibischer Meeresschwämme N2 - Während gewinnbringende Assoziationen von Schwämmen mit Mikroorganismen in den letzten Jahren viel Aufmerksamkeit erhalten haben, wurde weit weniger in die Interaktion von Schwämmen mit möglicherweise pathogenen Mikroben investiert. Somit war es das Ziel dieser Studie zwei ausgewählte Karibische Schwammkrankheiten namens „Sponge Orange Band“ und „Sponge White Patch“ mittels ökologischer und molekularer Methoden zu untersuchen. Die Sponge Orange Band (SOB) Erkrankung befällt den bedeutenden karibischen Fass-Schwamm Xestospongia muta, der zu den bakterienhaltigen (HMA) Schwämmen gezählt wird, während die Sponge White Patch (SWP) Erkrankung den häufig vorkommenden Seil-Schwamm Amphimedon compressa betrifft, der zu den bakterienarmen (LMA) Schwämmen gehört. Für beide Karibischen Schwammkrankheiten konnte ich einen Krankheitsverlauf beschreiben, der mit massiver Gewebszerstörung und dem Verlust charakteristischer mikrobieller Signaturen einhergeht. Obwohl ich zeigen konnte, dass zusätzliche Bakterienarten die gebleichten Schwammbereiche kolonisieren, lieferten meine Infektionsversuche in beiden Fällen keinen Beweis für die Beteiligung eines mikrobiellen Pathogens als Krankheitserreger. Somit liegen die eigentlichen Auslöser der Erkrankungen Sponge Orange Band als auch Sponge White Patch noch immer im Dunkeln. N2 - While beneficial sponge-microbe associations have received much attention in recent years, less effort has been undertaken to investigate the interactions of sponges with potentially pathogenic microorganisms. Thus, the aim of this study was to examine two selected Caribbean disease conditions, termed “Sponge Orange Band” and “Sponge White Patch”, via ecological and molecular methods. Sponge Orange Band (SOB) disease affects the prominent Caribbean barrel sponge Xestospongia muta that is counted among the high-microbial-abundance (HMA) sponges, whereas Sponge White Patch (SWP) disease affects the abundant rope sponge Amphimedon compressa that belongs to the low-microbial-abundance (LMA) sponges. I have documented for both Caribbean sponge diseases a disease progression going along with massive tissue destruction as well as loss of the characteristic microbial signatures. Even though new bacteria were shown to colonize the bleached areas, the infection trials revealed in both cases no indication for the involvement of a microbial pathogen as an etiologic agent of disease leaving us still in the dark about the cause of Sponge Orange Band as well as Sponge White Patch disease. KW - Meeresschwämme KW - Pathogene Bakterien KW - Porifera KW - Schwamm KW - Bakterien KW - Cyanobakterien KW - Pathologie KW - Mikroskopie KW - Denaturierende Gradienten-Gelelektrophorese KW - Symbiose KW - Sponge diseases KW - Porifera KW - Bacteria KW - Cyanobacteria KW - Pathogens KW - Symbionts KW - Pathology KW - Microscopy KW - Denaturing Gradient Gel Electrophoresis Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-56855 N1 - weitere Originalveröffentlichungen: Angermeier H, Glöckner V, Pawlik JR, Lindquist NL & Hentschel U (2012). Sponge white patch disease affecting the Caribbean sponge Amphimedon compressa. Diseases of Aquatic Organisms 99 (2): 95-102. ER - TY - THES A1 - Seo, Ean Jeong T1 - Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins T1 - Konstruktion von rekombinanten E. coli Nissle 1917 (EcN) Stämmen, die Defensine exprimieren bzw. sekretieren N2 - Der probiotische Escherichia coli Stamm Nissle 1917 (EcN) ist eines der wenigen Probiotika, die als aktive Komponente eines Medikaments in mehreren Ländern zugelassen sind. Am besten ist die Wirksamkeit des EcN für die Remissionserhaltung von an Colitis Ulcerosa leidenden Patienten dokumentiert. Diese Fähigkeit ist vermutlich darauf zurückzuführen, 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 Stämme konstruiert, die ein Defensin zu produzieren vermögen. 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 Stämme demonstriert. Das rekombinante reife HBD2 mit einem N-terminalen His-Tag konnte mittels Ni-Säulen-Chromatographie aufgereinigt werden. Das so gewonnene HBD2 zeigte antimikrobielle Aktivität 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 Präsenz von YebFMHBD2 im Medium war nicht das Ergebnis von Zellyse wie Western-Blots spezifisch für die -Galaktosidase und das Maltose-Bindeprotein mit dem Kulturüberstand zeigten. Dieser Kulturüberstand inhibierte das Wachstum von E. coli, Salmonella enterica Serovar Typhimurium und Listeria monocytogenes nach Dialyse und Aufkonzentration sowohl in Agardiffusionsassays als auch in Flüssigcokultur. Damit konnte gezeigt werden, dass EcN ein für 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 Unfähigkeit zur ausreichenden Defensinproduktion solcher Individuen begründet. Als ein erster Schritt in der Entwicklung von alternativen Ansätzen zur Behandlung Morbus Crohn Patienten wurden in dieser Arbeit EcN Stämme konstruiert, die in der Lage sind HD5 oder HBD2 zu produzieren. N2 - The probiotic Escherichia coli strain Nissle 1917 (EcN) is one of the few probiotics licensed as a medication in several countries. Best documented is its effectiveness in keeping patients suffering from ulcerative colitis (UC) in remission. This might be due to its ability to induce the production of human beta defensin 2 (HBD2) in a flagellin-dependent way in intestinal epithelial cells. In contrast to ulcerative colitis, for Crohn´s disease (CD) convincing evidence is lacking that EcN might be clinically effective, most likely due to the genetically based inability of sufficient defensin production in CD patients. As a first step in the development of an alternative approach for the treatment of CD patients, EcN strains were constructed which were able to produce human alpha-defensin 5 (HD5) or beta-defensin 2 (HBD2). For that purpose codon-optimized defensin genes encoding either the proform with the signal sequence or the mature form of human alpha defensin 5 (HD5) or the gene encoding HBD2 with or without the signal sequence were cloned in an expression vector plasmid under the control of the T7 promoter. Synthesis of the encoded defensins was shown by Western blots after induction of expression and lysis of the recombinant EcN strains. Recombinant mature HBD2 with an N-terminal His-tag could be purified by Ni-column chromatography and showed antimicrobial activity against E. coli, Salmonella enterica serovar Typhimurium and Listeria monocytogenes. In a second approach, that part of the HBD2-gene which encodes mature HBD2 was fused with yebF gene. The resulting fusion protein YebFMHBD2 was secreted from the encoding EcN mutant strain after induction of expression. Presence of YebFMHBD2 in the medium was not the result of leakage from the bacterial cells, as demonstrated in the spent culture supernatant by Western blots specific for ß-galactosidase and maltose-binding protein. The dialyzed and concentrated culture supernatant inhibited the growth of E. coli, Salmonella enterica serovar Typhimurium and Listeria monocytogenes in radial diffusion assays as well as in liquid coculture. This demonstrates EcN to be a suitable probiotic E. coli strain for the production of certain defensins. KW - Escherichia coli KW - Probiotikum KW - Rekombinante DNS KW - Genexpression KW - Defensine KW - Probiotic KW - Recombinant defensins KW - E. coli Nissle 1917 KW - HBD2 KW - HD5 KW - Antimicrobial activity KW - Secretion Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-72005 ER - TY - THES A1 - Schubert, Sabrina T1 - Funktionelle Analyse des „Multidrug-Resistance“-Regulators MRR1 im humanpathogenen Hefepilz Candida albicans T1 - Functional analysis of the multidrug resistance regulator MRR1 in the pathogenic yeast Candida albicans N2 - Der Hefepilz Candida albicans gehört zu den fakultativ pathogenen Infektionserregern und ist Teil der natürlichen Mikroflora der Schleimhäute des Verdauungs- und Urogenitaltraktes der meisten gesunden Menschen. Ist das Gleichgewicht der Flora gestört, kann es zu oberflächlichen Mykosen kommen, wie z.B. der oropharyngealen Candidiasis (Mundsoor), die in der Regel durch die Gabe eines Antimykotikums in wenigen Tagen zu behandeln sind. In seltenen Fällen kann es auch zu schwerwiegenden Infektionsverläufen bis hin zu lebensbedrohlichen systemischen Mykosen kommen. Hauptsächlich immunsupprimierte Patienten, wie z.B. AIDS-Patienten oder Personen, die kürzlich einer Organ- oder Knochenmarkstransplantation unterzogen wurden, leiden häufig an oberflächlichen C. albicans-Infektionen. Insbesondere bei wiederkehrenden Infektionen ist der Pilz in der Lage, gegen das häufig verabreichte Medikament Fluconazol eine Resistenz zu entwickeln. Ein wichtiger Mechanismus dieser Resistenzentwicklung ist die Überexpression von Effluxpumpen, die das Medikament aus der Zelle heraustransportieren. Zwei Arten von Effluxpumpen, die eine Rolle in der Resistenzentwicklung in C. albicans spielen, konnten bisher identifiziert werden, die ABC (ATP binding cassette)-Transporter Cdr1 und Cdr2 sowie der MFS (major facilitator superfamily)-Transporter Mdr1. Der Zinc-Cluster Transkriptionsfaktor Mrr1 spielt eine wichtige Rolle in der Regulation der MDR1-E¬ffluxpumpe. Er kontrolliert die MDR1-Expression in Anwesenheit induzierender Substanzen und sogenannte "gain-of-function" Mutationen in MRR1 konnten als die Ursache der konstitutiven MDR1-Hochregulierung und der "Multidrug-Resistance" in C. albicans identifiziert werden. In dieser Arbeit konnte ein Ortholog zu MRR1 aus C. albicans in Candida dubliniensis, einer zu C. albicans nahe verwandten Hefe, identifiziert werden. Es wurde gezeigt, dass in den untersuchten klinischen und in vitro generierten Fluconazol-resistenten C. dubliniensis-Stämmen ebenfalls gain-of-funcion Mutationen in MRR1 die MDR1-Überexpression und eine Resistenz bewirken. Die Ergebnisse demonstrieren, dass der Transkriptionsfaktor Mrr1 eine wichtige Rolle in der Entwicklung der Resistenz in diesen humanpathogenen Pilzen spielt. Bisher ist nicht bekannt, wie der Zinc-Cluster Transkriptionsfaktor MRR1 durch induzierende Substanzen oder gain-of-function Mutationen aktiviert wird. Um zu verstehen, wie die Mrr1- Aktivität reguliert wird, wurden in dieser Arbeit durch Deletionsstudien funktionelle Domänen des Transkriptionsfaktors identifiziert. Um einen besseren Einblick in die Regulation der MDR1-vermittelten Resistenz in C. albicans zu bekommen, wurde in dieser Arbeit die gegenseitige Abhängigkeit von Mrr1 und Cap1 bzw. Upc2 in Bezug auf die MDR1-Expression untersucht. Es wurden ChIP-on-chip Analysen und Transkriptionsprofile mit aktiviertem Mrr1 durchgeführt, um direkte Targets von Mrr1 zu identifizieren. Mit der vorliegenden Arbeit wurde ein wichtiger Beitrag zum Verständnis der Entwicklung der Multidrug-Resistenz in C. albicans geleistet. E¬ffluxpumpen und deren Regulatoren stellen in der Bekämpfung von C. albicans-Infektionen ein interessantes Angriffsziel für die Entwicklung neuer Medikamente und die Weiterentwicklung bereits vorhandender Antimykotika dar. N2 - The yeast Candida albicans is a human fungal pathogen and is part of the microflora of mucosal surfaces of the gastrointestinal and urogenital tract in most healthy people. If the balance of the flora is disturbed C. albicans can cause super cial mycoses, e.g. oropharyngeal Candidiasis, also known as "thrush", which are usually easy to cure within a few days by treatment with antimycotic drugs. Infections with the yeast can also result in serious as well as life-threatening systemic mycoses. However, immunocompromised patients, e.g. AIDS patients, often suffer from super cial C. albicans infections and especially in recurrent infections the yeast can develop resistance to the commonly used antifungal drug fluconazole. An important mechanism of drug resistance is the overexpression of e¬ux pumps, which mediate the transport of toxic compounds out of the cell. Two types of e¬fflux pumps, which play a role in die development of resistance in C. albicans, have been described so far, the ABC (ATP binding cassette) transporters Cdr1 and Cdr2, and the MFS (major facilitator superfamily) transporter Mdr1. The zinc cluster transcription factor Mrr1 plays an important role in the regulation of the MDR1 gene. It controls the MDR1 expression in response to inducing chemicals and gain-of function mutations in MRR1 are responsible for the constitutive upregulation of MDR1 and fluconazole resistance. In this work a CaMRR1 ortholog was found in Candida dubliniesis, a yeast closely related to C. albicans. It could be shown that gain-of-function mutations in CdMRR1 were the cause of MDR1 overexpression and drug resistance in all investigated clinical and in vitro generated strains. The results showed that Mrr1 plays an important role in the development of drug resistence in these human fungal pathogens. Currently it is not understood how these zinc cluster transcription factors are activated under inducing conditions or by gain-of-function mutations. To better understand the regulation of Mrr1 activation, in this work deletion studies were performed to identify functional domains of the transcription factor. To gain better insight into the regulation of MDR1-mediated drug resistance in C. albicans, the interdependence of Mrr1 and two other MDR1 regulators, Cap1 and Upc2, was studied in this work. ChIP-on-chip analyses and transcriptional profiles with acitvated Mrr1 were performed to identify direct targets of Mrr1. This thesis contributes to the understanding of the development of multidrug resistance in C. albicans. Efflux pumps and their transcriptional regulators provide an interesting target for the development of new antifungal drugs or the further development of available drugs against C. albicans infections. KW - Candida albicans KW - Resistenz KW - Effluxpumpen KW - Candida albicans KW - resistance KW - efflux pump Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-70916 ER - TY - THES A1 - Aminake, Makoah Nigel T1 - 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 T1 - Die Entwicklung von Malaria-Kombinationstherapien: Die Charakterisierung von Hybridmolekülen für eine HIV/Malaria-Kombinationstherapie und von Thiostrepton als ein gegen das Proteasom-gerichtetes Antibiotikum mit dualem Wirkmodus N2 - 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. N2 - Malaria und HIV gehören zu den wichtigsten weltweiten Gesundheitsproblemen unserer Zeit und verursachen jährlich zusammen fast drei Millionen Todesfälle. Das Verbreitungsgebiet beider Krankheit überschneidet sich in vielen Weltregionen wie Afrika südlich der Sahara, Südostasien und Südamerika, was zu einem erhöhten Risiko für Koinfektionen führt. Während der vorliegenden Arbeit stellten wir Hybridmoleküle her und charakterisierten diese in Bezug auf ihre gleichzeitige Wirksamkeit gegen P. falciparum und HIV mit dem Ziel einer möglichen Kombinationstherapie gegen beide Krankheiten. Diese Hybridmoleküle wurden durch kovalente Verbindung von Azidothymidin (AZT) mit Dihydroartemisinin (DHA), Tetraoxan und Chloroquin (CQ) hergestellt. Die dabei hergestellte kleine Molekülsammlung wurde auf antivirale Wirkung und Wirkung gegen Malaria getestet. In vitro ist, gemäß unserer Daten, Dihyate das wirksamste Molekül, mit einer dem DHA vergleichbaren Wirksamkeit gegen Plasmodium (IC50 = 26 nM, SI > 3000), einer mittelmäßigen Wirksamkeit gegen HIV (IC50 = 2.9 µM; SI > 35) und keiner Wirkung auf HeLa-Zellen bei den im Versuch verwendeten Konzentrationen (CC50 > 100 µM). Weiterhin ergaben pharmakokinetische Studien, dass Dihyate metabolisch instabil ist und nach einer O-Dealkylierung gespalten wird, sobald es in Kontakt mit Cytochrom P450 Enzymen kommt. Dies erklärt auch die Unwirksamkeit von Dihyate gegen dem CQ-sensitiven P. berghei N Stamm im Mausversuch bei oraler Gabe von 20mg/kg. Wir berichten hier von einem ersten Ansatz Hybridmoleküle gegen Malaria/ HIV zu entwickeln. Zukünftige Verbesserungen werden darauf abzielen Hybridmoleküle der zweiten Generation herzustellen um sowohl die Wirksamkeit gegen HIV als auch die Bioverfügbarkeit zu verbessern. Auf Grund der Entwicklung von Resistenzen gegenüber sämtliche Substanzen, die zusammen mit Artemisinin in Kombinationstherapien genutzt werden, hat die Verwendung von Antibiotika bei der Behandlung der Malaria das Interesse daran neu geweckt, Antibiotika mit starker Wirksamkeit gegenüber Plasmodium aufzuspüren. Während der vorliegenden Studie untersuchten wir die Wirksamkeit von Thiostrepton und seinen Derivaten gegenüber Plasmodium. Diese Derivate wurden durch Kombinationen von Verkürzung der Seitenkette, Oxidation und der Anbringung von lipophilen Thiolen an die endständige Dehydroaminosäure hergestellt. Wir konnten zeigen, dass die Derivate SS231 und SS234 (IC50 = 1 µM SI > 59 und SI > 77) eine bessere Wirksamkeit gegen Plasmodium besitzen als Thiostrepton (IC50 = 8.95 µM, SI = 1.7). Diese Wirksamkeit konnte bei Konzentrationen beobachtet werden, die nicht hämolytisch sind und ungiftig gegenüber menschlichen Zelllinien. Thiostrepton und seine Derivate zeigten transmissionsblockierende Eigenschaften, wenn sie in Konzentrationen, die ihren IC50- oder IC90-Werten entsprachen, eingesetzt wurden. Unsere Daten zeigen auch, dass diese Substanzen die Aktivität des Proteasoms von Plasmodium abschwächen, was zu einer Anreicherung von ubiquitinierten Proteinen führte, wenn die Parasiten mit den Substanzen in IC80-Konzentrationen inkubiert wurden. Unsere Ergebnisse sprechen dafür, dass das Proteasom ein attraktives Ziel für therapeutische Maßnahmen sein kann. In diesem Zusammenhang sind die Derivate des Thiostreptons vielversprechende Kandidaten, da sie gleichzeitig an zwei unabhängigen Zielstrukturen angreifen, dem Proteasom und dem Apicoplasten und die Fähigkeit besitzen, sowohl die asexuellen Blutstadien als auch diejenigen Blutstadien, die für die Weitergabe des Parasiten verantwortlich sind, zu beseitigen. Um unsere Ergebnisse weiter zu untermauern, untersuchten wir die Wirkung von Peptidyl-Sulfonyl-Fluoriden, einer neuen Klasse von Substanzen mit Wirksamkeit gegen Malaria und hemmender Wirkung gegenüber dem Proteasom auf die Reifung von Gametozyten. Die Substanzen AJ34 und AJ38 unterdrückten die Bildung von Gametozyten vollständig, wenn sie in Konzentrationen, die ihren IC50-Werten (0.23 µM und 0.17 µM) entsprachen, eingesetzt wurden. Dies spricht für ein starkes transmissionsblockierendes Potential dieser Substanzen. Das Proteasom, ein bedeutender proteinabbauender Komplex, der für den Abbau und die Wiedergewinnung nicht funktioneller Proteine verantwortlich ist, wurde bisher nur indirekt in P. falciparum untersucht. Zusätzlich wurde die Existenz eines, dem Proteasom-ähnlichen, Proteins mit Ähnlichkeiten zu bakteriellen ClpQ/hslV Threonin-Peptidasen in Plasmodium vermutet. Gegen die Untereinheiten alpha 5 (α5-SU), beta 5 (β5-SU) und gegen pfhslV wurden in Mäusen Antikörper generiert. Mit diesen konnten wir zeigen, dass das Proteasom sowohl in den asexuellen als auch in den sexuellen Blutstadien von P. falciparum exprimiert wird und im Zellkern und im Zytoplasma lokalisiert sind. Die Expression von PfhslV konnte jedoch nur in Trophozoiten und Schizonten beobachtet werden. Der Transport der Proteasomuntereinheiten wurde weiterhin durch die Herstellung von transgenen Parasiten, die GFP-markierte Proteine bilden, untersucht. Die Expression von α5-SU-GFP in transgenen Parasiten schien im Zytoplasma aller Blutstadien lokalisiert zu sein, wobei durch diese Parasiten keine zusätzlichen Informationen gewonnen werden konnten. Zusammengefasst sprechen unsere Daten für zwei neue Werkzeuge für Kombinationstherapien. Hybridmoleküle sind vielversprechende Werkzeuge zur Heilung von gleichzeitig mit Malaria und HIV infizierten Patienten. Sehr wirksame Antibiotika mit einem breiten Wirkungsspektrum könnten in Artemisinin-Kombinationstherapien nützlich werden, wenn es darum geht, resistente Parasiten zu beseitigen und die Übertragung sowohl der Resistenz als auch der Krankheit zu verringern. KW - Malaria KW - HIV KW - Thiostrepton KW - Arzneimitteldesign KW - Malaria KW - HIV KW - co-infection KW - drug KW - screening KW - hybrid KW - proteasome KW - thiostrepton Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-71841 ER - TY - JOUR A1 - Sasse, Christoph A1 - Schillig, Rebecca A1 - Dierolf, Franziska A1 - Weyler, Michael A1 - Schneider, Sabrina A1 - Mogavero, Selene A1 - Rogers, David P. A1 - Morschhäuser, Joachim T1 - The Transcription Factor Ndt80 Does Not Contribute to Mrr1-, Tac1-, and Upc2-Mediated Fluconazole Resistance in Candida albicans N2 - 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. KW - Candida albicans Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-69201 ER - TY - JOUR A1 - Eulalio, Ana A1 - Fröhlich, Kathrin S. A1 - Mano, Miguel A1 - Giacca, Mauro A1 - Vogel, Jörg T1 - A Candidate Approach Implicates the Secreted Salmonella Effector Protein SpvB in P-Body Disassembly N2 - 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. KW - Salmonella KW - RNS Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68928 ER - TY - THES A1 - Menzel, Thomas Michael T1 - Studien zum Wirkungsmechanismus neuer antiinfektiver Bisnaphthalimide gegen Staphylococcus aureus und Transkriptomanalysen zur Auswirkung von Antibiotika auf S. epidermidis T1 - Mode-of-action studies of novel antiinfective bisnaphthalimides on Staphylococcus aureus and transcriptional analysis of the effect of antibiotics on S. epidermidis N2 - Die Therapie von bakteriellen Infektionen beruht heutzutage zum Großteil auf dem Einsatz von Antibiotika. Die schnelle Entwicklung und rasche Verbreitung von resistenten Stämmen mancher Erreger gegen diese Antibiotika stellt ein enormes Problem für das Gesundheitswesen dar. Da momentan zur Antibiotikatherapie keine Alternativen bestehen, kommt der Erforschung neuer potenzieller Wirkstoffe eine sehr große Bedeutung zu. In einem Screening-Verfahren lagen die minimalen Hemmkonzentrationen einiger bisquartärer Bisnaphthalimide gegen Staphylococcus aureus und S. epidermidis im Bereich von 0,6 bis 2,5 µg/ml. Die Substanz mit den geringsten minimalen Hemmkonzentrationen war MT02. Daraufhin wurde das Wirkungsspektrum von MT02 gegen Bakterien detaillierter untersucht und gefunden, dass die Substanz vorwiegend gegen Gram-positive Erreger und nicht gegen Gram-negative Bakterien wirksam ist. Zytotoxizitätstests ergaben eine geringe bis nicht nachweisbare Toxizität gegen verschiedene Zelllinien im Bereich von 73 bis mehr als 150 µg/ml. Um die Wirkungsweise von MT02 genauer zu untersuchen wurden zunächst DNA-Microarray-Untersuchungen an S. aureus durchgeführt. Deren Ergebnisse ließen einen Einfluss der Substanz auf viele Gene des DNA-Metabolismus erkennen. Inkorporationsstudien mittels radioaktiver Ganzzellmarkierung bestätigten die Auswirkung von MT02 auf den DNA-Stoffwechsel. Durch kompetitive Inkubation wurde festgestellt, dass MT02 in der Lage ist Ethidiumbromid von DNA zu verdrängen bzw. dessen Bindung zu verhindern. Genauere Untersuchungen mittels Oberflächen-Plasmon-Resonanz ergaben, dass MT02 konzentrationsabhängig, reversibel und sequenzunspezifisch an DNA bindet. Die thermodynamischen Dissoziationskonstanten lagen im Mittel bei ca. 4 x 10-8 mol/l und beschrieben somit eine relativ starke Bindung von MT02 an DNA. Neben diesem primären Wirkungsmechanismus der DNA-Bindung gaben mehrere Befunde Hinweise auf einen sekundären Wirkmechanismus, der die Zellwand-Struktur bzw. Zellwand-Biosynthese beinhaltet. Eine MT02-resistente Mutante von S. aureus HG001 konnte durch vielfaches Passagieren in MT02-haltigem Medium generiert werden. Diese erzeugte bei Wachstum mit hohen Konzentrationen an MT02 einen roten Phänotyp. Die Natur dieses roten Farbstoffes konnte bislang nicht aufgeklärt werden, jedoch gibt es Hinweise, dass dieser auf Abbauprodukte von MT02 zurückzuführen ist. In einem weiteren Projekt wurde mittels Transkriptionsstudien die Auswirkung von verschiedenen bekannten Antibiotika sowie von neuen Wirkstoffen auf das Transkriptom von S. epidermidis untersucht. Die Ergebnisse dieser Studien können durch vergleichende Analysen als Grundlage für die Einordnung des Wirkmechanismus neuer Substanzen dienen. N2 - The treatment of bacterial infections is nowadays mostly accomplished by the application of antibiotics. However, the rapid development and vast distribution of resistant strains of some pathogens against a variety of antibiotics form an enormous challenge for public health care systems worldwide. As until now there are no applicable alternatives to antibiotic therapy against most pathogens, there is an urgent need for the discovery of new antibacterial substances. Screening several newly synthesized compounds, the minimal inhibitory concentrations of some bisquaternary bisnaphthalimides ranged from 0.6 to 2.5 µg/ml against Staphylococcus aureus and S. epidermidis. Thereof one substance, designated MT02, revealed the lowest minimal inhibitory concentrations of this compound class. Following these susceptibility tests, the antibacterial spectrum of MT02 was determined and revealed a broad spectrum against Gram-positive bacteria but almost no activity against Gram-negative species. In cytotoxicity tests with several cell lines MT02 exhibited low or undetectable toxicity in concentrations ranging from 73 to more than 150 µg/ml. Microarray studies were conducted to further elucidate the mode of action of MT02 against S. aureus. The results suggested that MT02 has an impact on the bacterial DNA-metabolism. To verify this, radioactive whole cell labeling experiments were performed which clearly provided evidence for the inhibition of incorporation of [3H]-thymidine into bacteria by MT02 and thus for an interference of MT02 with DNA-metabolism. Coincubation and gel retardation studies showed that MT02 is able to directly interact with DNA and to displace ethidiumbromide which has intercalated into the DNA before. Surface plasmon resonance experiments pinpointed the binding of MT02 to doublestranded DNA and revealed binding constants in the range of 4 x 10-8 mol/l describing a strong binding affinity of MT02 to DNA. In addition to this primary mode of action, several results suggested a secondary mode of action comprising the structure and / or the biosynthesis of the bacterial cell wall. By passaging S. aureus HG001 in broth with increasing concentrations of MT02, a MT02-resistant mutant could be obtained. This mutant produced a red phenotype when grown with high concentrations of MT02. Studies to determine the nature of this red dye are still in progress. However, first results indicate that the red color is a degradation product of MT02. Another project dealt with the comparative analysis of transcriptomes of S. epidermidis under the influence of antibiotics with known modes of action as well as new active compounds. The results of these studies can be the basis for the assignment of new antibacterial substances to classes of antibiotics with known modes of action. KW - MRSA KW - Naphthalinderivate KW - Quartäre Bisnaphthalimide KW - MT02 KW - Antibiotikum KW - MRSA KW - Antibiotic KW - Quaternary Bisnaphthalimide KW - MT02 Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-56362 ER - TY - JOUR A1 - Weibel, Stephanie A1 - Raab, Viktoria A1 - Yu, Yong A. A1 - Worschech, Andrea A1 - Wang, Ena A1 - Marincola, Francesco M. A1 - Szalay, Aladar A. T1 - Viral-mediated oncolysis is the most critical factor in the late-phase of the tumor regression process upon vaccinia virus infection N2 - 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. KW - Virusinfektion KW - Krebs Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68691 ER - TY - JOUR A1 - Zdziarski, Jaroslaw A1 - Brzuszkiewicz, Elzbieta A1 - Wullt, Bjorn A1 - Liesegang, Heiko A1 - Biran, Dvora A1 - Voigt, Birgit A1 - Gronberg-Hernandez, Jenny A1 - Ragnarsdottir, Bryndis A1 - Hecker, Michael A1 - Ron, Eliora Z. A1 - Daniel, Rolf A1 - Gottschalk, Gerhard A1 - Hacker, Joerg A1 - Svanborg, Catharina A1 - Dobrindt, Ulrich T1 - Host Imprints on Bacterial Genomes-Rapid, Divergent Evolution in Individual Patients N2 - 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. KW - Proteomanalyse KW - Bakterien Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68594 ER - TY - JOUR A1 - Moll, Heidrun T1 - Epidermal Langerhans cells are critical for immunoregulation of cutaneous leishmaniasis N2 - 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. KW - Biologie KW - Immunologie Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61323 ER - TY - JOUR A1 - Moll, Heidrun A1 - Müller, Christoph A1 - Gillitzer, Reinhard A1 - Fuchs, Harald A1 - Röllinghoff, Martin A1 - Simon, Markus M. A1 - Kramer, Michael D. T1 - Expression of T-cell-associated serine proteinase-1 during murine Leishmania major infection correlates with susceptibility to disease N2 - 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. KW - Biologie KW - Immunologie Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61311 ER - TY - JOUR A1 - Moll, Heidrun A1 - Röllinghoff, Martin T1 - Resistance to murine cutaneous leishmaniasis is mediated by T\(_H\)1 cells, but disease-promoting CD4\(^+\) cells are different from T\(_H\)2 cells N2 - No abstract available KW - Biologie KW - Immunologie Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61305 ER - TY - JOUR A1 - Moll, Heidrun A1 - Binöder, Kerstin A1 - Bogdan, Christian A1 - Solbach, Werner A1 - Röllinghoff, Martin T1 - Production of tumour necrosis factor during murine cutaneous leishmaniasis N2 - 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. KW - Immunologie Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61291 ER - TY - JOUR A1 - Hacker, Jörg A1 - Ott, M. A1 - Hof, H. T1 - 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 N2 - No abstract available KW - Infektionsbiologie Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59874 ER - TY - JOUR A1 - Zingler, G. A1 - Blum, G. A1 - Falkenhagen, U. A1 - Orskov, I. A1 - Orskov, F. A1 - Hacker, Jörg A1 - Ott, M T1 - Clonal differentiation of uropathogenic E. coli isolates of serotype O6:K5 by fimbrial antigen typing and DNA long-range mapping techniques N2 - 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. KW - Infektionsbiologie Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59865 ER - TY - JOUR A1 - Hacker, Jörg A1 - Kestler, H. A1 - Hoschützky, H. A1 - Jann, K. A1 - Lottspeich, F. A1 - Korhonen, T. K. T1 - Cloning and characterization of the S fimbrial adhesin (SfaII) complex of an Escherichia coli O18:K1 meningitis isolate N2 - 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. KW - Infektionsbiologie Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59853 ER - TY - JOUR A1 - Morschhäuser, J. A1 - Uhlin, B. E. A1 - Hacker, Jörg T1 - Transcriptional analysis and regulation of the sfa determinant coding for S fimbria of pathogenic E. coli strains N2 - 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 prömoters, 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. KW - Infektionsbiologie KW - Gene regulation KW - Fimbriae KW - Adhesion KW - Transcription KW - trans-activation Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59844 ER - TY - JOUR A1 - Schroten, H. A1 - Steinig, M. A1 - Plogmann, R. A1 - Hanisch, F. G. A1 - Hacker, Jörg A1 - Herzig, P. A1 - Wahn, V T1 - S-fimbriae mediated adhesin of Escherichia coli to human buccal epithelial cells is age independent N2 - 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 N2 - Die S-Fimbrien vermittelte Adhiision von Escherichia coli an menschliche Mundschleimhautzellen ist altersunabhängig. S-Fimbrien tragende Escherichia coli, die Sepsis und Meningitis . im Neugeborenenalter verursachen, binden an sialinsäurehaltige Glycoproteine atif der Oberfläche menschlicher Mundschleimhautzellen. Wir untersuchten die Abhängigkeit · der Bindung vom Alter des Schleimhautzellenspenders. S-Fimbrien tragende. E. coli banden in vergleichbarer Zahl an Zellen von Neugeborenen, Säuglingen, älteren · Kindern und Erwachsenen (23,0 ± 8,6; 23,1 ± 11,5; 24,7 ± 7,9; 28,9 ± 8,8). Die vermehrte Empfänglichkeit von Neugeborenen für Infektionen, die durch S- Fimbrien tragende E. coli verursacht werden, kann nicht mit einer verstärkten Adhäsion an Mundschleimhautzellen erklärt wer.den. KW - Infektionsbiologie Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59830 ER - TY - JOUR A1 - Ott, M. A1 - Bender, L. A1 - Lück, P. C. A1 - Meyer, P. A1 - Hacker, Jörg T1 - Distribution of Legionellae in a hospital water system: prevalence of immunologically and genetically related Legionella pneumophila serogroup 6 isolates N2 - 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. KW - Infektionsbiologie KW - Legionella pneumophila KW - Hospital water system KW - Environmental isolate KW - Serogroup KW - Genomic profile Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59827 ER - TY - JOUR A1 - Linhardt, F. A1 - Ziebuhr, W. A1 - Meyer, P. A1 - Witte, W. A1 - Hacker, Jörg T1 - Pulsed-field gel electrophoresis of genomic restriction fragments as a tool for the epidemiological analysis of Staphylococcus aureus and coagulase-negative Staphylococci N2 - 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. epidermülis 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. KW - Infektionsbiologie Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59811 ER - TY - JOUR A1 - Schroten, H. A1 - Lethen, A. A1 - Hanisch, F., G. A1 - Plogmann, R. A1 - Hacker, Jörg A1 - Nobis-Bosch, R. A1 - Wahn, V. T1 - 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 N2 - 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. KW - Infektionsbiologie Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59804 ER - TY - JOUR A1 - Zingler, G. A1 - Ott, M. A1 - Blum, G. A1 - Falkenhagen, U. A1 - Naumann, G. A1 - Sokolowska-Köhler, W. A1 - Hacker, Jörg T1 - Clonal analysis of Escherichia coli serotype O6 strains from urinary tract infections N2 - 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. KW - Infektionsbiologie KW - E. coli serotype 06 KW - urinary tract infection KW - virulence factors KW - clonal analysis KW - molecular epidemiology Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59786 ER - TY - JOUR A1 - Schroten, H. A1 - Hanisch, F. G. A1 - Plogmann, R. A1 - Hacker, Jörg A1 - Uhlenbruck, G. A1 - Wahn, V. T1 - 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 N2 - 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. KW - Infektionsbiologie Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59793 ER - TY - JOUR A1 - Fischer, G. A1 - Bang, H. A1 - Ludwig, B. A1 - Mann, K. H. A1 - Hacker, Jörg T1 - Mip protein of Legionella pneumophila exhibits peptidyl-prolyl cis-trans-isomerase (PPIase) activity N2 - 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. KW - Infektionsbiologie Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59778 ER - TY - JOUR A1 - Ott, M. A1 - Bender, L. A1 - Chirinos, E. A1 - Ehret, W. A1 - Hacker, Jörg T1 - Phenotype versus genotype of the 19 kd peptido-glycan associated protein of Legionella (PplA) among Legionellae and other gram-negative bacteria N2 - 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. KW - Infektionsbiologie KW - Legionellae KW - peptido-glycan associated protein KW - ppl KW - Southern hybridization KW - stringency KW - polymerase chain reaction (PCR) Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59768 ER - TY - JOUR A1 - Ott, M. A1 - Messner, P. A1 - Heesemann, J. A1 - Marre, R. A1 - Hacker, Jörg T1 - Temperature dependent expression of flagella in Legionella N2 - 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. KW - Infektionsbiologie Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59755 ER - TY - JOUR A1 - Bender, L. A1 - Ott, M. A1 - Debes, A. A1 - Rdest, U. A1 - Heesemann, J. A1 - Hacker, Jörg T1 - Distribution, expression and long range mapping of legiolysin gene (lly) specific DNA sequences in Legionellae N2 - 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. pneumophiüz 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. KW - Infektionsbiologie Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59744 ER - TY - JOUR A1 - Ott, M. A1 - Bender, L. A1 - Blum, G. A1 - Schmittroth, M. A1 - Achtmann, M. A1 - Tschäpe, H. A1 - Hacker, Jörg T1 - Virulence patterns and long range mapping of extraintestinal Escherichia coli K1, K5 and K100 isolates: Use of pulse field gel electrophoresis N2 - 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. KW - Infektionsbiologie Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59738 ER - TY - JOUR A1 - Ludwig, B. A1 - Schmid, A. A1 - Marre, R. A1 - Hacker, Jörg T1 - Cloning, genetic analysis and nucleotide sequence of a determinant coding for a 19 kd peptidoglycan-associated protein (Ppl) of Legionella pneumophila N2 - 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. KW - Infektionsbiologie Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59721 ER - TY - JOUR A1 - Blum, G. A1 - Ott, M. A1 - Cross, A. A1 - Hacker, Jörg T1 - Virulence determinants of Escherichia coli O6 extraintestinal isolates analysed by Southern hybridizations and DNA long range mapping techniques N2 - 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. KW - Infektionsbiologie KW - E. coli serotype 06 KW - extraintestinal isolates KW - virulence factors KW - gene probes KW - DNA lang range mapping KW - epidemiology Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59717 ER - TY - JOUR A1 - Wintermeyer, E. A1 - Rdest, U. A1 - Ludwig, B. A1 - Debes, A. A1 - Hacker, Jörg T1 - Characterization of legiolysin (lly); responsible for hemolytic activity, colour production and fluorescence of Legionella pneumophila N2 - No abstract available KW - Infektionsbiologie Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59706 ER - TY - JOUR A1 - Ott, M. A1 - Hacker, Jörg T1 - Analysis of the variability of S fimbriae expression in an Escherichia coli pathogen. N2 - 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. KW - Infektionsbiologie KW - Escherichia coli KW - S-fimbria KW - Variability KW - Expression Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59695 ER - TY - JOUR A1 - Hacker, Jörg A1 - Ott, M. A1 - Ludwig, B. A1 - Rdest, U. T1 - Intracellular survival and expression of virulence determinants of Legionella pneumophila N2 - 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: Intrazelluläres Überleben N2 - Intrazelluläres Überleben und Expression der Virulenzdeterminanten vonLegionella pneumophila. L. pneumophila, der Erreger der Legionärskrankheit, hat die Fähigkeit, sowohl in Makrophagen als auch in Protozoen zu leben und sich dort zu vermehret;t. Legionellen inhibieren die Fusion von Phagosom und Lysosom und hemmen die Ansäuerung des Phagosoms. Mit Hilfe von zwei unterschiedlichen Zellkultur-Systemen konnte gezeigt werden, daß Legionella-Stämme ihre Virulenz nach Laborpassage verlieren. Um die Mechanismen zu studieren, die für das intrazelluläre Überleben von Legionellen verantwortlich sind, haben wir eine Genbank des Legionella pneumophila-Stammes Philadelphia I in Escherichia coli K-12angelegt. Mit Hilfe der Cosmid-Klonierungstechnik war es möglich, fünf putative Virulenzfaktoren zu klonieren. Zwei von diesen Faktoren haben hämolytische Eigenschaften und drei sind Membran-assoziierte Proteine mit Molekulargewichten von 19, 26 und 60 kilodalton. Eines der hämolytischen Proteine, das Legiolysin, lysiert spezifisch humane Erythrozyten. Das zweite Hämolysin zeigt zusätzlich proteolytische Eigenschaften und schädigt sowohl Vero- als auch CHO-Zellen. Weitere Studien sind notwendig, um die Rolle der klonierten Proteine in der Pathogenese von Legionella exakt zu bestimmen. KW - Infektionsbiologie Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59681 ER - TY - JOUR A1 - Ott, M. A1 - Bender, L. A1 - Marre, R. A1 - Hacker, Jörg T1 - Pulsed field electrophoresis of genomic restriction fragments for the detection of nosocomial Legionella pneumophila in hospital water supplies N2 - 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 Lü~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. KW - Infektionsbiologie Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59672 ER - TY - JOUR A1 - Schmoll, T. A1 - Morschhäuser, J. A1 - Ott, M. A1 - Ludwig, B. A1 - Van Die, I. A1 - Hacker, Jörg T1 - Complete genetic organization and functional aspects of the Escherichia coli S fimbrial adhesin determinant: nucleotide sequence of the genes sfaB, C, D, E, F. N2 - 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. KW - Infektionsbiologie KW - Escherichia coli KW - S fimbrial adhesin (Sfa) KW - genetic organization KW - gene regulation KW - nucleotide sequence Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59661 ER - TY - JOUR A1 - Bender, L. A1 - Ott, M. A1 - Marre, R. A1 - Hacker, Jörg T1 - Genome analysis of Legionella spp. by orthogonal field alternation gel electrophoresis (OFAGE) N2 - 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. KW - Infektionsbiologie KW - Legionella ssp. KW - Genome analysis KW - Orthogonal field attenuation gel electrophoresis Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59657 ER - TY - JOUR A1 - Marre, R. A1 - Kreft, B. A1 - Hacker, Jörg T1 - Genetically engineered S and F1C fimbriae differ in their contribution to adherence of Escherichia coli to cultured renal tubular cells N2 - 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. KW - Infektionsbiologie Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59644 ER - TY - JOUR A1 - Ventur, Y. A1 - Scheffer, J. A1 - Hacker, Jörg A1 - König, W. T1 - Effects of adhesins from mannose-resistant Escherichia coli on mediator release from human lymphocytes, monocytes and basophils and from polymorphonuclear granulo-cytes N2 - 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. KW - Infektionsbiologie Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59636 ER - TY - JOUR A1 - Schmoll, T. A1 - Ott, M. A1 - Ougeda, B. A1 - Hacker, Jörg T1 - 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 N2 - 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. KW - Infektionsbiologie Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59625 ER - TY - JOUR A1 - Morschhäuser, J. A1 - Hoschützky, H. A1 - Jann, K. A1 - Hacker, Jörg T1 - Functional analysis of the Sialic acid-binding adhesin SfaS of pathogenic Escherichia coli by site-specific mutagenesis N2 - 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. KW - Infektionsbiologie Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59613 ER - TY - JOUR A1 - Hacker, Jörg A1 - Bender, L. A1 - Ott, M. A1 - Wingeder, J. A1 - Lund, B. A1 - Marre, R. A1 - Goebel, W. T1 - Deletions of chromosomal regions coding for fimbriae and hemolysins occur in vivo and in vitro in various extraintestinal Escherichia coli isolates N2 - No abstract available KW - Infektionsbiologie KW - P-fimbriae KW - hemolysin KW - genomic deletions KW - extraintestinal E. coli KW - virulence modulation Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59608 ER - TY - JOUR A1 - Riegmann, N. A1 - Kusters, R. A1 - Van Veggel, H. A1 - Bergmans, H. A1 - Van Bergen en Henegouwen, P. A1 - Hacker, Jörg A1 - Van Die, I. T1 - F1C fimbriae of an uropathogenic Escherichia coli: Genetic and functional organization of the foc gene cluster and identification of minor subunits N2 - 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. KW - Infektionsbiologie Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59597 ER - TY - JOUR A1 - Schmoll, T. A1 - Hoschützky, H. A1 - Morschhäuser, J. A1 - Lottspeich, F. A1 - Jann, K. A1 - Hacker, Jörg T1 - 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 N2 - 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. KW - Infektionsbiologie Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59585 ER - TY - JOUR A1 - Marre, R. A1 - Hacker, Jörg A1 - Braun, V. T1 - The cell-bound hemolysin of Serratia marcescens contributes to uropathogenicity N2 - No abstract available KW - Infektionsbiologie KW - Serratia marcescens KW - uropathogenicity KW - hemolysin KW - rat Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59576 ER - TY - JOUR A1 - König, W. A1 - König, B. A1 - Scheffer, J. A1 - Hacker, Jörg A1 - Goebel, W. T1 - Role of cloned virulence factors (mannose-resistant hemagglutination, mannose-resistant adhesins) from uropathogenic Escherichia coli strains in release of inflammatory mediators from neutrophils and mast cells N2 - Genetically cloned E. co/i strains expressing cloned virulence factors were studied with regard to their capability to induce inflammatory mediator release from various target cells. Among the strains were E. co/i strains with mannose-resistant haemagglutination (MRH +) and mannose-resistant adhesins, e.g. E. coli 536/21 pANN 80 I /4, E. coli 536/21 pANN 921 and E. coli 536/21 pANN 801-1. In comparison, E. coli 536/21, E. coli 536/21 pGB 30 int and E. coli Kl2, without and with mannosesensitive haemagglutination (MSH±), and adhesins were studied. The properties of the various strains for human PMN with regard to adherence and phagocytosis, chemiluminescence, 5-lipoxygenase activation of arachidonic acid, leukotriene formation, granular enzyme release and release of histamine from rat mast cells were analysed. It is evident that the various 'biochemical processes of cell activation are dissociated events. The highest chemiluminescence response is obtained with strains expressing MSH+, P-M RH+ or S-M RH+; the presence of S-adhesins suppressed the response. Highest leukotriene formation is obtained with E. coli 536/21 pANN 801-4, while E. coli with MSH was inactive. The concomitant presence of haemolysin secretion enhanced mediator release significantly. Our data suggest a potent role for mannose-resistant haemagglutination (MRH), adhesins and haemolysin as virulence factors in inducing the release of inflammatory mediators. KW - Infektionsbiologie Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59564 ER - TY - JOUR A1 - Marre, R. A1 - Hacker, Jörg A1 - Wood, G. A1 - Schmidt, G. T1 - Oral vaccination of rats with live avirulent Salmonella derivatives expressing adhesive fimbrial agents of uropathogenic Escherichia coli N2 - The avirulent Salmonella typhimurium F885 was transformed with a plasmid carrying the cloned S fimbriae genes of a uropathogenic Escherichia coli. The resulting transformant (F885-1) produced efficiently E. coli S fimbriae and was used for live oral vaccination of rats. For comparison rats were immunized subcutaneously with isolated S fimbriae. Both routes of vaccination resulted in a significant lgG antibody response to S fimbriae. In addition live oral vaccination induced a serum lgA response against S fimbriae. After transurethral infection of rats with a S fimbriae producing E. coli a 10-fold reduction of bacterial counts in the kidney was observed in rats orally vaccinated with F885-1 as compared to unvaccinated controls. This study suggests that the avirulent Salmonella F885 may be used as a fimbrial antigen carrier for oral vaccination against renal infections. KW - Infektionsbiologie KW - Avirulent Salmonella KW - S-Fimbriae KW - Uropatbcgenie E. coli Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59559 ER - TY - JOUR A1 - Krallmann-Wenzel, U. A1 - Ott, M. A1 - Hacker, Jörg A1 - Schmidt, G T1 - Chromosomal mapping of genes encoding mannose-sensitive (type I) and mannose-resistant F8(P) fimbriae of Escherichia coli O18:K5:H5 N2 - DNA hybridization experiments demonstrated that the gene clusters encoding the F8 fimbriae (fei) as well as the type I fimbriae (pi/) exist in a single copy on the chromosome of E. coli 018:K5 strain 2980. In conjugation experiments with appropriate donors, the chromosomal site of these gene clusters was determined. The pil genes were mapped close to the gene clusters thr and Jeu controlling the biosynthesis of threonine and leucine, respectively. The fei genes were found to be located close to the galactose operon (gal) between the position 17 and 21 of the E. coli chromosomallinkage map. KW - Infektionsbiologie KW - Escherichia coli KW - F1 KW - F8 fimbriae KW - Gene cloning KW - Gene mapping Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59545 ER - TY - JOUR A1 - Munoa, F. A1 - Hacker, Jörg A1 - Juarez, A. T1 - Characterization of a chromosomal mutant that blocks hemolysin excretion in Escherichia coli N2 - We analyzed an Escherichia coli strain which harbours a chromosomal mutation that blocks the hemolysin excretion. Compartmentation studies showed that hemolysin accumulates in the cytoplasm and not in the periplasm. The mutation did not affect the SDS-PAGE protein pattern of the outer membrane, although some alterations were apparent in the periplasmic protein pattern. The mutant strain, E. coli Hsb-1 also failed to export a cloned fimbrial adhesin. The mutation maps in the min. 3.5 of the E. coli genetic map. KW - Infektionsbiologie KW - Hemolysin excretion KW - Escherichia coli KW - Mutant Y1 - 1988 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59534 ER - TY - JOUR A1 - Pawelzik, M. A1 - Heesemann, J. A1 - Hacker, Jörg A1 - Opferkuch, W. T1 - Cloning and characterization of a new type of fimbria (S/F1C-related fimbria) expressed by an Escherichia coli O75:K1:H7 blood culture isolate N2 - The Escherichia coli blood culture isolate BK658 (07S:K1:H7) expresses F1A and F1B fimbriae as weil as a third fimbrial type which reacts with anti-S-fimbrial antiserum but fails to show S-specific binding properlies (i.e., agglutination of bovine erythrocytes). To characterize these fimbriae, we cloned the respective genetic determinant in E. coli K-12. The resulting recombinant clone HB101(pMMP658-6) expresses fimbriae of 1.2-p.m length and a diameter of approximately 7 nm. The determinant codes for the fimbrillin subunit, a protein of 17 kUodaltons in size, and for at least five other proteins of 87, 31, 23, 14.3, and 13.8 kUodaltons. By restriction analysis and by DNA-DNA hybridization, it could be shown that the cloned fimbrial determinant of strain BK658 exhibits a high degree of sequence homology to the gene clusters coding for S fimbrial adhesins (sfa) and F1C fimbriae (/oc). By using the Western blot (immunoblot) technique and a quantitative enzyme-linked immunosorbent assay, it could be further demonstrated that the cloned fimbriae of BK658, S fimbriae, and FlC fimbriae share cross-reactive epitopes as weil as antigenic determinants specific for each fimbrial type. No antigenic cross-reactivity with F1C fimbriae could be detected. The results indicate a genetical and serological relatedness of the cloned fimbriae toS fimbriae and F1C fimbriae. Therefore, this new type of fimbriae is preliminarily termed SIF1C-related fimbriae (Sfr). KW - Infektionsbiologie Y1 - 1988 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59529 ER - TY - JOUR A1 - Ott, M. A1 - Hoschützky, H. A1 - Jann, K. A1 - Van Die, I. A1 - Hacker, Jörg T1 - Gene clusters for S fimbrial adhesin (sfa) and F1C Fimbriae (foc) of Escherichia coli: Comparative aspects of structure and function N2 - Fimbrial 8dhesins en8ble b8cteria to 8ttach t9 eucaryotic ceU~. The genetic determin8nts for S fimbrial 8dhesins (sja) an.d for FlC ("pseudotype I") fimbri8e ifoc) were compared. Sfa and FlC represent functionally distinct 8dbesins in tbeir receptor specificities. Nevertheless, 8 high degree of bomology between both determin8nts was found on the basis of DNA-DNA hybridizations. Characteristic difl'erences in the restriCtion maps of tbe corresponding gene clusters, bowever, were visible in regions coding for the fimbrial subunits and for the S-specific 8dhesin. While a plasmid carrying the geneiic deternlinant for FlC fimbri8e was 8ble to complement transposon-induced sfa mutants, 8 plasmid carrying tbe genetic determin8nt for 8 tbird 8dht$in type, termed P fimbriae, was un8ble to do so. Proximal sfa-specific sequences carrying the S fimbrial st'"uctural gene were fused to sequences representing tbe di$tal part of the foc gene cluster to form 8 hybrid cluster, and tbe foc proxim~ region coding for tbe structural protein was Iigated to sfa distal sequences to form 8 second hybrid. Botb hybrid clones produced intact fimbriae. Anti-FlC monoclonal8ntibodies (MAbs) only recognized clones which produced FlC fimbriae, and an ~ti-S 8dhesin MAb marked clones whicb expressed the S adhesin. Bowever, one of four other anti-S fimbri8e-specific MAbs reacted witb both fimbrial structures, S and FlC, indicating 8 common epitope on both antigens. The results presented bere ~upport tbe view th8t sfa and foc determinants code for fimbri8e tb8t 8re simil8r in several aspects, wbile the P fimbri8e are members of 8 more distantly rel8ted group. KW - Infektionsbiologie Y1 - 1988 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59519 ER - TY - JOUR A1 - Parkkinen, J. A1 - Korhonen, T. K. A1 - Pere, A. A1 - Hacker, Jörg A1 - Soinila, S. T1 - Binding sites in the rat brain for Escherichia coli S fimbriae associated with neontal meningitis N2 - Escherichia coli strains that cause sepsis and meningitis in neonatal infants carry S fimbriae that bind to sialyl galactoside units of cell surface glycoproteins. To investigate the possible role of S fimbriae in determining the tissue tropism of neonatal menlngitis, we have studied the preselice of binding sites for S fimbriae in different tissues of the neonatal rat which is susceptible to meningitis caused by S-fimbriated E. coli. Purified S fimbriae were incubated on cryostat sections of different rat oipns and their bindina was assessed by indirect immunofluorescence. In the bnin of the neonatal rat, S fimbriae specifically bound to the luminal surfaces of the vascular endothelium and of the epithelium lining the choroid plexuses and bnin ventricles. The · bindlog W.s completely inhibited by the trisaccharide NeuAca2-3Ga)ßl-4Gic, a receptor analogue of S fimbriae, and by a preceding neuraminidase treatment of the sections. A recombinant E. coli strain expressina S fimbriae adhered in large numbers to the same tissue sites in the neonatal brain sections as did the purified fimbriae, · whereas the nonfimbriated host strahi and a recombiiuuit strain expresslog P fi.mbriae did not adhere to brain tissues. The results soggest that adhesion of S-fimbriated bacteria to the binding sites observed in the neonatai bnin has a pathogenetic roJe durlog bacterial Invasion from cii'culation into the cerebrospinal fluid. KW - Infektionsbiologie Y1 - 1988 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59500 ER - TY - JOUR A1 - Ott, M. A1 - Schmoll, T. A1 - Goebel, W. A1 - Van Die, I. A1 - Hacker, Jörg T1 - Comparison of the genetic determinant coding for the S-fimbrial adhesin (sfa) of Escherichia coli to other chromosomally encoded fimbrial determinants N2 - DNA probes specific for different regions of the S-fimbrial adhesin (sja) determinant were constructed and hybridized with DNA sequences coding for P (F8 and F13), mannose-sensitive hemagglutinating type 1 (FlA), and FlC fimbriae. While the sfa and F1C DNA determinants exhibited homology along their entire lengths, the P-fimbrial and type 1-fimbrial determinants exhibited homology to regions of the sfa duster responsible for the control of transcription and, to a minor extent, to regions coding for proteins involved in biogenesis and/or adhesion of the fimbriae and for the N-terminal part of the fimbrillin subunit. KW - Infektionsbiologie Y1 - 1987 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59499 ER -