TY - JOUR A1 - Wang, Huiqiang A1 - Chen, Nanhai G. A1 - Minev, Boris R. A1 - Szalay, Aladar A. T1 - Oncolytic vaccinia virus GLV-1h68 strain shows enhanced replication in human breast cancer stem-like cells in comparison to breast cancer cells JF - Journal of Translational Medicine N2 - Background: Recent data suggest that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence, novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy. Methods: Cancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance, irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover, we identified and isolated CD44\(^+\)CD24\(^+\)ESA\(^+\) cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models. Results: We demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore, GLV-1h68 also showed preferential replication in CD44\(^+\)CD24\(^+\)ESA\(^+\) cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44\(^+\)CD24\(^-\)ESA\(^+\) cells. Conclusions: Taken together, our findings indicate that GLV-1h68 efficiently replicates and kills cancer stem-like cells. Thus, GLV-1h68 may become a promising agent for eradicating both primary and metastatic tumors, especially tumors harboring cancer stem-like cells that are resistant to chemo and/or radiotherapy and may be responsible for recurrence of tumors. KW - tumors KW - therapy KW - metastasis KW - identification KW - lines KW - gene expression KW - in-vitro propagation KW - acute myeloid leukemia KW - epithelial-mesenchymal transition KW - subpopulation Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130019 VL - 10 IS - 167 ER - TY - JOUR A1 - Gentschev, Ivaylo A1 - Adelfinger, Marion A1 - Josupeit, Rafael A1 - Rudolph, Stephan A1 - Ehrig, Klaas A1 - Donat, Ulrike A1 - Weibel, Stephanie A1 - Chen, Nanhai G. A1 - Yu, Yong A. A1 - Zhang, Qian A1 - Heisig, Martin A1 - Thamm, Douglas A1 - Stritzker, Jochen A1 - MacNeill, Amy A1 - Szalay, Aladar A. T1 - Preclinical Evaluation of Oncolytic Vaccinia Virus for Therapy of Canine Soft Tissue Sarcoma JF - PLoS One N2 - Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS) using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS. KW - breast-tumors KW - animal-model KW - nude-mice KW - cell-line KW - in-vitro KW - glv-1h68 KW - cancer KW - virotherapy KW - dogs KW - neutrophils Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129998 VL - 7 IS - 5 ER - TY - JOUR A1 - Sass, Andrea M. A1 - Van Acker, Heleen A1 - Förstner, Konrad U. A1 - Van Nieuwerburgh, Filip A1 - Deforce, Dieter A1 - Vogel, Jörg A1 - Coenye, Tom T1 - Genome-wide transcription start site profiling in biofilm-grown Burkholderia cenocepacia J2315 JF - BMC Genomics N2 - Background: Burkholderia cenocepacia is a soil-dwelling Gram-negative Betaproteobacterium with an important role as opportunistic pathogen in humans. Infections with B. cenocepacia are very difficult to treat due to their high intrinsic resistance to most antibiotics. Biofilm formation further adds to their antibiotic resistance. B. cenocepacia harbours a large, multi-replicon genome with a high GC-content, the reference genome of strain J2315 includes 7374 annotated genes. This study aims to annotate transcription start sites and identify novel transcripts on a whole genome scale. Methods: RNA extracted from B. cenocepacia J2315 biofilms was analysed by differential RNA-sequencing and the resulting dataset compared to data derived from conventional, global RNA-sequencing. Transcription start sites were annotated and further analysed according to their position relative to annotated genes. Results: Four thousand ten transcription start sites were mapped over the whole B. cenocepacia genome and the primary transcription start site of 2089 genes expressed in B. cenocepacia biofilms were defined. For 64 genes a start codon alternative to the annotated one was proposed. Substantial antisense transcription for 105 genes and two novel protein coding sequences were identified. The distribution of internal transcription start sites can be used to identify genomic islands in B. cenocepacia. A potassium pump strongly induced only under biofilm conditions was found and 15 non-coding small RNAs highly expressed in biofilms were discovered. Conclusions: Mapping transcription start sites across the B. cenocepacia genome added relevant information to the J2315 annotation. Genes and novel regulatory RNAs putatively involved in B. cenocepacia biofilm formation were identified. These findings will help in understanding regulation of B. cenocepacia biofilm formation. KW - persistence KW - genomic islands KW - pathogen KW - identification KW - bacteria KW - small RNAs KW - translation initiation KW - cepedia complex KW - global gene expression KW - SEQ KW - resistance KW - burkholderia cenocepacia KW - biofilms KW - dRNA-Seq KW - transcription start site KW - antisense RNA Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-139748 VL - 16 IS - 775 ER - TY - JOUR A1 - von Bohl, Andreas A1 - Kuehn, Andrea A1 - Simon, Nina A1 - Nkwouano Ngongang, Vanesa A1 - Spehr, Marc A1 - Baumeister, Stefan A1 - Przyborski, Jude M. A1 - Fischer, Rainer A1 - Pradel, Gabriele T1 - A WD40-repeat protein unique to malaria parasites associates with adhesion protein complexes and is crucial for blood stage progeny JF - Malaria Journal N2 - Background During development in human erythrocytes, Plasmodium falciparum parasites display a remarkable number of adhesive proteins on their plasma membrane. In the invasive merozoites, these include members of the PfMSP1 and PfAMA1/RON complexes, which facilitate contact between merozoites and red blood cells. In gametocytes, sexual precursor cells mediating parasite transmission to the mosquito vector, plasma membrane-associated proteins primarily belong to the PfCCp and 6-cys families with roles in fertilization. This study describes a newly identified WD40-repeat protein unique to Plasmodium species that associates with adhesion protein complexes of both merozoites and gametocytes. Methods The WD40-repeat protein-like protein PfWLP1 was identified via co-immunoprecipitation assays followed by mass spectrometry and characterized using biochemical and immunohistochemistry methods. Reverse genetics were employed for functional analysis. Results PfWLP1 is expressed both in schizonts and gametocytes. In mature schizonts, the protein localizes underneath the merozoite micronemes and interacts with PfAMA1, while in gametocytes PfWLP1 primarily accumulates underneath the plasma membrane and associates with PfCCp1 and Pfs230. Reverse genetics failed to disrupt the pfwlp1 gene, while haemagglutinin-tagging was feasible, suggesting a crucial function for PfWLP1 during blood stage replication. Conclusions This is the first report on a plasmodial WD40-repeat protein associating with cell adhesion proteins. Since WD40 domains are known to mediate protein–protein contact by serving as a rigid scaffold for protein interactions, the presented data suggest that PfWLP1 supports the stability of adhesion protein complexes of the plasmodial blood stages. KW - PfCCp protein KW - Pfs230 KW - PfAMA1 KW - WD40 KW - gametocyte KW - microneme KW - merozoite KW - plasmodium falciparum KW - malaria Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-139728 VL - 14 IS - 435 ER - TY - JOUR A1 - Fan, Ben A1 - Li, Lei A1 - Chao, Yanjie A1 - Förstner, Konrad A1 - Vogel, Jörg A1 - Borriss, Rainer A1 - Wu, Xiao-Qin T1 - dRNA-Seq Reveals Genomewide TSSs and Noncoding RNAs of Plant Beneficial Rhizobacterium Bacillus amyloliquefaciens FZB42 JF - PLoS One N2 - Bacillus amyloliquefaciens subsp. plantarum FZB42 is a representative of Gram-positive plant-growth-promoting rhizobacteria (PGPR) that inhabit plant root environments. In order to better understand the molecular mechanisms of bacteria-plant symbiosis, we have systematically analyzed the primary transcriptome of strain FZB42 grown under rhizospheremimicking conditions using differential RNA sequencing (dRNA-seq). Our analysis revealed 4,877 transcription start sites for protein-coding genes, identified genes differentially expressed under different growth conditions, and corrected many previously mis-annotated genes. We also identified a large number of riboswitches and cis-encoded antisense RNAs, as well as trans-encoded small noncoding RNAs that may play important roles in the gene regulation of Bacillus. Overall, our analyses provided a landscape of Bacillus primary transcriptome and improved the knowledge of rhizobacteria-host interactions. KW - gene expression KW - subtilis genome KW - enterica serovar thphimurium KW - small regulatory RNAs KW - binding protein HFQ KW - escherichia coli KW - messenger RNA KW - transcriptional landscape KW - mycobacterium tuberculosis KW - listeria monocytogenes Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-138369 VL - 10 IS - 11 ER - TY - JOUR A1 - Beiss, Veronique A1 - Spiegel, Holger A1 - Boes, Alexander A1 - Scheuermayer, Matthias A1 - Reimann, Andreas A1 - Schillberg, Stefan A1 - Fischer, Rainer T1 - Plant expression and characterization of the transmission-blocking vaccine candidate PfGAP50 JF - BMC Biotechnology N2 - Background: Despite the limited success after decades of intensive research and development efforts, vaccination still represents the most promising strategy to significantly reduce the disease burden in malaria endemic regions. Besides the ultimate goal of inducing sterile protection in vaccinated individuals, the prevention of transmission by so-called transmission blocking vaccines (TBVs) is being regarded as an important feature of an efficient malaria eradication strategy. Recently, Plasmodium falciparum GAP50 (PfGAP50), a 44.6 kDa transmembrane protein that forms an essential part of the invasion machinery (glideosome) multi-protein complex, has been proposed as novel potential transmission-blocking candidate. Plant-based expression systems combine the advantages of eukaryotic expression with a up-scaling potential and a good product safety profile suitable for vaccine production. In this study we investigated the feasibility to use the transient plant expression to produce PfGAP50 suitable for the induction of parasite specific inhibitory antibodies. Results: We performed the transient expression of recombinant PfGAP50 in Nicotiana benthamiana leaves using endoplasmatic reticulum (ER) and plastid targeting. After IMAC-purification the protein yield and integrity was investigated by SDS-PAGE and Western Blot. Rabbit immune IgG derived by the immunization with the plastidtargeted variant of PfGAP50 was analyzed by immune fluorescence assay (IFA) and zygote inhibition assay (ZIA). PfGAP50 could be produced in both subcellular compartments at different yields IMAC (Immobilized Metal Affinity Chromatography) purification from extract yielded up to 4.1 mu g/g recombinant protein per fresh leaf material for ER-retarded and 16.2 mu g/g recombinant protein per fresh leave material for plasmid targeted PfGAP50, respectively. IgG from rabbit sera generated by immunization with the recombinant protein specifically recognized different parasite stages in immunofluorescence assay. Furthermore up to 55 % inhibition in an in vitro zygote inhibition assay could be achieved using PfGAP50-specific rabbit immune IgG. Conclusions: The results of this study demonstrate that the plant-produced PfGAP50 is functional regarding the presentation of inhibitory epitopes and could be considered as component of a transmission-blocking malaria vaccine formulation. KW - PFS25 KW - plastid targeting KW - plant-made vaccines KW - agroinfiltration KW - gametes KW - sexual stage KW - plasmodium falciparum KW - membrane KW - antibodies KW - immunization KW - RTS,S/AS01 malaria vaccine KW - recombinant proteins KW - cost-effectiveness KW - purification Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-137327 VL - 15 IS - 108 ER - TY - JOUR A1 - Abda, Ebrahim M. A1 - Krysciak, Dagmar A1 - Krohn-Molt, Ines A1 - Mamat, Uwe A1 - Schmeisser, Christel A1 - Förstner, Konrad U. A1 - Schaible, Ulrich E. A1 - Kohi, Thomas A. A1 - Nieman, Stefan A1 - Streit, Wolfgang R. T1 - Phenotypic Heterogeneity Affects Stenotrophomonas maltophilia K279a Colony Morphotypes and \(\beta\)-Lactamase Expression JF - Frontiers in Microbiology N2 - Phenotypic heterogeneity at the cellular level in response to various stresses, e.g., antibiotic treatment has been reported for a number of bacteria. In a clonal population, cell-to-cell variation may result in phenotypic heterogeneity that is a mechanism to survive changing environments including antibiotic therapy. Stenotrophomonas rnaltophilia has been frequently isolated from cystic fibrosis patients, can cause numerous infections in other organs and tissues, and is difficult to treat due to antibiotic resistances. S. maltophilia K279a produces the Li and L2 beta-lactamases in response to beta-lactam treatment. Here we report that the patient isolate S. rnaltophilia K279a diverges into cellular subpopulations with distinct but reversible morphotypes of small and big colonies when challenged with ampicillin. This observation is consistent with the formation of elongated chains of bacteria during exponential growth phase and the occurrence of mainly rod-shaped cells in liquid media. RNA-seq analysis of small versus big colonies revealed differential regulation of at least seven genes among the colony morphotypes. Among those, bleu and bla(L2) were transcriptionally the most strongly upregulated genes. Promoter fusions of b/a(L1) and b/a(L2) genes indicated that expression of both genes is also subject to high levels of phenotypic heterogeneous expression on a single cell level. Additionally, the comE homolog was found to be differentially expressed in homogenously versus heterogeneously bla(L2) expressing cells as identified by RNA(seq) analysis. Overexpression of cornE in S. maltophilia K279a reduced the level of cells that were in a bla(L2)-ON mode to 1% or lower. Taken together, our data provide strong evidence that S. maltophilia K279a populations develop phenotypic heterogeneity in an ampicillin challenged model. This cellular variability is triggered by regulation networks including b/a(L1), b/a(L2), and comE. KW - xanthomonas maltophilia KW - gram-negative bacteria KW - RNA-seq KW - pseudomas aeruginosa KW - antibiotic resistance KW - colony morphotypes KW - beta-lactamases KW - K279a KW - Stenotrophomonas maltophilia KW - phenotypic heterogeneity KW - persister cells KW - streptococcus pneumoniae KW - nosocomial pathogen KW - membrane vesicles KW - sinorhizobium fredii NGR234 KW - red fluorescent protein KW - escherichia coli Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-136446 VL - 6 IS - 1373 ER - TY - JOUR A1 - Fröhlich, Kathrin S. A1 - Papenfort, Kai A1 - Berger, Allison A. A1 - Vogel, Jörg T1 - A conserved RpoS-dependent small RNA controls the synthesis of major porin OmpD JF - Nucleic Acids Research N2 - A remarkable feature of many small non-coding RNAs (sRNAs) of Escherichia coli and Salmonella is their accumulation in the stationary phase of bacterial growth. Several stress response regulators and sigma factors have been reported to direct the transcription of stationary phase-specific sRNAs, but a widely conserved sRNA gene that is controlled by the major stationary phase and stress sigma factor, Sigma(S) (RpoS), has remained elusive. We have studied in Salmonella the conserved SdsR sRNA, previously known as RyeB, one of the most abundant stationary phase-specific sRNAs in E. coli. Alignments of the sdsR promoter region and genetic analysis strongly suggest that this sRNA gene is selectively transcribed by Sigma(S). We show that SdsR down-regulates the synthesis of the major Salmonella porin OmpD by Hfq-dependent base pairing; SdsR thus represents the fourth sRNA to regulate this major outer membrane porin. Similar to the InvR, MicC and RybB sRNAs, SdsR recognizes the ompD mRNA in the coding sequence, suggesting that this mRNA may be primarily targeted downstream of the start codon. The SdsR-binding site in ompD was localized by 3'-RACE, an experimental approach that promises to be of use in predicting other sRNA-target interactions in bacteria. KW - shock sigma factor KW - general stress response KW - down regulation KW - stationary phase KW - salmonella enterica KW - messenger RNA KW - escherichia coli KW - enterica serovar typhimurium KW - outer-membrane proteins KW - small noncoding RNAs Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134230 VL - 40 IS - 8 ER - TY - JOUR A1 - Makoah Nigel, Animake A1 - Arndt, Hans-Dieter A1 - Pradel, Gabriele T1 - The proteasome of malaria parasites: A multi-stage drug target for chemotherapeutic intervention? JF - International Journal for Parasitology: Drugs and Drug Resistance N2 - The ubiquitin/proteasome system serves as a regulated protein degradation pathway in eukaryotes, and is involved in many cellular processes featuring high protein turnover rates, such as cell cycle control, stress response and signal transduction. In malaria parasites, protein quality control is potentially important because of the high replication rate and the rapid transformations of the parasite during life cycle progression. The proteasome is the core of the degradation pathway, and is a major proteolytic complex responsible for the degradation and recycling of non-functional ubiquitinated proteins. Annotation of the genome for Plasmodium falciparum, the causative agent of malaria tropica, revealed proteins with similarity to human 26S proteasome subunits. In addition, a bacterial ClpQ/hslV threonine peptidase-like protein was identified. In recent years several independent studies indicated an essential function of the parasite proteasome for the liver, blood and transmission stages. In this review, we compile evidence for protein recycling in Plasmodium parasites and discuss the role of the 26S proteasome as a prospective multi-stage target for antimalarial drug discovery programs. KW - plasmodium falciparum KW - proteasome KW - ubiquitin KW - inhibitor Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-137777 VL - 2 ER - TY - THES A1 - Frank, Benjamin T1 - Untersuchungen zur Autophagieinduktion in Leishmania major-infizierten Knochenmarksmakrophagen T1 - Analyses of autophagy induction in Leishmania major-infected bone marrow-derived macrophages N2 - Die von der WHO zu den 17 wichtigsten NTDs gezählte Leishmaniose wird durch intrazelluläre Parasiten der Gattung Leishmania hervorgerufen. Der Lebenszyklus der Parasiten besteht aus zwei Phasen. Die länglichen und beweglichen Promastigoten kennzeichnen die Phase in der Sandmücke – der Vektor der Leishmaniose. Hingegen ist die Phase im Säugerwirt durch runde unbewegliche Amastigoten charakterisiert. Aufgrund des Mangels an potenten antileishmanialen Therapien wurde in der vorliegenden Arbeit die Interaktion zwischen L. m. Parasiten und der Hauptwirtszelle, der Makrophage, v. a. in Hinblick auf autophage Prozesse in den infizierten Makrophagen näher untersucht, um demgemäß neue Erkenntnisse zu gewinnen, welche bei der Herstellung zukünftiger anti-leishmanialer Medikamente helfen könnten. Bei der Autophagie handelt es sich um einen katabolen Prozess, wodurch Zellen bei Nahrungsmangel oder zellulärem Stress ihre Homöostase erhalten können. Durch diesen Prozess können überflüssige oder beschädigte Organellen recycelt werden, um die Funktionen der Zelle aufrechtzuerhalten. Daneben übernimmt Autophagie auch eine essenzielle Rolle bei der Abwehr von ins Zytosol eindringenden Pathogenen. Mittels des neu etablierten totalen Autophagiescore konnte festgestellt werden, dass Autophagie in L. m.-infizierten BMDM induziert wird. Die intrazellulären Amastigoten werden durch Autophagie in den BMDM verdaut. Die erhöhte autophage Aktivität konnte zudem durch Western-Blot-Analysen der autophagierelevanten Proteine ATG5, LC3B und UB bestätigt werden. Die molekulargenetischen Untersuchungen von L. m.-infizier-ten BMDM mithilfe von Affymetrix Microarrays führten zu einem Netzwerk aus autophagierelevanten und infektionsspezifischen Genen, welches als LISA bezeichnet worden ist. Hier hat sich ebenfalls eine starke Verknüpfung von autophagierelevanten Genen und den Genen der Glykolyse, einem zweiten katabolen Prozess, gezeigt. Zudem konnten zwei weitere autophagierelevante und infektionsspezifische Gene außerhalb von LISA identifiziert werden, nämlich Bnip3 und Ctse, welche im Anschluss genauer untersucht worden sind. Bei beiden Genen konnte auf Proteinebene gezeigt werden, dass sie in L. m.-infizierten BMDM signifikant erhöht sind. Durch siRNA-Analysen konnte überdies beobachtet werden, dass beide für die erfolgreiche Elimination der Amastigoten essenziell sind. Somit konnte mit den Proteinen BNIP3 und CTSE zwei potenzielle neue Ansatzpunkte für mögliche zukünftige antileishmaniale Therapien gefunden werden. Auch die in LISA enthaltenen Gene stellen prinzipiell vielversprechende Ziele für künftige Medikamente gegen Leishmaniose dar. Durch all diese Untersuchungen kommt man dem Ziel einer neuen, gezielten und nebenwirkungsärmeren Behandlung der Leishmaniose einen Schritt näher. N2 - Leishmaniasis, listed by the WHO to be one of the 17 most important NTDs, is caused by intracellular parasites of the genus Leishmania. The life cycle of the parasites consists of two stages. The oblong and motile promastigotes characterize the stage in the sand fly, the vector of leishmaniasis. However, the stage in the vertebrate host is characterized by round immotile amastigotes. Due to a lack of capable antileishmanial therapies, the interaction between L. m. parasites and their main host cell, the macrophage, was investigated in the present work, huge focus on autophagic processes in infected macrophages. Our goal was to get new insights for the future production of antileishmanial drugs. Autophagy is a catabolic process whereby cells are able to maintain their homeostasis in times of starvation or cellular stress. During to this process, redundant or damaged organelles are recycled in order to sustain cellular viability. Furthermore, autophagy has an essential role in the defense of pathogens invading the cytosol. The newly established total autophagy score showed an autophagy induction in L. m.-infected BMDM. Intracellular amastigotes are digested by autophagy in BMDM. The increased autophagic activity could also be confirmed by western-blot analyses of the autophagy-relevant proteins ATG5, LC3B, and UB. Molecular genetic investigations of L. m.-infected BMDM by Affymetrix microarrays led to a network of autophagy-relevant and infection-specific genes, which was called LISA. Additionally, it showed a strong connection between autophagy-relevant genes and genes of the glycolysis, a second catabolic process. Moreover, we identified and further characterized two additional autophagy-relevant genes, Bnip3 and Ctse, which were not included in LISA. Both genes were significantly overexpressed on protein level in L. m.-infected BMDM. By siRNA analyses we also demonstrated their importance for successful elimination of amastigotes. Therefore, both proteins, BNIP3 and CTSE, could be new potential targets for possible future antileishmanial therapies. In addition, the genes included in LISA might be promising targets for future drugs against leishmaniasis. Due to all these investigations we are one step closer to our goal of a targeted and safe therapy of leishmaniasis. KW - Autophagie KW - Leishmania major KW - Cathepsin E KW - BNIP3 KW - Elektronenmikroskopie KW - Autophagie KW - Leishmania major KW - Cathepsin E KW - BNIP3 KW - Elektronenmikroskopie KW - Microarray KW - siRNA KW - Autophagiescore Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-137277 ER - TY - JOUR A1 - Lioliou, Efthimia A1 - Sharma, Cynthia M. A1 - Caldelari, Isabelle A1 - Helfer, Anne-Catherine A1 - Fechter, Pierre A1 - Vandenesch, François A1 - Vogel, Jörg A1 - Romby, Pascale T1 - Global Regulatory Functions of the Staphylococcus aureus Endoribonuclease III in Gene Expression JF - PLoS Genetics N2 - RNA turnover plays an important role in both virulence and adaptation to stress in the Gram-positive human pathogen Staphylococcus aureus. However, the molecular players and mechanisms involved in these processes are poorly understood. Here, we explored the functions of S. aureus endoribonuclease III (RNase III), a member of the ubiquitous family of double-strand-specific endoribonucleases. To define genomic transcripts that are bound and processed by RNase III, we performed deep sequencing on cDNA libraries generated from RNAs that were co-immunoprecipitated with wild-type RNase III or two different cleavage-defective mutant variants in vivo. Several newly identified RNase III targets were validated by independent experimental methods. We identified various classes of structured RNAs as RNase III substrates and demonstrated that this enzyme is involved in the maturation of rRNAs and tRNAs, regulates the turnover of mRNAs and non-coding RNAs, and autoregulates its synthesis by cleaving within the coding region of its own mRNA. Moreover, we identified a positive effect of RNase III on protein synthesis based on novel mechanisms. RNase III–mediated cleavage in the 5′ untranslated region (5′UTR) enhanced the stability and translation of cspA mRNA, which encodes the major cold-shock protein. Furthermore, RNase III cleaved overlapping 5′UTRs of divergently transcribed genes to generate leaderless mRNAs, which constitutes a novel way to co-regulate neighboring genes. In agreement with recent findings, low abundance antisense RNAs covering 44% of the annotated genes were captured by co-immunoprecipitation with RNase III mutant proteins. Thus, in addition to gene regulation, RNase III is associated with RNA quality control of pervasive transcription. Overall, this study illustrates the complexity of post-transcriptional regulation mediated by RNase III. KW - staphylococcus aureus KW - ribonucleases KW - messenger RNA KW - RNA sequencing KW - antisense RNA KW - RNA structure KW - RNA synthesis KW - RNA denaturation Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127219 VL - 8 IS - 6 ER - TY - JOUR A1 - Wilms, Ina A1 - Overlöper, Aaron A1 - Nowrousian, Minou A1 - Sharma, Cynthia M. A1 - Narberhaus, Franz T1 - Deep sequencing uncovers numerous small RNAs on all four replicons of the plant pathogen Agrobacterium tumefaciens JF - RNA Biology N2 - Agrobacterium species are capable of interkingdom gene transfer between bacteria and plants. The genome of Agrobacterium tumefaciens consists of a circular and a linear chromosome, the At-plasmid and the Ti-plasmid, which harbors bacterial virulence genes required for tumor formation in plants. Little is known about promoter sequences and the small RNA (sRNA) repertoire of this and other α-proteobacteria. We used a differential RNA sequencing (dRNA-seq) approach to map transcriptional start sites of 388 annotated genes and operons. In addition, a total number of 228 sRNAs was revealed from all four Agrobacterium replicons. Twenty-two of these were confirmed by independent RNA gel blot analysis and several sRNAs were differentially expressed in response to growth media, growth phase, temperature or pH. One sRNA from the Ti-plasmid was massively induced under virulence conditions. The presence of 76 cis-antisense sRNAs, two of them on the reverse strand of virulence genes, suggests considerable antisense transcription in Agrobacterium. The information gained from this study provides a valuable reservoir for an in-depth understanding of sRNA-mediated regulation of the complex physiology and infection process of Agrobacterium. KW - regulatory RNA KW - plant-microbe interaction KW - deep sequencing KW - RNA-seq KW - small RNA Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127101 VL - 9 IS - 446-457 ER - TY - JOUR A1 - Röhrich, Christian Rene A1 - Ngwa, Che Julius A1 - Wiesner, Jochen A1 - Schmidtberg, Henrike A1 - Degenkolb, Thomas A1 - Kollewe, Christian A1 - Fischer, Rainer A1 - Pradel, Gabriele A1 - Vilcinskas, Andreas T1 - Harmonine, a defence compound from the harlequin ladybird, inhibits mycobacterial growth and demonstrates multi-stage antimalarial activity JF - Biology Letters N2 - The harlequin ladybird beetle Harmonia axyridis has been introduced in many countries as a biological control agent, but has become an invasive species threatening the biodiversity of native ladybirds. Its invasive success has been attributed to its vigorous resistance against diverse pathogens. This study demonstrates that harmonine ((17R,9Z)-1,17-diaminooctadec-9-ene), which is present in H. axyridis haemolymph, displays broad-spectrum antimicrobial activity that includes human pathogens. Antibacterial activity is most pronounced against fast-growing mycobacteria and Mycobacterium tuberculosis, and the growth of both chloroquine-sensitive and -resistant Plasmodium falciparum strains is inhibited. Harmonine displays gametocytocidal activity, and inhibits the exflagellation of microgametocytes and zygote formation. In an Anopheles stephensi mosquito feeding model, harmonine displays transmission-blocking activity. KW - insect immunity KW - antimicrobial activity KW - harmonine KW - harmonia axyridis Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127079 VL - 8 ER - TY - JOUR A1 - Bandyra, Katarzyna J. A1 - Said, Nelly A1 - Pfeiffer, Verena A1 - Górna, Maria W. A1 - Vogel, Jörg A1 - Luisi, Ben F. T1 - The Seed Region of a Small RNA Drives the Controlled Destruction of the Target mRNA by the Endoribonuclease RNase E JF - Molecular Cell N2 - Numerous small non-coding RNAs (sRNAs) in bacteria modulate rates of translation initiation and degradation of target mRNAs, which they recognize through base-pairing facilitated by the RNA chaperone Hfq. Recent evidence indicates that the ternary complex of Hfq, sRNA and mRNA guides endoribonuclease RNase E to initiate turnover of both the RNAs. We show that a sRNA not only guides RNase E to a defined site in a target RNA, but also allosterically activates the enzyme by presenting a monophosphate group at the 5′-end of the cognate-pairing “seed.” Moreover, in the absence of the target the 5′-monophosphate makes the sRNA seed region vulnerable to an attack by RNase E against which Hfq confers no protection. These results suggest that the chemical signature and pairing status of the sRNA seed region may help to both ‘proofread’ recognition and activate mRNA cleavage, as part of a dynamic process involving cooperation of RNA, Hfq and RNase E. KW - medicine Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126202 VL - 47 IS - 6 ER - TY - JOUR A1 - Schwarz, Tobias A1 - Remer, Katharina A. A1 - Nahrendorf, Wiebke A1 - Masic, Anita A1 - Siewe, Lisa A1 - Müller, Werner A1 - Roers, Axel A1 - Moll, Heidrun T1 - T Cell-Derived IL-10 Determines Leishmaniasis Disease Outcome and Is Suppressed by a Dendritic Cell Based Vaccine JF - PLoS Pathogens N2 - Abstract In the murine model of Leishmania major infection, resistance or susceptibility to the parasite has been associated with the development of a Th1 or Th2 type of immune response. Recently, however, the immunosuppressive effects of IL-10 have been ascribed a crucial role in the development of the different clinical correlates of Leishmania infection in humans. Since T cells and professional APC are important cellular sources of IL-10, we compared leishmaniasis disease progression in T cell-specific, macrophage/neutrophil-specific and complete IL-10-deficient C57BL/6 as well as T cell-specific and complete IL-10-deficient BALB/c mice. As early as two weeks after infection of these mice with L. major, T cell-specific and complete IL-10-deficient animals showed significantly increased lesion development accompanied by a markedly elevated secretion of IFN-γ or IFN-γ and IL-4 in the lymph nodes draining the lesions of the C57BL/6 or BALB/c mutants, respectively. In contrast, macrophage/neutrophil-specific IL-10-deficient C57BL/6 mice did not show any altered phenotype. During the further course of disease, the T cell-specific as well as the complete IL-10-deficient BALB/c mice were able to control the infection. Furthermore, a dendritic cell-based vaccination against leishmaniasis efficiently suppresses the early secretion of IL-10, thus contributing to the control of parasite spread. Taken together, IL-10 secretion by T cells has an influence on immune activation early after infection and is sufficient to render BALB/c mice susceptible to an uncontrolled Leishmania major infection. Author Summary The clinical symptoms caused by infections with Leishmania parasites range from self-healing cutaneous to uncontrolled visceral disease and depend not only on the parasite species but also on the type of the host's immune response. It is estimated that 350 million people worldwide are at risk, with a global incidence of 1–1.5 million cases of cutaneous and 500,000 cases of visceral leishmaniasis. Murine leishmaniasis is the best-characterized model to elucidate the mechanisms underlying resistance or susceptibility to Leishmania major parasites in vivo. Using T cell-specific and macrophage-specific mutant mice, we demonstrate that abrogating the secretion of the immunosuppressive cytokine IL-10 by T cells is sufficient to render otherwise susceptible mice resistant to an infection with the pathogen. The healing phenotype is accompanied by an elevated specific inflammatory immune response very early after infection. We further show that dendritic cell-based vaccination against leishmaniasis suppresses the early secretion of IL-10 following challenge infection. Thus, our study unravels a molecular mechanism critical for host immune defense, aiding in the development of an effective vaccine against leishmaniasis. KW - cytokines KW - mouse models KW - T cells KW - lymph nodes KW - leishmania major KW - secretion KW - parasitic diseases KW - immune response Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130385 VL - 9 IS - 6 ER - TY - JOUR A1 - Amich, Jorge A1 - Schafferer, Lukas A1 - Haas, Hubertus A1 - Krappmann, Sven T1 - Regulation of Sulphur Assimilation Is Essential for Virulence and Affects Iron Homeostasis of the Human-Pathogenic Mould Aspergillus fumigatus JF - PLoS Pathogens N2 - Abstract Sulphur is an essential element that all pathogens have to absorb from their surroundings in order to grow inside their infected host. Despite its importance, the relevance of sulphur assimilation in fungal virulence is largely unexplored. Here we report a role of the bZIP transcription factor MetR in sulphur assimilation and virulence of the human pathogen Aspergillus fumigatus. The MetR regulator is essential for growth on a variety of sulphur sources; remarkably, it is fundamental for assimilation of inorganic S-sources but dispensable for utilization of methionine. Accordingly, it strongly supports expression of genes directly related to inorganic sulphur assimilation but not of genes connected to methionine metabolism. On a broader scale, MetR orchestrates the comprehensive transcriptional adaptation to sulphur-starving conditions as demonstrated by digital gene expression analysis. Surprisingly, A. fumigatus is able to utilize volatile sulphur compounds produced by its methionine catabolism, a process that has not been described before and that is MetR-dependent. The A. fumigatus MetR transcriptional activator is important for virulence in both leukopenic mice and an alternative mini-host model of aspergillosis, as it was essential for the development of pulmonary aspergillosis and supported the systemic dissemination of the fungus. MetR action under sulphur-starving conditions is further required for proper iron regulation, which links regulation of sulphur metabolism to iron homeostasis and demonstrates an unprecedented regulatory crosstalk. Taken together, this study provides evidence that regulation of sulphur assimilation is not only crucial for A. fumigatus virulence but also affects the balance of iron in this prime opportunistic pathogen. Author Summary Invasive pulmonary aspergillosis (IPA) is a life-threatening disease that affects primarily immunosuppressed patients. During the last decades the incidence of this disease that is accompanied by high mortality rates has increased. Since opportunistic pathogenic fungi, unlike other pathogens, do not express specific virulence factors, it is becoming more and more clear that the elucidation of fungal metabolism is an essential task to understand fungal pathogenicity and to identify novel antifungal targets. In this work we report genetic inactivation of the sulphur transcription regulator MetR in Aspergillus fumigatus and subsequent study of the resulting phenotypes and transcriptional deregulation of the mutant. Here we show that regulation of sulphur assimilation is an essential process for the manifestation of IPA. Moreover, a regulatory connection between sulphur metabolism and iron homeostasis, a further essential virulence determinant of A. fumigatus, is demonstrated in this study for the first time. A deeper knowledge of sulphur metabolism holds the promise of increasing our understanding of fungal virulence and might lead to improved antifungal therapy. KW - gene regulation KW - transcription factors KW - DNA transcription KW - aspergillus fumigatus KW - methionine KW - sulfur KW - fungal pathogens KW - sulfates Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130372 VL - 9 IS - 8 ER - TY - JOUR A1 - Schoenfelder, Sonja M. K. A1 - Marincola, Gabriella A1 - Geiger, Tobias A1 - Goerke, Christiane A1 - Wolz, Christiane A1 - Ziebuhr, Wilma T1 - Methionine Biosynthesis in Staphylococcus aureus Is Tightly Controlled by a Hierarchical Network Involving an Initiator tRNA-Specific T-box Riboswitch JF - PLoS Pathogens N2 - Abstract In line with the key role of methionine in protein biosynthesis initiation and many cellular processes most microorganisms have evolved mechanisms to synthesize methionine de novo. Here we demonstrate that, in the bacterial pathogen Staphylococcus aureus, a rare combination of stringent response-controlled CodY activity, T-box riboswitch and mRNA decay mechanisms regulate the synthesis and stability of methionine biosynthesis metICFE-mdh mRNA. In contrast to other Bacillales which employ S-box riboswitches to control methionine biosynthesis, the S. aureus metICFE-mdh mRNA is preceded by a 5′-untranslated met leader RNA harboring a T-box riboswitch. Interestingly, this T-box riboswitch is revealed to specifically interact with uncharged initiator formylmethionyl-tRNA \((tRNA_i^{fMet})\)while binding of elongator \(tRNA^{Met}\) proved to be weak, suggesting a putative additional function of the system in translation initiation control. met leader RNA/metICFE-mdh operon expression is under the control of the repressor CodY which binds upstream of the met leader RNA promoter. As part of the metabolic emergency circuit of the stringent response, methionine depletion activates RelA-dependent (p)ppGpp alarmone synthesis, releasing CodY from its binding site and thereby activating the met leader promoter. Our data further suggest that subsequent steps in metICFE-mdh transcription are tightly controlled by the 5′ met leader-associated T-box riboswitch which mediates premature transcription termination when methionine is present. If methionine supply is limited, and hence \((tRNA_i^{fMet})\) becomes uncharged, full-length met leader/metICFE-mdh mRNA is transcribed which is rapidly degraded by nucleases involving RNase J2. Together, the data demonstrate that staphylococci have evolved special mechanisms to prevent the accumulation of excess methionine. We hypothesize that this strict control might reflect the limited metabolic capacities of staphylococci to reuse methionine as, other than Bacillus, staphylococci lack both the methionine salvage and polyamine synthesis pathways. Thus, methionine metabolism might represent a metabolic Achilles' heel making the pathway an interesting target for future anti-staphylococcal drug development. Author Summary Prokaryote metabolism is key for our understanding of bacterial virulence and pathogenesis and it is also an area with huge opportunity to identify novel targets for antibiotic drugs. Here, we have addressed the so far poorly characterized regulation of methionine biosynthesis in S. aureus. We demonstrate that methionine biosynthesis control in staphylococci significantly differs from that predicted for other Bacillales. Notably, involvement of a T-box instead of an S-box riboswitch separates staphylococci from other bacteria in the order. We provide, for the first time, direct experimental proof for an interaction of a methionyl-tRNA-specific T-box with its cognate tRNA, and the identification of initiator \((tRNA_i^{fMet})\) as the specific binding partner is an unexpected finding whose exact function in Staphylococcus metabolism remains to be established. The data further suggest that in staphylococci a range of regulatory elements are integrated to form a hierarchical network that elegantly limits costly (excess) methionine biosynthesis and, at the same time, reliably ensures production of the amino acid in a highly selective manner. Our findings open a perspective to exploit methionine biosynthesis and especially its T-box-mediated control as putative target(s) for the development of future anti-staphylococcal therapeutics. KW - ribonucleases KW - transfer RNA KW - staphylococcus aureus KW - staphylococcus KW - biosynthesis KW - methionine KW - RNA synthesis KW - DNA transcription Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130365 VL - 9 IS - 9 ER - TY - JOUR A1 - Weibel, Stephanie A1 - Basse-Luesebrink, Thomas Christian A1 - Hess, Michael A1 - Hofmann, Elisabeth A1 - Seubert, Carolin A1 - Langbein-Laugwitz, Johanna A1 - Gentschev, Ivaylo A1 - Sturm, Volker Jörg Friedrich A1 - Ye, Yuxiang A1 - Kampf, Thomas A1 - Jakob, Peter Michael A1 - Szalay, Aladar A. T1 - Imaging of Intratumoral Inflammation during Oncolytic Virotherapy of Tumors by \(^{19}\)F-Magnetic Resonance Imaging (MRI) JF - PLoS ONE N2 - Background Oncolytic virotherapy of tumors is an up-coming, promising therapeutic modality of cancer therapy. Unfortunately, non-invasive techniques to evaluate the inflammatory host response to treatment are rare. Here, we evaluate \(^{19}\)F magnetic resonance imaging (MRI) which enables the non-invasive visualization of inflammatory processes in pathological conditions by the use of perfluorocarbon nanoemulsions (PFC) for monitoring of oncolytic virotherapy. Methodology/Principal Findings The Vaccinia virus strain GLV-1h68 was used as an oncolytic agent for the treatment of different tumor models. Systemic application of PFC emulsions followed by \(^1H\)/\(^{19}\)F MRI of mock-infected and GLV-1h68-infected tumor-bearing mice revealed a significant accumulation of the \(^{19}\)F signal in the tumor rim of virus-treated mice. Histological examination of tumors confirmed a similar spatial distribution of the \(^{19}\)F signal hot spots and \(CD68^+\)-macrophages. Thereby, the \(CD68^+\)-macrophages encapsulate the GFP-positive viral infection foci. In multiple tumor models, we specifically visualized early inflammatory cell recruitment in Vaccinia virus colonized tumors. Furthermore, we documented that the \(^{19}\)F signal correlated with the extent of viral spreading within tumors. Conclusions/Significance These results suggest \(^{19}\)F MRI as a non-invasive methodology to document the tumor-associated host immune response as well as the extent of intratumoral viral replication. Thus, \(^{19}\)F MRI represents a new platform to non-invasively investigate the role of the host immune response for therapeutic outcome of oncolytic virotherapy and individual patient response. KW - inflammation KW - fluorescence microscopy KW - oncolytic viruses KW - fluorescence imaging KW - macrophages KW - magnetic resonance imaging KW - histology KW - in vivo imaging Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130311 VL - 8 IS - 3 ER - TY - JOUR A1 - Hertlein, Tobias A1 - Sturm, Volker A1 - Jakob, Peter A1 - Ohlsen, Knut T1 - \(^{19}\)F Magnetic Resonance Imaging of Perfluorocarbons for the Evaluation of Response to Antibiotic Therapy in a Staphylococcus aureus Infection Model JF - PLoS ONE N2 - Background The emergence of antibiotic resistant bacteria in recent decades has highlighted the importance of developing new drugs to treat infections. However, in addition to the design of new drugs, the development of accurate preclinical testing methods is essential. In vivo imaging technologies such as bioluminescence imaging (BLI) or magnetic resonance imaging (MRI) are promising approaches. In a previous study, we showed the effectiveness of \(^{19}\)F MRI using perfluorocarbon (PFC) emulsions for detecting the site of Staphylococcus aureus infection. In the present follow-up study, we investigated the use of this method for in vivo visualization of the effects of antibiotic therapy. Methods/Principal findings Mice were infected with S. aureus Xen29 and treated with 0.9% NaCl solution, vancomycin or linezolid. Mock treatment led to the highest bioluminescence values during infection followed by vancomycin treatment. Counting the number of colony-forming units (cfu) at 7 days post-infection (p.i.) showed the highest bacterial burden for the mock group and the lowest for the linezolid group. Administration of PFCs at day 2 p.i. led to the accumulation of \(^{19}\)F at the rim of the abscess in all mice (in the shape of a hollow sphere), and antibiotic treatment decreased the \(^{19}\)F signal intensity and volume. Linezolid showed the strongest effect. The BLI, cfu, and MRI results were comparable. Conclusions \(^{19}\)F-MRI with PFCs is an effective non-invasive method for assessing the effects of antibiotic therapy in vivo. This method does not depend on pathogen specific markers and can therefore be used to estimate the efficacy of antibacterial therapy against a broad range of clinically relevant pathogens, and to localize sites of infection. KW - staphylococcus aureus KW - abscesses KW - vancomycin KW - antibiotics KW - magnetic resonance imaging KW - emulsions KW - bioluminescence imaging KW - in vivo imaging Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130113 VL - 8 IS - 5 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 JF - Nucleic Acids Research 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 - Molekulare Infektionsbiologie Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129765 VL - 41 IS - 1 ER - TY - JOUR A1 - Mielich-Süss, Benjamin A1 - Schneider, Johannes A1 - Lopez, Daniel T1 - Overproduction of Flotillin Influences Cell Differentiation and Shape in Bacillus subtilis JF - mBio N2 - ABSTRACT Bacteria organize many membrane-related signaling processes in functional microdomains that are structurally and functionally similar to the lipid rafts of eukaryotic cells. An important structural component of these microdomains is the protein flotillin, which seems to act as a chaperone in recruiting other proteins to lipid rafts to facilitate their interaction. In eukaryotic cells, the occurrence of severe diseases is often observed in combination with an overproduction of flotillin, but a functional link between these two phenomena is yet to be demonstrated. In this work, we used the bacterial model Bacillus subtilis as a tractable system to study the physiological alterations that occur in cells that overproduce flotillin. We discovered that an excess of flotillin altered specific signal transduction pathways that are associated with the membrane microdomains of bacteria. As a consequence of this, we detected significant defects in cell division and cell differentiation. These physiological alterations were in part caused by an unusual stabilization of the raft-associated protease FtsH. This report opens the possibility of using bacteria as a working model to better understand fundamental questions related to the functionality of lipid rafts. IMPORTANCE The identification of signaling platforms in the membrane of bacteria that are functionally and structurally equivalent to eukaryotic lipid rafts reveals a level of sophistication in signal transduction and membrane organization unexpected in bacteria. It opens new and promising venues to address intricate questions related to the functionality of lipid rafts by using bacteria as a more tractable system. This is the first report that uses bacteria as a working model to investigate a fundamental question that was previously raised while studying the role of eukaryotic lipid rafts. It also provides evidence of the critical role of these signaling platforms in orchestrating diverse physiological processes in prokaryotic cells. KW - bacillus subtilis Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129653 VL - 4 IS - 6 ER - TY - JOUR A1 - Ehrig, Klaas A1 - Kilinc, Mehmet O. A1 - Chen, Nanhai G. A1 - Stritzker, Jochen A1 - Buckel, Lisa A1 - Zhang, Qian A1 - Szalay, Aladar A. T1 - Growth inhibition of different human colorectal cancer xenografts after a single intravenous injection of oncolytic vaccinia virus GLV-1h68 JF - Journal of Translational Medicine N2 - Background: Despite availability of efficient treatment regimens for early stage colorectal cancer, treatment regimens for late stage colorectal cancer are generally not effective and thus need improvement. Oncolytic virotherapy using replication-competent vaccinia virus (VACV) strains is a promising new strategy for therapy of a variety of human cancers. Methods: Oncolytic efficacy of replication-competent vaccinia virus GLV-1h68 was analyzed in both, cell cultures and subcutaneous xenograft tumor models. Results: In this study we demonstrated for the first time that the replication-competent recombinant VACV GLV-1h68 efficiently infected, replicated in, and subsequently lysed various human colorectal cancer lines (Colo 205, HCT-15, HCT-116, HT-29, and SW-620) derived from patients at all four stages of disease. Additionally, in tumor xenograft models in athymic nude mice, a single injection of intravenously administered GLV-1h68 significantly inhibited tumor growth of two different human colorectal cell line tumors (Duke’s type A-stage HCT-116 and Duke’s type C-stage SW-620), significantly improving survival compared to untreated mice. Expression of the viral marker gene ruc-gfp allowed for real-time analysis of the virus infection in cell cultures and in mice. GLV-1h68 treatment was well-tolerated in all animals and viral replication was confined to the tumor. GLV-1h68 treatment elicited a significant up-regulation of murine immune-related antigens like IFN-γ, IP-10, MCP-1, MCP-3, MCP-5, RANTES and TNF-γ and a greater infiltration of macrophages and NK cells in tumors as compared to untreated controls. Conclusion: The anti-tumor activity observed against colorectal cancer cells in these studies was a result of direct viral oncolysis by GLV-1h68 and inflammation-mediated innate immune responses. The therapeutic effects occurred in tumors regardless of the stage of disease from which the cells were derived. Thus, the recombinant vaccinia virus GLV-1h68 has the potential to treat colorectal cancers independently of the stage of progression. KW - oncolytic virotherapy KW - colorectal KW - vaccinia virus KW - cancer KW - metastasis Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129619 VL - 11 IS - 79 ER - TY - JOUR A1 - Becker, Svetlana A1 - Oelschlaeger, Tobias A. A1 - Wullaert, Andy A1 - Pasparakis, Manolis A1 - Wehkamp, Jan A1 - Stange, Eduard F. A1 - Gersemann, Michael T1 - Bacteria Regulate Intestinal Epithelial Cell Differentiation Factors Both In Vitro and In Vivo JF - PLoS ONE N2 - Background: The human colon harbours a plethora of bacteria known to broadly impact on mucosal metabolism and function and thought to be involved in inflammatory bowel disease pathogenesis and colon cancer development. In this report, we investigated the effect of colonic bacteria on epithelial cell differentiation factors in vitro and in vivo. As key transcription factors we focused on Hes1, known to direct towards an absorptive cell fate, Hath1 and KLF4, which govern goblet cell. Methods: Expression of the transcription factors Hes1, Hath1 and KLF4, the mucins Muc1 and Muc2 and the defensin HBD2 were measured by real-time PCR in LS174T cells following incubation with several heat-inactivated E. coli strains, including the probiotic E. coli Nissle 1917+/- flagellin, Lactobacilli and Bifidobacteria. For protein detection Western blot experiments and chamber-slide immunostaining were performed. Finally, mRNA and protein expression of these factors was evaluated in the colon of germfree vs. specific pathogen free vs. conventionalized mice and colonic goblet cells were counted. Results: Expression of Hes1 and Hath1, and to a minor degree also of KLF4, was reduced by E. coli K-12 and E. coli Nissle 1917. In contrast, Muc1 and HBD2 expression were significantly enhanced, independent of the Notch signalling pathway. Probiotic E. coli Nissle 1917 regulated Hes1, Hath1, Muc1 and HBD2 through flagellin. In vivo experiments confirmed the observed in vitro effects of bacteria by a diminished colonic expression of Hath1 and KLF4 in specific pathogen free and conventionalized mice as compared to germ free mice whereas the number of goblet cells was unchanged in these mice. Conclusions: Intestinal bacteria influence the intestinal epithelial differentiation factors Hes1, Hath1 and KLF4, as well as Muc1 and HBD2, in vitro and in vivo. The induction of Muc1 and HBD2 seems to be triggered directly by bacteria and not by Notch. KW - stem cells KW - inflammatory-bowel-disease KW - Ileal Crohns-disease KW - coli nissel 1917 KW - ulcreative colitis KW - escherichia coli KW - porphyromonas gingivalis KW - antimicrobial peptides KW - colorectal cancer KW - alpha defensins Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131168 VL - 8 IS - 2 ER - TY - JOUR A1 - Palige, Katja A1 - Linde, Jörg A1 - Martin, Ronny A1 - Böttcher, Bettina A1 - Citiulo, Francesco A1 - Sullivan, Derek J. A1 - Weber, Johann A1 - Staib, Claudia A1 - Rupp, Steffen A1 - Hube, Bernhard A1 - Morschhäuser, Joachim A1 - Staib, Peter T1 - Global Transcriptome Sequencing Identifies Chlamydospore Specific Markers in Candida albicans and Candida dubliniensis JF - PLoS ONE N2 - Candida albicans and Candida dubliniensis are pathogenic fungi that are highly related but differ in virulence and in some phenotypic traits. During in vitro growth on certain nutrient-poor media, C. albicans and C. dubliniensis are the only yeast species which are able to produce chlamydospores, large thick-walled cells of unknown function. Interestingly, only C. dubliniensis forms pseudohyphae with abundant chlamydospores when grown on Staib medium, while C. albicans grows exclusively as a budding yeast. In order to further our understanding of chlamydospore development and assembly, we compared the global transcriptional profile of both species during growth in liquid Staib medium by RNA sequencing. We also included a C. albicans mutant in our study which lacks the morphogenetic transcriptional repressor Nrg1. This strain, which is characterized by its constitutive pseudohyphal growth, specifically produces masses of chlamydospores in Staib medium, similar to C. dubliniensis. This comparative approach identified a set of putatively chlamydospore-related genes. Two of the homologous C. albicans and C. dubliniensis genes (CSP1 and CSP2) which were most strongly upregulated during chlamydospore development were analysed in more detail. By use of the green fluorescent protein as a reporter, the encoded putative cell wall related proteins were found to exclusively localize to C. albicans and C. dubliniensis chlamydospores. Our findings uncover the first chlamydospore specific markers in Candida species and provide novel insights in the complex morphogenetic development of these important fungal pathogens. KW - NRG1 KW - staib agar KW - gene KW - morphogenesis KW - expression KW - regulator KW - virulence KW - growth KW - UME6 KW - epidemiology Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131007 VL - 8 IS - 4 ER - TY - JOUR A1 - Makgotlho, Phuti E. A1 - Marincola, Gabriella A1 - Schäfer, Daniel A1 - Liu, Quian A1 - Bae, Taeok A1 - Geiger, Tobias A1 - Wasserman, Elizabeth A1 - Wolz, Christine A1 - Ziebuhr, Wilma A1 - Sinha, Bhanu T1 - SDS Interferes with SaeS Signaling of Staphylococcus aureus Independently of SaePQ JF - PLOS ONE N2 - The Staphylococcus aureus regulatory saePQRS system controls the expression of numerous virulence factors, including extracellular adherence protein (Eap), which amongst others facilitates invasion of host cells. The saePQRS operon codes for 4 proteins: the histidine kinase SaeS, the response regulator SaeR, the lipoprotein SaeP and the transmembrane protein SaeQ. S. aureus strain Newman has a single amino acid substitution in the transmembrane domain of SaeS (L18P) which results in constitutive kinase activity. SDS was shown to be one of the signals interfering with SaeS activity leading to inhibition of the sae target gene eap in strains with SaeS(L) but causing activation in strains containing SaeS(P). Here, we analyzed the possible involvement of the SaeP protein and saePQ region in SDS-mediated sae/eap expression. We found that SaePQ is not needed for SDS-mediated SaeS signaling. Furthermore, we could show that SaeS activity is closely linked to the expression of Eap and the capacity to invade host cells in a number of clinical isolates. This suggests that SaeS activity might be directly modulated by structurally non-complex environmental signals, as SDS, which possibly altering its kinase/phosphatase activity. KW - host-cell invasion KW - 2-component system KW - strain Newman KW - allelic replacement KW - genome sequence KW - locus KW - gene KW - activation KW - expression KW - infection Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-128469 SN - 1932-6203 VL - 8 IS - 8 ER - TY - JOUR A1 - Maudet, Claire A1 - Sourisce, Adèle A1 - Dragin, Loïc A1 - Lahouassa, Hichem A1 - Rain, Jean-Christopher A1 - Bouaziz, Serge A1 - Ramirez, Bertha Cécilia A1 - Margottin-Goguet, Florence T1 - HIV-1 Vpr Induces the Degradation of ZIP and sZIP, Adaptors of the NuRD Chromatin Remodeling Complex, by Hijacking DCAF1/VprBP JF - PLOS ONE N2 - The Vpr protein from type 1 and type 2 Human Immunodeficiency Viruses (HIV-1 and HIV-2) is thought to inactivate several host proteins through the hijacking of the DCAF1 adaptor of the Cul4A ubiquitin ligase. Here, we identified two transcriptional regulators, ZIP and sZIP, as Vpr-binding proteins degraded in the presence of Vpr. ZIP and sZIP have been shown to act through the recruitment of the NuRD chromatin remodeling complex. Strikingly, chromatin is the only cellular fraction where Vpr is present together with Cul4A ubiquitin ligase subunits. Components of the NuRD complex and exogenous ZIP and sZIP were also associated with this fraction. Several lines of evidence indicate that Vpr induces ZIP and sZIP degradation by hijacking DCAF1: (i) Vpr induced a drastic decrease of exogenously expressed ZIP and sZIP in a dose-dependent manner, (ii) this decrease relied on the proteasome activity, (iii) ZIP or sZIP degradation was impaired in the presence of a DCAF1-binding deficient Vpr mutant or when DCAF1 expression was silenced. Vpr-mediated ZIP and sZIP degradation did not correlate with the growth-related Vpr activities, namely G2 arrest and G2 arrest-independent cytotoxicity. Nonetheless, infection with HIV-1 viruses expressing Vpr led to the degradation of the two proteins. Altogether our results highlight the existence of two host transcription factors inactivated by Vpr. The role of Vpr-mediated ZIP and sZIP degradation in the HIV-1 replication cycle remains to be deciphered. KW - immunodeficiency-virus type-1 KW - MI-2/NURD complex KW - cell-cycle arrest KW - CUL4-DDB1 ubiquitin ligase KW - viral protein-R KW - NF-KAPPA-B KW - macrophage infection KW - enzyme APOBEC3G KW - in-vivo KW - transcription Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-128316 SN - 1932-6203 VL - 8 IS - 10 ER - TY - JOUR A1 - Feller, Tatjana A1 - Thom, Pascal A1 - Koch, Natalie A1 - Spiegel, Holger A1 - Addai-Mensah, Otchere A1 - Fischer, Rainer A1 - Reimann, Andreas A1 - Pradel, Gabriele A1 - Fendel, Rolf A1 - Schillberg, Stefan A1 - Scheuermayer, Matthias A1 - Schinkel, Helga T1 - Plant-Based Production of Recombinant Plasmodium Surface Protein Pf38 and Evaluation of its Potential as a Vaccine Candidate JF - PLOS ONE N2 - Pf38 is a surface protein of the malarial parasite Plasmodium falciparum. In this study, we produced and purified recombinant Pf38 and a fusion protein composed of red fluorescent protein and Pf38 (RFP-Pf38) using a transient expression system in the plant Nicotiana benthamiana. To our knowledge, this is the first description of the production of recombinant Pf38. To verify the quality of the recombinant Pf38, plasma from semi-immune African donors was used to confirm specific binding to Pf38. ELISA measurements revealed that immune responses to Pf38 in this African subset were comparable to reactivities to AMA-1 and \(MSP1_{19}\). Pf38 and RFP-Pf38 were successfully used to immunise mice, although titres from these mice were low (on average 1:11.000 and 1:39.000, respectively). In immune fluorescence assays, the purified IgG fraction from the sera of immunised mice recognised Pf38 on the surface of schizonts, gametocytes, macrogametes and zygotes, but not sporozoites. Growth inhibition assays using \(\alpha Pf38\) antibodies demonstrated strong inhibition \((\geq 60 \% ) \) of the growth of blood-stage P. falciparum. The development of zygotes was also effectively inhibited by \(\alpha Pf38\) antibodies, as determined by the zygote development assay. Collectively, these results suggest that Pf38 is an interesting candidate for the development of a malaria vaccine. KW - malaria vaccine KW - balancing selection KW - N-glycans KW - falciparum KW - expression KW - antibodies KW - identification KW - transmission KW - tobacco KW - antigen Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-128221 SN - 1932-6203 VL - 8 IS - 11 ER - TY - THES A1 - Westermann, Alexander J. T1 - Dual RNA-seq of pathogen and host T1 - Duale RNA-Sequenzierung eines Pathogens und seines Wirts N2 - The infection of a eukaryotic host cell by a bacterial pathogen is one of the most intimate examples of cross-kingdom interactions in biology. Infection processes are highly relevant from both a basic research as well as a clinical point of view. Sophisticated mechanisms have evolved in the pathogen to manipulate the host response and vice versa host cells have developed a wide range of anti-microbial defense strategies to combat bacterial invasion and clear infections. However, it is this diversity and complexity that makes infection research so challenging to technically address as common approaches have either been optimized for bacterial or eukaryotic organisms. Instead, methods are required that are able to deal with the often dramatic discrepancy between host and pathogen with respect to various cellular properties and processes. One class of cellular macromolecules that exemplify this host-pathogen heterogeneity is given by their transcriptomes: Bacterial transcripts differ from their eukaryotic counterparts in many aspects that involve both quantitative and qualitative traits. The entity of RNA transcripts present in a cell is of paramount interest as it reflects the cell’s physiological state under the given condition. Genome-wide transcriptomic techniques such as RNA-seq have therefore been used for single-organism analyses for several years, but their applicability has been limited for infection studies. The present work describes the establishment of a novel transcriptomic approach for infection biology which we have termed “Dual RNA-seq”. Using this technology, it was intended to shed light particularly on the contribution of non-protein-encoding transcripts to virulence, as these classes have mostly evaded previous infection studies due to the lack of suitable methods. The performance of Dual RNA-seq was evaluated in an in vitro infection model based on the important facultative intracellular pathogen Salmonella enterica serovar Typhimurium and different human cell lines. Dual RNA-seq was found to be capable of capturing all major bacterial and human transcript classes and proved reproducible. During the course of these experiments, a previously largely uncharacterized bacterial small non-coding RNA (sRNA), referred to as STnc440, was identified as one of the most strongly induced genes in intracellular Salmonella. Interestingly, while inhibition of STnc440 expression has been previously shown to cause a virulence defect in different animal models of Salmonellosis, the underlying molecular mechanisms have remained obscure. Here, classical genetics, transcriptomics and biochemical assays proposed a complex model of Salmonella gene expression control that is orchestrated by this sRNA. In particular, STnc440 was found to be involved in the regulation of multiple bacterial target mRNAs by direct base pair interaction with consequences for Salmonella virulence and implications for the host’s immune response. These findings exemplify the scope of Dual RNA-seq for the identification and characterization of novel bacterial virulence factors during host infection. N2 - Die Infektion einer eukaryontischen Wirtszelle mit einem bakteriellen Pathogen ist eines der komplexesten Beispiele einer Domänen-überschreitenden Wechselwirkung zweier Organismen. Infektionsprozesse sind in höchstem Maße relevant, sowohl in der Grundforschung als auch von einem klinischen Blickwinkel aus betrachtet. Im Laufe der Evolution entstanden komplizierte Mechanismen, die es einem Pathogen erlauben, die Wirtsantwort zu manipulieren. Umgekehrt haben potentielle Wirtszellen eine Reihe von anti-mikrobiellen Verteidigungsstrategien entwickelt, um bakterielle Infektionen zu bekämpfen und letztlich zu beseitigen. Es sind jedoch genau diese Verschiedenheit und Komplexität, welche die Infektionsforschung so anspruchsvoll und technisch schwer analysierbar machen. Gängige Analysemethoden wurden zumeist entweder für bakterielle oder aber eukaryontische Organismen entwickelt. Dagegen werden Techniken benötigt, welche es erlauben, mit den mitunter extremen Unterschieden zwischen Wirt und Pathogen umzugehen, die sich in etlichen zellulären Eigenschaften und Prozessen manifestieren. Eine Klasse zellulärer Makromoleküle, die diese Heterogenität zwischen Wirt und Pathogen widerspiegelt, sind ihre jeweiligen Transkriptome: Bakterielle Transkripte unter-scheiden sich von ihren eukaryontischen Pendants in vielerlei Hinsicht, was sowohl quantitative als auch qualitative Aspekte miteinschließt. Die Gesamtheit zellulärer Transkripte ist von größter Bedeutung, da sie den physiologischen Zustand der jeweiligen Zelle unter den gegebenen Bedingungen reflektiert. Aus diesem Grund werden Genom-weite Transkriptom-techniken wie etwa die RNA-Sequenzierung seit etlichen Jahren erfolgreich angewandt, um biologische Prozesse zu untersuchen – jedoch ist deren Eignung für Infektionsstudien in starkem Maße limitiert. Die vorliegende Arbeit beschreibt die Etablierung eines neuartigen Ansatzes, „Duale RNA-Sequenzierung“ genannt, der Transkriptomstudien mit der Infektionsbiologie kompatibel macht. Mithilfe dieser Technologie wurde hier im Besonderen versucht, die Rolle nicht-proteinkodierender RNA-Moleküle für die Virulenz zu beleuchten, da die Charakterisierung dieser RNA-Klassen bisherigen Infektionsstudien weitgehend verwehrt blieb. Die Anwendbar-keit der Dualen RNA-Sequenzierung wurde innerhalb eines In-vitro-Infektionsmodells getestet, welches auf dem wichtigen, fakultativ intrazellulären Pathogen Salmonella enterica serovar Tyhimurium und verschiedenen humanen Zelllinien basiert. Die Duale RNA-Sequenzierung zeigte sich dabei in der Lage alle wesentlichen bakteriellen sowie humanen Transkriptklassen zu erfassen und erwies sich als reproduzierbar. Im Zuge dieser Experimente wurde ein Gen für eine zuvor kaum beschriebene kleine nicht-kodierende RNA (STnc440) als eines der am stärksten induzierten Gene intrazellulärer Salmonellen identifiziert. Interessanterweise hatten vorherige Studien gezeigt, dass die Inaktivierung dieses Gens zu einem Virulenzdefizit innerhalb unterschiedlicher Tiermodelle für Salmonellose führt. Die zugrunde liegenden molekularen Mechanismen blieben jedoch unbekannt. In der vorliegenden Arbeit wurden genetische, Transkriptom- sowie biochemische Analysen eingesetzt um das komplexe Regulationsnetzwerk dieser kleinen RNA erstmals näher zu beleuchten. Im Einzelnen konnte gezeigt werden, dass STnc440 die Expression mehrerer bakterieller mRNAs durch das Ausbilden zwischen-molekularer Basenpaarungen reguliert, was weitreichende Konsequenzen sowohl für die Virulenz des Pathogens als auch die Immunantwort des Wirts hat. Diese Ergebnisse veranschaulichen das Potential der Dualen RNA-Sequenzierung für das Auffinden und Charakterisieren neuer bakterieller Virulenzfaktoren während der Wirtsinfektion. KW - Transkriptomanalyse KW - Dual RNA-seq KW - Salmonella enterica KW - Wirtszelle Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112462 ER - TY - JOUR A1 - Berghoff, Bork A. A1 - Konzer, Anne A1 - Mank, Nils N. A1 - Looso, Mario A1 - Rische, Tom A1 - Förstner, Konrad U. A1 - Krüger, Marcus A1 - Klug, Gabriele T1 - Integrative "Omics"-Approach Discovers Dynamic and Regulatory Features of Bacterial Stress Responses JF - PLOS Genetics N2 - Bacteria constantly face stress conditions and therefore mount specific responses to ensure adaptation and survival. Stress responses were believed to be predominantly regulated at the transcriptional level. In the phototrophic bacterium Rhodobacter sphaeroides the response to singlet oxygen is initiated by alternative sigma factors. Further adaptive mechanisms include post-transcriptional and post-translational events, which have to be considered to gain a deeper understanding of how sophisticated regulation networks operate. To address this issue, we integrated three layers of regulation: (1) total mRNA levels at different time-points revealed dynamics of the transcriptome, (2) mRNAs in polysome fractions reported on translational regulation (translatome), and (3) SILAC-based mass spectrometry was used to quantify protein abundances (proteome). The singlet oxygen stress response exhibited highly dynamic features regarding short-term effects and late adaptation, which could in part be assigned to the sigma factors RpoE and RpoH2 generating distinct expression kinetics of corresponding regulons. The occurrence of polar expression patterns of genes within stress-inducible operons pointed to an alternative of dynamic fine-tuning upon stress. In addition to transcriptional activation, we observed significant induction of genes at the post-transcriptional level (translatome), which identified new putative regulators and assigned genes of quorum sensing to the singlet oxygen stress response. Intriguingly, the SILAC approach explored the stress-dependent decline of photosynthetic proteins, but also identified 19 new open reading frames, which were partly validated by RNA-seq. We propose that comparative approaches as presented here will help to create multi-layered expression maps on the system level ("expressome"). Finally, intense mass spectrometry combined with RNA-seq might be the future tool of choice to re-annotate genomes in various organisms and will help to understand how they adapt to alternating conditions. KW - singlet oxygen stress KW - genome-wide analysis KW - anti-sigma factor KW - rhodobacter sphaeroides KW - gene expression KW - quanititative proteomics KW - photooxidative stress KW - in-vivo KW - photosynthesis genes KW - mass spectrometry Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127587 SN - 1553-7404 VL - 9 IS - 6 ER - TY - JOUR A1 - Müller, Sara A1 - Windhof, Indra M. A1 - Maximov, Vladimir A1 - Jurkowski, Tomasz A1 - Jeltsch, Albert A1 - Förstner, Konrad U. A1 - Sharma, Cynthia M. A1 - Gräf, Ralph A1 - Nellen, Wolfgang T1 - Target recognition, RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA) JF - Nucleic Acids Research N2 - Although the DNA methyltransferase 2 family is highly conserved during evolution and recent reports suggested a dual specificity with stronger activity on transfer RNA (tRNA) than DNA substrates, the biological function is still obscure. We show that the Dictyostelium discoideum Dnmt2-homologue DnmA is an active tRNA methyltransferase that modifies C38 in \(tRNA^{Asp(GUC)}\) in vitro and in vivo. By an ultraviolet-crosslinking and immunoprecipitation approach, we identified further DnmA targets. This revealed specific tRNA fragments bound by the enzyme and identified \(tRNA^{Glu(CUC/UUC)}\) and \(tRNA^{Gly(GCC)}\) as new but weaker substrates for both human Dnmt2 and DnmA in vitro but apparently not in vivo. Dnmt2 enzymes form transient covalent complexes with their substrates. The dynamics of complex formation and complex resolution reflect methylation efficiency in vitro. Quantitative PCR analyses revealed alterations in dnmA expression during development, cell cycle and in response to temperature stress. However, dnmA expression only partially correlated with tRNA methylation in vivo. Strikingly, dnmA expression in the laboratory strain AX2 was significantly lower than in the NC4 parent strain. As expression levels and binding of DnmA to a target in vivo are apparently not necessarily accompanied by methylation, we propose an additional biological function of DnmA apart from methylation. KW - DNA methylferase homolog KW - drospophila KW - TRNA(ASP) KW - mechanism KW - binding Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-123149 SN - 1362-4962 VL - 41 IS - 18 ER - TY - JOUR A1 - Gholami, Sepideh A1 - Chen, Chun-Hao A1 - Belin, Laurence J. A1 - Lou, Emil A1 - Fujisawa, Sho A1 - Antonacci, Caroline A1 - Carew, Amanda A1 - Chen, Nanhai G. A1 - De Brot, Marina A1 - Zanzonico, Pat B. A1 - Szalay, Aladar A. A1 - Fong, Yuman T1 - Vaccinia virus GLV-1h153 is a novel agent for detection and effective local control of positive surgical margins for breast cancer JF - Breast Cancer Research N2 - Introduction: Surgery is currently the definitive treatment for early-stage breast cancer. However, the rate of positive surgical margins remains unacceptably high. The human sodium iodide symporter (hNIS) is a naturally occurring protein in human thyroid tissue, which enables cells to concentrate radionuclides. The hNIS has been exploited to image and treat thyroid cancer. We therefore investigated the potential of a novel oncolytic vaccinia virus GLV1h-153 engineered to express the hNIS gene for identifying positive surgical margins after tumor resection via positron emission tomography (PET). Furthermore, we studied its role as an adjuvant therapeutic agent in achieving local control of remaining tumors in an orthotopic breast cancer model. Methods: GLV-1h153, a replication-competent vaccinia virus, was tested against breast cancer cell lines at various multiplicities of infection (MOIs). Cytotoxicity and viral replication were determined. Mammary fat pad tumors were generated in athymic nude mice. To determine the utility of GLV-1h153 in identifying positive surgical margins, 90% of the mammary fat pad tumors were surgically resected and subsequently injected with GLV-1h153 or phosphate buffered saline (PBS) in the surgical wound. Serial Focus 120 microPET images were obtained six hours post-tail vein injection of approximately 600 mu Ci of I-124-iodide. Results: Viral infectivity, measured by green fluorescent protein (GFP) expression, was time-and concentrationdependent. All cell lines showed less than 10% of cell survival five days after treatment at an MOI of 5. GLV-1h153 replicated efficiently in all cell lines with a peak titer of 27 million viral plaque forming units (PFU) ( < 10,000-fold increase from the initial viral dose) by Day 4. Administration of GLV-1h153 into the surgical wound allowed positive surgical margins to be identified via PET scanning. In vivo, mean volume of infected surgically resected residual tumors four weeks after treatment was 14 mm(3) versus 168 mm(3) in untreated controls (P < 0.05). Conclusions: This is the first study to our knowledge to demonstrate a novel vaccinia virus carrying hNIS as an imaging tool in identifying positive surgical margins of breast cancers in an orthotopic murine model. Moreover, our results suggest that GLV-1h153 is a promising therapeutic agent in achieving local control for positive surgical margins in resected breast tumors. KW - conservation KW - carcinoma KW - mastectomy KW - metastases KW - stage-i KW - thyroid-cancer KW - radiation-therapy KW - conserving surgery KW - sodium-iodide symporter Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-122140 VL - 15 IS - R26 ER - TY - JOUR A1 - Neumann, Yvonne A1 - Ohlsen, Knut A1 - Donat, Stefanie A1 - Engelmann, Susanne A1 - Kusch, Harald A1 - Albrecht, Dirk A1 - Cartron, Michael A1 - Hurd, Alexander A1 - Foster, Simon J. T1 - The effect of skin fatty acids on Staphylococcus aureus JF - Archives of Microbiology N2 - Staphylococcus aureus is a commensal of the human nose and skin. Human skin fatty acids, in particular cis-6-hexadecenoic acid (C-6-H), have high antistaphylococcal activity and can inhibit virulence determinant production. Here, we show that sub-MIC levels of C-6-H result in induction of increased resistance. The mechanism(s) of C-6-H activity was investigated by combined transcriptome and proteome analyses. Proteome analysis demonstrated a pleiotropic effect of C-6-H on virulence determinant production. In response to C-6-H, transcriptomics revealed altered expression of over 500 genes, involved in many aspects of virulence and cellular physiology. The expression of toxins (hla, hlb, hlgBC) was reduced, whereas that of host defence evasion components (cap, sspAB, katA) was increased. In particular, members of the SaeRS regulon had highly reduced expression, and the use of specific mutants revealed that the effect on toxin production is likely mediated via SaeRS KW - S. aureus KW - skin fatty acid KW - C-6-H KW - resistance Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121657 VL - 197 ER - TY - JOUR A1 - Vembar, Shruti S. A1 - Scherf, Artur A1 - Siegel, T. Nicolai T1 - Noncoding RNAs as emerging regulators of Plasmodium falciparum virulence gene expression JF - Current Opinion in Microbiology N2 - The eukaryotic unicellular pathogen Plasmodium falciparum tightly regulates gene expression, both during development and in adaptation to dynamic host environments. This regulation is evident in the mutually exclusive expression of members of clonally variant virulence multigene families. While epigenetic regulators have been selectively identified at active or repressed virulence genes, their specific recruitment remains a mystery. In recent years, noncoding RNAs (ncRNAs) have emerged as lynchpins of eukaryotic gene regulation; by binding to epigenetic regulators, they provide target specificity to otherwise non-specific enzyme complexes. Not surprisingly, there is great interest in understanding the role of ncRNA in P. falciparum, in particular, their contribution to the mutually exclusive expression of virulence genes. The current repertoire of P. falciparum ncRNAs includes, but is not limited to, subtelomeric ncRNAs, virulence gene-associated ncRNAs and natural antisense RNA transcripts. Continued improvement in high-throughput sequencing methods is sure to expand this repertoire. Here, we summarize recent advances in P. falciparum ncRNA biology, with an emphasis on ncRNA-mediated epigenetic modes of gene regulation. Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121416 SN - 1369-5274 VL - 20 IS - 100 ER - TY - JOUR A1 - Jäger, Dominik A1 - Förstner, Konrad U. A1 - Sharma, Cynthia M. A1 - Santangelo, Thomas J. A1 - Reeve, John N. T1 - Primary transcriptome map of the hyperthermophilic archaeon Thermococcus kodakarensis JF - BMC Genomics N2 - Background Prokaryotes have relatively small genomes, densely-packed with protein-encoding sequences. RNA sequencing has, however, revealed surprisingly complex transcriptomes and here we report the transcripts present in the model hyperthermophilic Archaeon, Thermococcus kodakarensis, under different physiological conditions. Results Sequencing cDNA libraries, generated from RNA isolated from cells under different growth and metabolic conditions has identified >2,700 sites of transcription initiation, established a genome-wide map of transcripts, and consensus sequences for transcription initiation and post-transcription regulatory elements. The primary transcription start sites (TSS) upstream of 1,254 annotated genes, plus 644 primary TSS and their promoters within genes, are identified. Most mRNAs have a 5'-untranslated region (5'-UTR) 10 to 50 nt long (median = 16 nt), but ~20% have 5'-UTRs from 50 to 300 nt long and ~14% are leaderless. Approximately 50% of mRNAs contain a consensus ribosome binding sequence. The results identify TSS for 1,018 antisense transcripts, most with sequences complementary to either the 5'- or 3'-region of a sense mRNA, and confirm the presence of transcripts from all three CRISPR loci, the RNase P and 7S RNAs, all tRNAs and rRNAs and 69 predicted snoRNAs. Two putative riboswitch RNAs were present in growing but not in stationary phase cells. The procedure used is designed to identify TSS but, assuming that the number of cDNA reads correlates with transcript abundance, the results also provide a semi-quantitative documentation of the differences in T. kodakarensis genome expression under different growth conditions and confirm previous observations of substrate-dependent specific gene expression. Many previously unanticipated small RNAs have been identified, some with relative low GC contents (≤50%) and sequences that do not fold readily into base-paired secondary structures, contrary to the classical expectations for non-coding RNAs in a hyperthermophile. Conclusion The results identify >2,700 TSS, including almost all of the primary sites of transcription initiation upstream of annotated genes, plus many secondary sites, sites within genes and sites resulting in antisense transcripts. The T. kodakarensis genome is small (~2.1 Mbp) and tightly packed with protein-encoding genes, but the transcriptomes established also contain many non-coding RNAs and predict extensive RNA-based regulation in this model Archaeon. KW - riboswitch KW - hyperthermophile KW - hydrogen regulation KW - transcriptome KW - archaea KW - promoters KW - antisense RNAs KW - small non-coding RNAs Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-120966 SN - 1471-2164 VL - 15 IS - 684 ER - TY - JOUR A1 - Glaser, Jan A1 - Schurigt, Uta A1 - Suzuki, Brian M. A1 - Caffrey, Connor R. A1 - Holzgrabe, Ulrike T1 - Anti-Schistosomal Activity of Cinnamic Acid Esters: Eugenyl JF - Molecules N2 - Bornyl caffeate (1) was previously isolated by us from Valeriana (V.) wallichii rhizomes and identified as an anti-leishmanial substance. Here, we screened a small compound library of synthesized derivatives 1–30 for activity against schistosomula of Schistosoma (S.) mansoni. Compound 1 did not show any anti-schistosomal activity. However, strong phenotypic changes, including the formation of vacuoles, degeneration and death were observed after in vitro treatment with compounds 23 (thymyl cinnamate) and 27 (eugenyl cinnamate). Electron microscopy analysis of the induced vacuoles in the dying parasites suggests that 23 and 27 interfere with autophagy. KW - thymyl cinnamate KW - vacuoles KW - autophagy KW - anti-schistosomal activity KW - schistosoma KW - schistosomula KW - parasite KW - eugenyl cinnamate Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125712 VL - 20 ER - TY - JOUR A1 - Schneider, Johannes A1 - Klein, Teresa A1 - Mielich-Süss, Benjamin A1 - Koch, Gudrun A1 - Franke, Christian A1 - Kuipers, Oskar P. A1 - Kovács, Ákos T. A1 - Sauer, Markus A1 - Lopez, Daniel T1 - Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium JF - PLoS Genetics N2 - Lipid rafts are membrane microdomains specialized in the regulation of numerous cellular processes related to membrane organization, as diverse as signal transduction, protein sorting, membrane trafficking or pathogen invasion. It has been proposed that this functional diversity would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known. We recently discovered that bacterial membranes organize their signal transduction pathways in functional membrane microdomains (FMMs) that are structurally and functionally similar to the eukaryotic lipid rafts. In this report, we took advantage of the tractability of the prokaryotic model Bacillus subtilis to provide evidence for the coexistence of two distinct families of FMMs in bacterial membranes, displaying a distinctive distribution of proteins specialized in different biological processes. One family of microdomains harbors the scaffolding flotillin protein FloA that selectively tethers proteins specialized in regulating cell envelope turnover and primary metabolism. A second population of microdomains containing the two scaffolding flotillins, FloA and FloT, arises exclusively at later stages of cell growth and specializes in adaptation of cells to stationary phase. Importantly, the diversification of membrane microdomains does not occur arbitrarily. We discovered that bacterial cells control the spatio-temporal remodeling of microdomains by restricting the activation of FloT expression to stationary phase. This regulation ensures a sequential assembly of functionally specialized membrane microdomains to strategically organize signaling networks at the right time during the lifespan of a bacterium. KW - membrane proteins KW - gene expression KW - bacillus subtilis KW - fluorescence microscopy KW - cell fusion KW - signal transduction KW - gene regulation KW - lipids Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125577 VL - 11 IS - 4 ER - TY - JOUR A1 - Masic, Anita A1 - Valencia Hernandez, Ana Maria A1 - Hazra, Sudipta A1 - Glaser, Jan A1 - Holzgrabe, Ulrike A1 - Hazra, Banasri A1 - Schurigt, Uta T1 - Cinnamic Acid Bornyl Ester Derivatives from Valeriana wallichii Exhibit Antileishmanial In Vivo Activity in Leishmania major-Infected BALB/c Mice JF - PLoS One N2 - Human leishmaniasis covers a broad spectrum of clinical manifestations ranging from self-healing cutaneous leishmaniasis to severe and lethal visceral leishmaniasis caused among other species by Leishmania major or Leishmania donovani, respectively. Some drug candidates are in clinical trials to substitute current therapies, which are facing emerging drug-resistance accompanied with serious side effects. Here, two cinnamic acid bornyl ester derivatives (1 and 2) were assessed for their antileishmanial activity. Good selectivity and antileishmanial activity of bornyl 3-phenylpropanoate (2) in vitro prompted the antileishmanial assessment in vivo. For this purpose, BALB/c mice were infected with Leishmania major promastigotes and treated with three doses of 50 mg/kg/day of compound 2. The treatment prevented the characteristic swelling at the site of infection and correlated with reduced parasite burden. Transmitted light microscopy and transmission electron microscopy of Leishmania major promastigotes revealed that compounds 1 and 2 induce mitochondrial swelling. Subsequent studies on Leishmania major promastigotes showed the loss of mitochondrial transmembrane potential (ΔΨm) as a putative mode of action. As the cinnamic acid bornyl ester derivatives 1 and 2 had exhibited antileishmanial activity in vitro, and compound 2 in Leishmania major-infected BALB/c mice in vivo, they can be regarded as possible lead structures for the development of new antileishmanial therapeutic approaches. KW - leishmania major KW - promastigotes KW - apoptosis KW - mitochondria KW - parasitic diseases KW - leishmania KW - leishmaniasis KW - mouse models Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125354 VL - 10 IS - 11 ER - TY - JOUR A1 - Frank, Benjamin A1 - Marcu, Ana A1 - de Oliveira Almeida Petersen, Antonio Luis A1 - Weber, Heike A1 - Stigloher, Christian A1 - Mottram, Jeremy C. A1 - Scholz, Claus Jürgen A1 - Schurigt, Uta T1 - Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210 JF - Parasites & Vectors N2 - Background Autophagy participates in innate immunity by eliminating intracellular pathogens. Consequently, numerous microorganisms have developed strategies to impair the autophagic machinery in phagocytes. In the current study, interactions between Leishmania major (L. m.) and the autophagic machinery of bone marrow-derived macrophages (BMDM) were analyzed. Methods BMDM were generated from BALB/c mice, and the cells were infected with L. m. promastigotes. Transmission electron microscopy (TEM) and electron tomography were used to investigate the ultrastructure of BMDM and the intracellular parasites. Affymetrix® chip analyses were conducted to identify autophagy-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The protein expression levels of autophagy related 5 (ATG5), BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), cathepsin E (CTSE), mechanistic target of rapamycin (MTOR), microtubule-associated proteins 1A/1B light chain 3B (LC3B), and ubiquitin (UB) were investigated through western blot analyses. BMDM were transfected with specific small interfering RNAs (siRNAs) against autophagy-related genes and with mimics or inhibitors of autophagy-associated miRNAs. The infection rates of BMDM were determined by light microscopy after a parasite-specific staining. Results The experiments demonstrated autophagy induction in BMDM after in vitro infection with L. m.. The results suggested a putative MTOR phosphorylation-dependent counteracting mechanism in the early infection phase and indicated that intracellular amastigotes were cleared by autophagy in BMDM in the late infection phase. Transcriptomic analyses and specific downregulation of protein expression with siRNAs suggested there is an association between the infection-specific over expression of BNIP3, as well as CTSE, and the autophagic activity of BMDM. Transfection with mimics of mmu-miR-101c and mmu-miR-129-5p, as well as with an inhibitor of mmu-miR-210-5p, demonstrated direct effects of the respective miRNAs on parasite clearance in L. m.-infected BMDM. Furthermore, Affymetrix® chip analyses revealed a complex autophagy-related RNA network consisting of differentially expressed mRNAs and miRNAs in BMDM, which indicates high glycolytic and inflammatory activity in the host macrophages. Conclusions Autophagy in L. m.-infected host macrophages is a highly regulated cellular process at both the RNA level and the protein level. Autophagy has the potential to clear parasites from the host. The results obtained from experiments with murine host macrophages could be translated in the future to develop innovative and therapeutic antileishmanial strategies for human patients. KW - autophagy KW - BNIP3 KW - CTSE KW - electron tomography KW - leishmania major KW - macrophages KW - miRNAs KW - MTOR KW - siRNAs KW - transmission electron microscopy Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124997 VL - 8 IS - 404 ER - TY - JOUR A1 - Amich, Jorge A1 - Krappmann, Sven T1 - Deciphering metabolic traits of the fungal pathogen Aspergillus fumigatus: redundancy vs. essentiality JF - Frontiers in Microbiology N2 - Incidence rates of infections caused by environmental opportunistic fungi have risen over recent decades. Aspergillus species have emerged as serious threat for the immunecompromised, and detailed knowledge about virulence-determining traits is crucial for drug target identification. As a prime saprobe, A. fumigatus has evolved to efficiently adapt to various stresses and to sustain nutritional supply by osmotrophy, which is characterized by extracellular substrate digestion followed by efficient uptake of breakdown products that are then fed into the fungal primary metabolism. These intrinsic metabolic features are believed to be related with its virulence ability. The plethora of genes that encode underlying effectors has hampered their in-depth analysis with respect to pathogenesis. Recent developments in Aspergillus molecular biology allow conditional gene expression or comprehensive targeting of gene families to cope with redundancy. Furthermore, identification of essential genes that are intrinsically connected to virulence opens accurate perspectives for novel targets in antifungal therapy. KW - Aspergillus fumigatus KW - aspergillosis KW - virulence KW - conditional promoter replacement KW - nutrients KW - gene family targeting Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-123669 VL - 3 ER - TY - THES A1 - Sturm, Volker Jörg Friedrich T1 - \(^{19}F\) Magnetresonanztomographie zur Bildgebung von Infektionen im Zeitverlauf T1 - \(^{19}F\) magnetic resonance imaging to monitor the timecourse of bacterial infections in vivo N2 - Im Rahmen dieser Arbeit sollten die Möglichkeiten der MR Tomographie erkundet werden bakterielle Infektionen im Zeitverlauf darzustellen. Genauer gesagt sollte das Potential der MR Tomographie anhand eines durch eine Infektion induzierten lokalisierten Abszesses unter Verwendung dreier unterschiedlicher MRT Methoden untersucht werden: Mittels nativem \(T_2\) Kontrast; der Verwendung von superparamagnetischen Eisenoxid Partieln (USPIO) als \(T_2^*\) Kontrastmittel; und dem Einsatz von Perfluorkarbonen (PFC) als \(^{19}F\) MRT Marker (siehe Kapitel 3). Wie erwartet führte die durch die Infektion hervorgerufene Entzündung zu veränderten \(T_2\)-Zeiten, welche auf \(T_2\)-gewichteten MR Bildern eine Lokalisierung des Abszessbereiches erlauben. Jedoch eigneten sich diese Daten aufgrund der graduellen Änderung der \(T_2\)-Zeiten nicht, um eine klare Grenze zwischen Abszess und umliegendem Gewebe zu ziehen. Superparamagnetische Eisenoxidpartikel andererseit haben als MRT Kontrastmittel bereits in den letzten Jahren ihre Fähigkeit unter Beweis gestellt Entzündungen [53, 58, 64] darzustellen. Die Anreicherung dieser Partikel am Rande des Abszesses [53], wie sie auch in unseren MR Daten zu beobachten war, erlaubte eine relativ scharfe Abgrenzung gegenüber dem umgebenden Gewebe in der chronischen Phase der Infektion (Tag 9 p.i.). Hingegen genügte die nur sehr spärlichen Anreicherung von USPIO Partikeln in der akuten Phase der Infektion (Tag 3 p.i.) nicht für eine entsprechende Abgrenzung [58]. Aufgrund der sehr geringen biologischen Häufigkeit und den sehr kurzen Relaxationszeiten von endogenem Fluor eignen sich Perfluorkarbone als Markersubstanz in der MR Tomographie von biologischen Systemen. Insbesondere da PFC Emulsionen durch phagozytierende Zellen aufgenommen werden und im Bereich von Entzündungen akkumulieren [30, 59]. In dieser Arbeit konnte anhand der erhaltenen MRT Daten eine Akkumulation von Perfluorkarbonen nicht nur in der chronischen Phase, sondern auch in der akuten Phase nachgewiesen werden. Diese Daten erlauben somit zu allen untersuchten Zeitpunkten eine Abgrenzung zwischen Infektion und umliegenden Gewebe. Aufgrund der besagten Vorteile wurden die Perfluorkarbone gewählt, um die Möglichkeiten der MR Tomographie zu testen, quantitative Informationen über die schwere der Infektion zu liefern. Als Referenz für die Bakterienbelastung wurden die Biolumineszenzbildgebung (BLI) [49, 50] und die Standardmethode zur Bestimmung der Bakterienbelastung cfu (koloniebildenden Einheiten) herangezogen. Eine Gegenüberstellung der zeitlichen Verläufe der durch die Biolumineszenzbildgebung und durch die cfu erhaltenen Daten liefert eine qualitative Übereinstimmung mit den durch die 19F MR Tomographie erhaltenen Daten. Dies trifft hierbei sowohl auf die über den gesamten Infektionsbereich hinweg summierten Signalamplituden, als auch auf das Volumen zu, in dem Fluor am Ort der Infektion akkumuliert wurde. Im Gegensatz zur Methode der cfu Bestimmung sind die MR Tomographie und die Biolumineszenzbildgebung nicht invasiv und erlauben die Verfolgung des Infektionsverlaufes an einem einzelnen Individuum. Hierzu benötigt, im Gegensatz zur MR Tomographie, die Methode der Biolumineszenzbildgebung jedoch einen speziellen Pathogenstamm. Darüber hinaus ist hervorzuheben, dass die MR Tomographie zudem die Möglichkeit bietet auch morphologische Informationen über den Infektionsbereich und seine Umgebung zu akquirieren. Gerade weil jede dieser Methoden die mit der Infektion einhergehenden Prozesse aus einer leicht anderen Blickrichtung betrachtet, erscheint es sinnvoll diese etablierte Untersuchungsplattform bestehend aus MRT, BLI und cfu über die in dieser Arbeit bearbeitete Fragestellung hinaus näher zu untersuchen. Insbesondere der Aspekt inwieweit die drei Methoden sich gegenseitig ergänzen, könnte einen tieferen Einblick in die Wechselwirkung zwischen Pathogen und Wirt erlauben. Auch wenn für die betrachtete Fragestellung bereits der hierdurchgeführte semiquanitative Ansatz zur Bestimmung der relativen Fluormengen am Ort der Infektion ausreichte, so ist doch im Allgemeinen wünschenswert probenbezogen die Sensitivität der Spule und damit die Güte der Spulenabstimmung zu bestimmen. Hierzu ist jedoch die Aufnahme von \(B_1\)-Karten unabdingbar und wird entsprechend im Kapitel 4 \(Bloch-Siegert B_1^+-Mapping\) näher addressiert. Der Schwerpunkt liegt hierbei, wie der Kapitelname bereits andeutet, auf der Bloch-Siegert Methode, die insbesondere in der präsentierten Implementierung in einer Turbo/ Multi Spin Echo Sequenz eine effiziente Nutzung der relativ langen \(T_\)2-Zeiten der Perfluorkarbone erlaubt. Da zudem die Bloch-Siegert-Methode eine rein phasenbasierte Methode ist, kann neben der aus den Daten erzeugten \(B_1\)-Karte zugleich ein unverfälschtes Magnitudenbild generiert werden, wodurch eine sehr effiziente Nutzung der vorhandenen Messzeit ermöglicht wird. Diese Eigenschaft ist insbesondere für \(^{19}F\) Bildgebung von besonderem Interesse, da hier für jede Messung, aufgrund der üblicherweise relativ geringen Konzentration an Fluoratomen, lange Messzeiten benötigt werden. Zusammenfassend konnte anhand des untersuchten Tiermodells sowohl die Fähigkeit der MR Tomographie nachgewiesen werden Infektionen im Zeitverlauf darzustellen, als auch die Fähigkeit der MR Tomographie quantitative Informationen über den Verlauf der Infektion zu liefern. Desweiteren konnte eine Möglichkeit aufgezeigt werden, welche das Potential hat in vertretbarem Zeitrahmen auch in vivo B1+-Karten auf dem Fluorkanal zu erstellen und so einen zentralen Unsicherheitsfaktor, für Relaxometry und absolute Quantifizierung von \(^{19}F\) Daten in vivo, zu beseitigen. N2 - The main focus of this work is to investigate the potential of magnetic resonance imaging (MRI) to monitor the timecourse of bacterial infections in vivo. More specifically, it focuses on the ability to localize and assess an infection-induced localized bulky abscess using three different MRI methods: the utilization of native \(T_2\) contrast; the usage of super paramagnetic iron oxide nanoparticles (USPIO) as MRI \(T_2^*\) contrast agents; and the application of perfluorcarbons (PFC) as \(^{19}F\) MRI marker (see chapter 3). Study results demonstrated that, as expected the altered \(T_2\) values present in the abscess area permit localization of the infection when using \(T_2\) weighted data. The precise boundary of the abscess, however, could not be determined due to the gradual change of the \(T_2\) values in the area of the infection. Conforming to other studies [53, 58], the MR-detected accumulation of USPIO particles along the abscess rim allowed definition of a fairly exact demarcation line between the abscess and surrounding tissue during the chronic phase of the infection (day 9 p.i.). During the acute phase of the infection (day 3 p.i.), however, the particle accumulation at the abscess rim was too sparse for precise boundary definition [58]. Because of their extremely low biological abundance and the very short relaxation times of endogenous fluorine, PFCs can be imaged background-free in a biological system. Moreover, as emulsified PFCs were taken up by phagocytosing cells and accumulated at the site of inflammation [30, 59], the acquired MRI data showed PFC accumulation during both the chronic and acute phases of infection. It was thus possible to differentiate between the abscess and surrounding tissue at each examined time point. Due to the described advantages, PFCs were chosen to evaluate with MRI the infection severity. As a bacterial burden reference, colony forming units (cfu) and bioluminescence imaging (BLI) [49, 50] were selected. Observation of BLI, cfu and \(^{19}F\) MRI data showed qualitative correlation during the investigated time course. This was true for the accumulated \(^{19}F\) MR signal in the area of infection and for the \(^{19}F\) MR signal volume. Additionally, unlike the cfu method MRI and BLI are non-invasive and thus data can be gathered at multiple time points. However, contrary to BLI, MRI does not require a special pathogen strain. Moreover, it can provide morphological data from an abscess and the surrounding tissue. Because the data delivered by each of these three methods (MRI, BLI and cfu), are based on alternative approaches, additional examinations of the established platform are suggested. For example, the extent to which the methods supplement each other may provide deeper insight into the interaction between pathogen and host. Even though the chosen semi quantitative approach was sufficient in the context of the evaluated issues to estimate the relative fluorine amount at the site of infection, it is in general desirable for each quantification to determine the sensitivity of the coil per sample. To address this issue the Bloch Siegert (BS) based \(B_1\) mapping method implemented in a turbo/ multi spin echo (TSE/MSE) sequence is presented in Chapter 4 Bloch-Siegert \(B_1^+\)-Mapping. Such a sequence allows effective use of the relatively long PFC \(T_2\) times and encodes BS information solely into the phase data. Thus, a \(B_1\) map can be created in addition to the unaltered TSE/MSE magnitude image. In the context of \(^{19}F\) imaging, this is of special interest due to the usually low amounts of fluorine resulting in long measurement times. In conclusion, it was shown that MRI not only enables visualization of the temporal behavior of infections on the investigated animal model, but it can also provide quantitative information about the progress of the infection. Additionally, a method potentially allowing in vivo B1+ mapping was introduced. This is an important step to improve the reliability of relaxometry and absolute quantification of in vivo \(^{19}F\) MRI. KW - Kernspintomografie KW - Bakterielle Infektion KW - 19F MR KW - Perfluorkarbon KW - Infektionsbildgebung KW - Bloch Siegert KW - B1 Mapping KW - Kontrastmittel Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-122851 ER - 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 -