@phdthesis{Ye2004, author = {Ye, Fang}, title = {The role of DNA supercoiling in the coordinated regulation of gene expression in Helicobacter pylori}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-9878}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2004}, abstract = {Summary Mechanisms of global gene regulation in bacteria are not well characterized yet. Changes in global or local supercoiling of chromosomal DNA are thought to play a role in global gene silencing and gene activation. In Helicobacter pylori, a bacterium with few dedicated transcriptional regulators, the structure of some promoters indicates a dependency on DNA topology. For example, the promoter of the major flagellar subunit gene flaA ({\´o}28-dependent) has a shorter spacing of 13 nucleotides (nt) in comparison to the consensus promoter (15 nt). Supercoiling changes might be a mechanism of gene-specific and global transcriptional regulation in this bacterium. The aim of this study was to elucidate, if changes in global supercoiling have an influence on global gene regulation in H. pylori, and on the temporal regulation of the flagellar biosynthesis pathway in this organism. In the present work, global DNA supercoiling in H. pylori was visualized for the first time, by determining the supercoiling state of plasmids under different growth conditions. Using this method, we showed that cellular supercoiling was clearly growth phase-dependent in H. pylori. Coinciding with increased supercoiling during the growth phases, transcription of the flaA gene was increased, while the transcription of a second {\´o}28-dependent gene with regular promoter spacing (HP0472) was reduced, supporting the hypothesis that growth phase-dependency of promoters might be mediated by changes of DNA topology. Supercoiling in H. pylori could be influenced in a reproducible fashion by inhibition of gyrase using novobiocin, which led to DNA relaxation and to a concomitant decrease of flaA transcript levels. Promoter spacer mutagenesis of the flaA promoter was performed. With flaA promoters of increased or reduced length, transcription of flaA was reduced, less susceptible to supercoiling changes, and, under specific conditions, inverted as compared to the wild type promoter. Transcriptional interdependence between the coupled topA-flaB genes and flaA was found by analysis of the flaA promoter mutants. Chromosomally linked gyrA-flgR, and topA-flaB genes were all dependent on supercoiling and coregulated with each other. Comprehensive transcript profiling (DNA microarrays) of wildtype H. pylori with and without novobiocin treatment identified a number of genes (10\% of total genes), including flagellin, virulence and housekeeping genes, which were strongly dependent on and appeared to be synchronized by supercoiling changes (transcriptional up- or downregulation). These findings indicate a tightly coupled temporal regulation of flagellar biogenesis and metabolism in H. pylori, dependent on global supercoiling. A specific group of genes was also regulated in H. pylori by overexpression of Topoisomerase I, as detected by genome-wide analysis (DNA microarray). The DNA-bending protein HU is thought to be responsible for influencing the negative supercoiling of DNA, through its ability to wrap DNA. HU is encoded by the hup single gene in H. pylori, and constitutively expressed during the whole growth curve. An H. pylori hup mutant was constructed. H. pylori cells lacking HU protein were viable, but exhibited a severe growth defect. Our data indicate that the lack of HU dramatically changes global DNA supercoiling, indicating an important function of HU in chromosome structuring in H. pylori. Transcriptome analyses were performed and demonstrated that a total of 66 genes were differentially transcribed upon hup deletion, which include virulence genes and many other cell functions. The data indicate that HU might act as further important global regulator in H. pylori. Increased gene expression of heat shock proteins and a decreased transcription of the urease gene cluster may indicate a co-ordinated response of H. pylori to changes of environmental conditions in its specific ecological niche, mediated by HU. After the whole genomic sequences of H. pylori strains 26695 and J99 were published, two ORFs (HP0116 and HP0440) were presumptively annotated as topoisomerase I orthologs. HP0116 is the functional H. pylori topoisomerase I (TopA). HP0440 (topA2) was found in only few (5 of 43) strains. Western blot analysis indicated that TopA2 is antigenically different from TopA. TopA2 is transcribed in H. pylori, but the protein must be functionally different from TopA, since it is lacking one functionally essential zinc finger motif, and was not able to functionally complement a TopA-deficient E. coli. Like topA, topA2 was also transcribed in a growth phase-dependent manner. We did not find a function of TopA2 in DNA structuring or topology, but, in the present study, we were able for the first time to establish a unique function for TopA2 in global gene regulation, by comprehensive transcriptome analysis (DNA microarray). Transcriptome analysis showed that a total of 46 genes were differentially regulated upon topA2 deletion, which included flagellar genes and urease genes. These results suggest that TopA2 might act as a novel important regulator of both flagellar biosynthesis and urease in H. pylori.}, subject = {Helicobacter pylori}, language = {en} } @phdthesis{Swiderek2005, author = {Swiderek, Halina}, title = {Typing and genome comparison of Neisseria meningitidis by DNA-microarrays}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-13374}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2005}, abstract = {In the present thesis, two projects on the use of microarray technology for molecular epidemiology of Neisseria meningitidis have been followed. The first one evaluated microarrays based on polymorphism-directed oligonucleotide design for typing of N. meningitidis adopting the multilocus sequence typing (MLST) concept. The number of oligonucleotides needed to cover all known polymorphisms was much lower compared to the number needed if a tiling strategy would have been chosen. Initial experiments using oligonucleotides 28-32 nucleotides in length, revealed that the applied hybridisation protocols were highly specific. However, despite of several optimisation steps, the rate of misidentification of oligonucleotides remained >1.8\% in consecutive validation experiments using arrays representing the genetic diversity at three MLST loci. This finding led to the assumption that the high density of polymorphic sites and extensive GC-content variations at N. meningitidis MLST loci hindered the successful implementation of MLST microarrays based on polymorphism-directed oligonucleotide design. In the 1980s, the ET-15 clone emerged within the ST-11 complex of N. meningitidis. This new clone was associated with severe meningococcal disease and outbreaks world-wide. Therefore, the goal of the second project was to identify genetic differences between ET-15 strains and other ST-11 strains using whole genome microarray technology. Three genes encoding hypothetical proteins were identified to be present in all ET-15 strains but absent in other ST-11 strains. This finding together with unpublished observation from our group suggested that several genome alterations occurred before the clonal expansion of the ET-15 clone started. The role that these three genes play in the pathogenicity of the ET-15 clone is unclear. The genome comparisons revealed furthermore that studies of the ET-15 clone displayed approximately two-fold less gene content variation than ST-11 strains not belonging to the ET-15 clone. This finding is in accordance with the recent emergence and clonal expansion of the ET-15 variant.}, subject = {Neisseria meningitis}, language = {en} } @phdthesis{Lee2006, author = {Lee, Sae Kyung}, title = {Interaction of Helicobacter pylori flagellins with the host innate immune system}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-19917}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {Helicobacter pylori (H. pylori) is a gram-negative, microaerophilic, spiral-shaped bacterium. It resides in the gastric mucous layer and epithelial lining of the stomach, often clustering at the junction of epithelial cells. H. pylori colonization usually occurs during childhood, and, when left untreated, generally persists for the host's lifetime. Persistent H. pylori infection can cause chronic superficial gastritis and gastric duodenal ulcers, which is possibly linked to the development of gastric carcinoma and primary gastric lymphoma, especially of the mucosa-associated lymphoid tissue (MALT) type. It was recently defined as a class 1 carcinogen. The gastric inflammatory response to H. pylori infection is characterized by infiltration of the mucosa by neutrophils, T and B cells, plasma cells and macrophages. This reaction is initially induced by H. pylori attachment, followed by cytokine release by gastric epithelial cells. Epidemiological studies revealed that more than 50\% of adults are infected with H. pylori all over the world. However, interestingly, only a subset of individuals develops serious H. pylori-related disease, while most infected individuals show no clinical symptoms. Gastric epithelial cells, like intestinal epithelial cells, express a subset of Toll-like receptors (TLRs) and similar pattern recognition receptors, which are important for the activation of the innate immune system. Bacterial components such as lipopeptides, peptidoglycan, LPS, flagellin, and CpG DNA are the ligands of TLRs. Thus, TLRs in gastric epithelial cells might be able to contribute to innate immune responses to H. pylori infection. However, there is scant knowledge about the mechanisms of innate immune response to acute and chronic H. pylori infection. This study is focused on host cell interaction with H. pylori flagellins, which are major components of the flagellar apparatus, and innate immune responses against them. The flagellins, which are essential for bacterial motility, are important for H. pylori to survive in the stomach mucus during the whole infectious cycle. Flagellins are known to act as the main determinant of many mucosal pathogenic bacteria that mediates proinflammatory signaling, including transcriptional factor NF-\&\#61547;B activation via TLR5. In the first part of the study, we investigated the effects of H. pylori flagellins on TLR5 expression, NF-\&\#61547;B activation and IL-8 production in various human intestinal and gastric epithelial cell lines by using Western blotting, semi-quantitative RT-PCR and ELISA. IL-8 is a potent neutrophil-activating chemokine expressed by gastric epithelial cells. When we stimulated the cells with the native form of or E. coli-expressed recombinant H. pylori flagellins, FlaA and FlaB, IL-8 was not induced in any case, while S. typhimurium flagellin (FliC) induced it significantly. H. pylori was able to modulate TLR5 protein expression and NF-\&\#61547;B activation in epithelial cells regardless of the presence of flagellins. Having established the finding that H. pylori flagellins have unusually low immune-stimulatory properties, we further investigated to find out possible reasons why H. pylori flagellins are distinct from other flagellins of pathogenic bacteria in terms of immune-stimulatory activity. From amino acid sequence comparisons, we found that some regions in the terminal D0D1 protein domains of H. pylori flagellins are different from flagellins of other pathogenic bacteria. D0D1 is the domain which is known to interact with TLR5 in Salmonella FliC. To examine whether the differences endow H. pylori flagellins with low immune-stimulatory properties, we created several mutated H. pylori flagellins (FlaA and FlaB) by site-directed mutagenesis that contain one to four epitopes of Salmonella flagellin D0D1 domain amino acid sequences. The mutant flagellins expressed both in H. pylori and E. coli were used to determine their influence on TLR5-signaling mediators and cytokines, such as MAPkinases, (ERK, p38), NF-\&\#61547;B, IL-8, and MIP-3\&\#61537;. Salmonella FliC expressed in E. coli induced activation of p38, I\&\#61547;B\&\#61537; and NF-\&\#61547;B leading to IL-8 and MIP-3\&\#61537; production in gastric epithelial cells. However, none of the H. pylori flagellin mutants activated MAP kinases or induced those cytokines. In a co-immunoprecipitation assay none of the recombinant wild type or mutated H. pylori flagellins showed any direct physical interaction with TLR5, while Salmonella FliC significantly co-precipitated with TLR5. Interestingly, we found H. pylori flagellins bind to the surface of gastric epithelial cells like FliC, although they do not bind to or stimulate TLR5. Based on the physical interaction of H. pylori flagellins and FliC with human gastric epithelial cells, we further analyzed transcriptional regulation by H. pylori flagellin in these host cells using microarray analysis. The result showed that H. pylori flagellins modulate host cell gene expression, and many of the identified regulation events overlap with the genes regulated by FliC. These findings imply that H. pylori flagellins do play a role in gene regulation of host cells probably through still unknown factors or receptors, although they do not trigger TLR5-related signaling pathways. The results of our study suggest that, in addition to the low immune-stimulatory activity of H. pylori LPS, the evolutionary reduction in stimulating activity of H. pylori flagellins on the local innate immune responses in the stomach in vivo might be a further strategy of this chronic mucosal pathogen to evade and minimize deleterious host responses, thereby promoting life-long persistence in the host, and possibly contributing to cancerogenesis.}, subject = {Helicobacter pylori}, language = {en} } @phdthesis{Konrad2007, author = {Konrad, Christian}, title = {Molecular analysis of insulin signaling mechanisms in Echinococcus multilocularis and their role in the host-parasite interaction in the alveolar echinococcosis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-22636}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2007}, abstract = {The insulin receptor ortholog EmIR of the fox-tapeworm Echinococcus multilocularis displays significant structural homology to the human insulin receptor (HIR) and has been suggested to be involved in insulin sensing mechanisms of the parasite's metacestode larval stage. In the present work, the effects of host insulin on Echinococcus metacestode vesicles and the proposed interaction between EmIR and mammalian insulin have been studied using biochemical and cell-biological approaches. Human insulin, exogenously added to in vitro cultivated parasite larvae, (i) significantly stimulated parasite survival and growth, (ii) induced DNA de novo synthesis in Echinococcus, (iii) affected overall protein phosphorylation in the parasite, and (iv) specifically induced the phosphorylation of the parasite's Erk-like MAP kinase orthologue EmMPK1. These results clearly indicated that Echinococcus metacestode vesicles are able to sense exogenous host insulin which induces a mitogenic response. To investigate whether EmIR mediates these effects, anti-EmIR antibodies were produced and utilized in biochemical assays and immunohistochemical analyses. EmIR was shown to be expressed in the germinal layer of the parasite both on the surface of glycogen storing cells and undifferentiated germinal cells. Upon addition of exogenous insulin to metacestode vesicles, the phosphorylation of EmIR was significantly induced, an effect which was suppressed in the presence of specific inhibitors of insulin receptor-like tyrosine kinases. Furthermore, upon expression of EmIR/HIR receptor chimera containing the extracellular ligand binding domain of EmIR in HEK 293 cells, a specific autophosphorylation of the chimera could be induced through the addition of exogenous insulin. These results indicated the capability of EmIR to sense and to transmit host insulin signals to the Echinococcus signaling machinery. The importance of insulin signaling mechanisms for parasite survival and growth were underscored by in vitro cultivation experiments in which the addition of an inhibitor of insulin receptor tyrosine kinases led to vesicle degradation and death. Based on the above outlined molecular data on the interaction between EmIR and mammalian insulin, the parasite's insulin receptor orthologue most probably mediates the insulin effects on parasite growth and is, therefore, a potential candidate factor for host-parasite communication via evolutionary conserved pathways. In a final set of experiments, signaling mechanisms that act downstream of EmIR have been analyzed. These studies revealed significant differences between insulin signaling in Echinococcus and the related cestode parasite Taenia solium. These differences could be associated with differences in the organo-tropism of both species.}, subject = {Fuchsbandwurm}, language = {en} } @phdthesis{vonSaintAndrevonArnim2007, author = {von Saint Andr{\´e} - von Arnim, Am{\´e}lie}, title = {The Role of Endosymbiotic Wolbachia Bacteria in the Pathogenesis of River Blindness}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-31560}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2007}, abstract = {Introduction: This study investigates the role of Wolbachia bacteria in the pathogenesis of O. volvulus keratitis in a mouse model. Wolbachia bacteria are essential symbionts of most filarial nematodes of importance for mankind. Methods: Using a mouse model for river blindness in which soluble extracts of filarial nematodes are injected in the corneal stroma, changes in stromal thickness and haze of the cornea are observed by in vivo confocal microscopy, followed by immunohistochemical staining for neutrophils and PECAM-1, as well as ELISA of corneal chemokines. Reactions to filarial extracts containing Wolbachia are compared to those without the endosymbiont. Results: The approach of characterizing Wolbachia's role in river blindness in this study is threefold. Firstly, Wolbachia-depleted extracts from doxycycline treated onchocerciasis patients led to a diminished inflammatory response in corneas of C57BL/6 mice compared to untreated, i.e. Wolbachia containing antigen. The decreased cell recruitment observed with doxycycline treated extracts involved neutrophils, but not eosinophils. This finding demonstrated that the presence of Wolbachia increases neutrophil recruitment. Secondly, extracts from Wolbachia-containing B. malayi revealed markedly more pathology than endosymbiont-free A. viteae antigen. This again pointed at the role of Wolbachia in development of disease. Thirdly, Toll-like Receptor 4 (TLR4) dependence was shown to exist for the inflammatory response to Wolbachia harboring O. volvulus antigen by looking at the corneal pathology in TLR4-mutant C3H/HeJ mice, compared to the wild-type C3H/HeN strain. Investigating further Wolbachia mediated mechanisms of neutrophil recruitment to the cornea, this study also showed that expression of the adhesion molecule PECAM-1 in limbal vessels, as well as upregulation of the CXC chemokines KC and MIP-2 were dependent on the presence of functional TLR4 and Wolbachia respectively. Conclusions: This study indicates that the innate immune system and Wolbachia endobacteria play an important role in the inflammatory response associated with the pathogenesis of onchocerca keratitis, suggesting a complete alteration in our understanding of the immunopathology of filariasis.}, subject = {Onchozerkose}, language = {en} } @phdthesis{Gelmedin2008, author = {Gelmedin, Verena Magdalena}, title = {Targeting flatworm signaling cascades for the development of novel anthelminthic drugs}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-33334}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2008}, abstract = {Echinococcus multilocularis verursacht die Alveol{\"a}re Echinokokkose (AE), eine lebendsbedrohliche Krankheit mit limitierten chemotherapeutischen M{\"o}glichkeiten. Die jetzige Anti-AE Chemotherapie basiert auf einer einzigen Wirkstoffklasse, den Benzimidazolen. Obwohl Benzimidazole in vitro parasitozid wirken, wirken sie in vivo bei AE-Behandlung lediglich parasitostatisch und rufen schwere Nebenwirkungen hervor. In F{\"a}llen operabler L{\"a}sionen erfordert die Resektion des Parasitengewebes {\"u}ber einen l{\"a}ngeren Zeitraum eine chemotherapeutische Unterst{\"u}tzung. Damit sind die jetzigen Behandlungsm{\"o}glichkeiten inad{\"a}quat und ben{\"o}tigen Alternativen. In der vorliegenden Arbeit wurden die Signalwege von Plattw{\"u}rmern analysiert, um potentielle Targets f{\"u}r neue therapeutische Ans{\"a}tze zu identifizieren. Dabei konzentrierte ich mich unter Anwendung von molekularbiologischer, biochemischer und zellbiologischer Methoden auf Faktoren, die an Entwicklung und Proliferation von E. multilocularis beteiligt sind. Darunter waren die drei MAP kinases des Parasiten EmMPK1, ein Erk1/2-Ortholog, EmMPK2, ein p38-Ortholog und EmMPK3, ein Erk7/8-Ortholog. Des Weiteren identifizierte und charakterisierte ich EmMKK2, ein MEK1/2-Ortholog des Parasiten, welches zusammen mit den bekannten Kinasen EmRaf und EmMPK1 ein Erk1/2-{\"a}hnliches MAPK Modul bildet. Ich konnte zudem verschiedene Einfl{\"u}sse von Wirtswachstumsfaktoren wie EGF (epidermal growth factor) und Insulin auf die Signalmechanismen des Parasiten und das Larvenwachstum zeigen, darunter die Phosphorylierung von Elp, ein Ezrin-Radixin-Moesin {\"a}hnliches Protein, die Aktivierung von EmMPK1 und EmMPK3 und eine gesteigerte mitotische Aktivit{\"a}t der Echinokokkenzellen. Zus{\"a}tzlich wurden verschiedene Substanzen auf ihre letale Wirkung auf den Parasiten untersucht, darunter befanden sich (1.) generelle Inhibitoren von Tyrosinkinasen (PP2, Leflunamid), (2.) gegen die Aktivit{\"a}t von Rezeptor-Tyrosin-Kinasen gerichtete Pr{\"a}parate, (3.) urspr{\"u}nglich anti-neoplastische Wirkstoffe wie Miltefosin und Perifosin, (4.) Inhibitoren von Serin/ Threonin-Kinasen, die die Erk1/2 MAPK Kaskade blockieren und (5.) Inhibitoren der p38 MAPK. In diesen Untersuchungen hat sich EmMPK2 aus den folgenden Gr{\"u}nden als vielversprechendes Target erwiesen. Aminos{\"a}uresequenz-Analysen offenbarten einige Unterschiede zu menschlichen p38 MAP Kinasen, welche sehr wahrscheinlich die beobachtete gesteigerte basale Aktivit{\"a}t des rekombinanten EmMPK2 verursachen, verglichen mit der Aktivit{\"a}t humaner p38 MAPK-\&\#945;. Zus{\"a}tzlich suggerieren die prominente Autophosphorylierungsaktivit{\"a}t von rekombinantem EmMPK2 und das Ausbleiben einer Interaktion mit den Echinococcus MKKs einen unterschiedlichen Regulierungsmechanismus im Vergleich zu den humanen Proteinen. Die Aktivit{\"a}t von EmMPK2 konnte sowohl in vitro als auch in kultivierten Metazestodenvesikeln durch die Behandlung mit SB202190 und ML3403, zwei ATP kompetitiven Pyridinylimidazolinhibitoren der p38 MAPK, in Konzentrations-abh{\"a}ngiger Weise inhibiert werden. Zudem verursachten beide Substanzen, insbesondere ML3403 die Inaktivierung von Parasitenvesikeln bei Konzentrationen, die kultivierte S{\"a}ugerzellen nicht beeintr{\"a}chtigten. Ebenso verhinderte die Anwesenheit von ML3403 die Generation von neuen Vesikeln w{\"a}hrend der Kultivierung von Echinococcus Prim{\"a}rzellen. Das Targeting von Mitgliedern des EGF-Signalwegs, insbesondere der Erk1/2-{\"a}hnlichen MAPK Kaskade mit Raf- und MEK- Inhibitoren verhinderte die Phosphorylierung von EmMPK1 in in vitro kultivierten Metazestoden. Obwohl das Parasitenwachstum unter diesen Konditionen verhindert wurde, blieb die strukturelle Integrit{\"a}t der Metazestodenvesikeln w{\"a}hrend der Langzeitkultivierung in Anwesenheit der MAPK Kaskade-Inhibitoren erhalten. {\"A}hnliche Effekte wurden beobachtet nach Behandlung mit den anderen zuvor aufgef{\"u}hrten Inhibitoren. Zusammenfassend l{\"a}sst sich festhalten, dass verschiedene Targets identifiziert werden konnten, die hoch sensibel auf die Anwesenheit der inhibitorischen Substanzen reagierten, aber nicht zum Absterben des Parasiten f{\"u}hrten, mit Ausnahme der Pyridinylimidazolen. Die vorliegenden Daten zeigen, dass EmMPK2 ein {\"U}berlebendsignal vermittelnden Faktor darstellt und dessen Inhibierung zur Behandlung der AE benutzt werden k{\"o}nnte. Dabei erwiesen sich p38 MAPK Inhibitoren der Pyridinylimidazolklasse als potentielle neue Substanzklasse gegen Echinokokken.}, subject = {Fuchsbandwurm}, language = {en} } @article{SchubertUnkmeirKonradSlaninaetal.2010, author = {Schubert-Unkmeir, Alexandra and Konrad, Christian and Slanina, Heiko and Czapek, Florian and Hebling, Sabrina and Frosch, Matthias}, title = {Neisseria meningitidis Induces Brain Microvascular Endothelial Cell Detachment from the Matrix and Cleavage of Occludin: A Role for MMP-8}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68589}, year = {2010}, abstract = {Disruption of the blood-brain barrier (BBB) is a hallmark event in the pathophysiology of bacterial meningitis. Several inflammatory mediators, such as tumor necrosis factor alpha (TNF-a), nitric oxide and matrix metalloproteinases (MMPs), contribute to this disruption. Here we show that infection of human brain microvascular endothelial cells (HBMEC) with Neisseria meningitidis induced an increase of permeability at prolonged time of infection. This was paralleled by an increase in MMP-8 activity in supernatants collected from infected cells. A detailed analysis revealed that MMP-8 was involved in the proteolytic cleavage of the tight junction protein occludin, resulting in its disappearance from the cell periphery and cleavage to a lower-sized 50-kDa protein in infected HBMEC. Abrogation of MMP-8 activity by specific inhibitors as well as transfection with MMP-8 siRNA abolished production of the cleavage fragment and occludin remained attached to the cell periphery. In addition, MMP-8 affected cell adherence to the underlying matrix. A similar temporal relationship was observed for MMP activity and cell detachment. Injury of the HBMEC monolayer suggested the requirement of direct cell contact because no detachment was observed when bacteria were placed above a transwell membrane or when bacterial supernatant was directly added to cells. Inhibition of MMP-8 partially prevented detachment of infected HBMEC and restored BBB permeability. Together, we established that MMP-8 activity plays a crucial role in disassembly of cell junction components and cell adhesion during meningococcal infection.}, subject = {Neisseria meningitidis}, language = {en} } @article{EliasSchoulsvandePoletal.2010, author = {Elias, Johannes and Schouls, Leo M. and van de Pol, Ingrid and Keijzers, Wendy C. and Martin, Diana R. and Glennie, Anne and Oster, Philipp and Frosch, Matthias and Vogel, Ulrich and van der Ende, Arie}, title = {Vaccine Preventability of Meningococcal Clone, Greater Aachen Region, Germany}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68083}, year = {2010}, abstract = {No abstract available}, subject = {IMD}, language = {en} } @article{BeckMorbachBeeretal.2010, author = {Beck, Christine and Morbach, Henner and Beer, Meinrad and Stenzel, Martin and Tappe, Dennis and Gattenl{\"o}hner, Stefan and Hofmann, Ulrich and Raab, Peter and Girschick, Hermann J.}, title = {Chronic nonbacterial osteomyelitis in childhood: prospective follow-up during the first year of anti-inflammatory treatment}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-67792}, year = {2010}, abstract = {Introduction: Chronic nonbacterial osteomyelitis (CNO) is an inflammatory disorder of unknown etiology. In children and adolescents CNO predominantly affects the metaphyses of the long bones, but lesions can occur at any site of the skeleton. Prospectively followed cohorts using a standardized protocol in diagnosis and treatment have rarely been reported. Methods: Thirty-seven children diagnosed with CNO were treated with naproxen continuously for the first 6 months. If assessment at that time revealed progressive disease or no further improvement, sulfasalazine and short-term corticosteroids were added. The aims of our short-term follow-up study were to describe treatment response in detail and to identify potential risk factors for an unfavorable outcome. Results: Naproxen treatment was highly effective in general, inducing a symptom-free status in 43\% of our patients after 6 months. However, four nonsteroidal anti-inflammatory drug (NSAID) partial-responders were additionally treated with sulfasalazine and short-term corticosteroids. The total number of clinical detectable lesions was significantly reduced. Mean disease activity estimated by the patient/physician and the physical aspect of health-related quality of life including functional ability (global assessment/childhood health assessment questionnaire and childhood health assessment questionnaire) and pain improved significantly. Forty-one percent of our patients showed radiological relapses, but 67\% of them were clinically silent. Conclusions: Most children show a favorable clinical course in the first year of anti-inflammatory treatment with NSAIDs. Relapses and new radiological lesions can occur at any time and at any site in the skeleton but may not be clinically symptomatic. Whole-body magnetic resonance imaging proved to be very sensitive for initial and follow-up diagnostics.}, subject = {Mikrobiologie}, language = {en} } @phdthesis{Schielke2010, author = {Schielke, Stephanie}, title = {Functional and molecular characterization of FarR - a transcriptional regulator of the MarR family in Neisseria meningitidis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-48550}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Neisseria meningitidis is a facultatively pathogenic human commensal and strictly adapted to its niche within the human host, the nasopharynx. Not much is known about the regulatory processes required for adaptation to this environment. Therefore the role of the transcriptional regulator NMB1843, one of the two predicted regulators of the MarR family in the meningococcal genome, was investigated. As this gene displayed a high sequence homology to FarR, the Fatty acid resistance Regulator in N. gonorrhoeae, we designated the meningococcal protein FarR (NmFarR). Homology modeling of this protein revealed a dimeric structure with the characteristic winged helix-turn-helix DNA binding motif of the MarR family. NmFarR is highly conserved among meningococcal strains and expression of farR during exponential growth is controlled post-transcriptionally, being highest in the late exponential phase. By means of electrophoretic mobility shift assays (EMSAs) the direct and specific binding of FarR to the farAB promoter region was shown, comparable to its homologue in gonococci. As FarR is involved in fatty acid resistance in N. gonorrhoeae, susceptibility assays with the medium chain lauric acid (C12:0), the long chain saturated palmitic acid (C16:0) and the long chain unsaturated linoleic acid (C18:2) were performed, testing a wide variety of strains of both species. In contrast to the unusually susceptible gonococci, a high intrinsic fatty acid resistance was detected in almost all meningococcal isolates. The molecular basis for this intrinsic resistance in N. meningitidis was elucidated, showing that both a functional FarAB efflux pump system as well as an intact lipopolysaccharide (LPS) are responsible for palmitic acid resistance. However, even despite circumvention of the intrinsic resistance, FarR could not be connected with fatty acid resistance in meningococci. Instead, FarR was shown to directly and specifically repress expression of the Neisseria adhesin A (nadA), a promising vaccine candidate absent in N. gonorrhoeae. Microarray analyses verified these results and disclosed no further similarly regulated genes, rendering the FarR regulon the smallest regulon in meningococci reported until now. The exact FarR binding site within the nadA promoter region was identified as a 16 bp palindromic repeat and its influence on nadA transcription was proved by reporter gene fusion assays. This repression was also shown to be relevant for infection as farR deficient mutant strains displayed an increased attachment to epithelial cells. Furthermore, farR transcription was attested to be repressed upon contact with active complement components within human serum. Concluding, it is shown that FarR adopted a role in meningococcal host niche adaptation, holding the balance between immune evasion by repressing the highly antigenic nadA and host cell attachment via this same adhesin.}, subject = {Transkription }, language = {en} } @article{NellBurgkartGradletal.2011, author = {Nell, Manuel and Burgkart, Rainer H. and Gradl, Guntmar and von Eisenhart-Rothe, R{\"u}diger and Schaeffeler, Christoph and Trappe, Dennis and Prazeres da Costa, Clarissa and Gradinger, Reiner and Kirchhoff, Chlodwig}, title = {Primary extrahepatic alveolar echinococcosis of the lumbar spine and the psoas muscle}, series = {Annals of Clinical Microbiology and Antimicrobials}, volume = {10}, journal = {Annals of Clinical Microbiology and Antimicrobials}, number = {13}, doi = {10.1186/1476-0711-10-13}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-141796}, pages = {1-6}, year = {2011}, abstract = {Alveolar echinococcosis (AE) of human being caused by Echinococcus multilocularis is a rare but important zoonosis especially in tempered zones of middle Europe and Northern America with endemic character in many countries. Due to the long incubation period, various clinical manifestations, critical prognosis, and outcome AE presents a serious and severe disease. The primary focus of infection is usually the liver. Although secondary affection of visceral organs is possible extrahepatic AE is highly uncommon. Moreover, the involvement of bone and muscle presents with an even lower incidence. In the literature numerous cases on hepatic AE have been reported. However, extrahepatic AE involving bones and/or muscles was described very rarely. We report a case of an 80-year-old man with primary extrahepatic alveolar Echinococcosis of the lumbar spine and the psoas muscle. The etiology, diagnosis, differential diagnoses, treatment options and outcome of this rare disease are discussed in context with the current literature.}, language = {en} } @article{TappeMeyerOesterleinetal.2011, author = {Tappe, Dennis and Meyer, Michael and Oesterlein, Anett and Jaye, Assan and Frosch, Matthias and Schoen, Christoph and Pantchev, Nikola}, title = {Transmission of Armillifer armillatus Ova at Snake Farm, The Gambia, West Africa}, series = {Emerging Infectious Diseases}, volume = {17}, journal = {Emerging Infectious Diseases}, number = {2}, doi = {10.3201/eid1702.101118}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142804}, pages = {251-254}, year = {2011}, abstract = {Visceral pentastomiasis caused by Armillifer armillatus larvae was diagnosed in 2 dogs in The Gambia. Parasites were subjected to PCR; phylogenetic analysis confirmed relatedness with branchiurans/crustaceans. Our investigation highlights transmission of infective A. armillatus ova to dogs and, by serologic evidence, also to 1 human, demonstrating a public health concern.}, language = {en} } @article{HeisigFrentzenBergmannetal.2011, author = {Heisig, Martin and Frentzen, Alexa and Bergmann, Birgit and Gentschev, Katharina Ivaylo and Hotz, Christian and Schoen, Christoph and Stritzker, Jochen and Fensterle, Joachim and Rapp, Ulf R. and Goebel, Werner}, title = {Specific antibody-receptor interactions trigger InlAB-independent uptake of Listeria monocytogenes into tumor cell lines}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68705}, year = {2011}, abstract = {Background: Specific cell targeting is an important, yet unsolved problem in bacteria-based therapeutic applications, like tumor or gene therapy. Here, we describe the construction of a novel, internalin A and B (InlAB)-deficient Listeria monocytogenes strain (Lm-spa+), which expresses protein A of Staphylococcus aureus (SPA) and anchors SPA in the correct orientation on the bacterial cell surface. Results: This listerial strain efficiently binds antibodies allowing specific interaction of the bacterium with the target recognized by the antibody. Binding of Trastuzumab (Herceptin®) or Cetuximab (Erbitux®) to Lm-spa+, two clinically approved monoclonal antibodies directed against HER2/neu and EGFR/HER1, respectively, triggers InlABindependent internalization into non-phagocytic cancer cell lines overexpressing the respective receptors. Internalization, subsequent escape into the host cell cytosol and intracellular replication of these bacteria are as efficient as of the corresponding InlAB-positive, SPA-negative parental strain. This specific antibody/receptormediated internalization of Lm-spa+ is shown in the murine 4T1 tumor cell line, the isogenic 4T1-HER2 cell line as well as the human cancer cell lines SK-BR-3 and SK-OV-3. Importantly, this targeting approach is applicable in a xenograft mouse tumor model after crosslinking the antibody to SPA on the listerial cell surface. Conclusions: Binding of receptor-specific antibodies to SPA-expressing L. monocytogenes may represent a promising approach to target L. monocytogenes to host cells expressing specific receptors triggering internalization.}, subject = {Listeria monocytogenes}, language = {en} } @article{BijuSchwarzLinkeetal.2011, author = {Biju, Joseph and Schwarz, Roland and Linke, Burkhard and Blom, Jochen and Becker, Anke and Claus, Heike and Goesmann, Alexander and Frosch, Matthias and M{\"u}ller, Tobias and Vogel, Ulrich and Schoen, Christoph}, title = {Virulence Evolution of the Human Pathogen Neisseria meningitidis by Recombination in the Core and Accessory Genome}, series = {PLoS One}, volume = {6}, journal = {PLoS One}, number = {4}, doi = {10.1371/journal.pone.0018441}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-137960}, pages = {e18441}, year = {2011}, abstract = {Background Neisseria meningitidis is a naturally transformable, facultative pathogen colonizing the human nasopharynx. Here, we analyze on a genome-wide level the impact of recombination on gene-complement diversity and virulence evolution in N. meningitidis. We combined comparative genome hybridization using microarrays (mCGH) and multilocus sequence typing (MLST) of 29 meningococcal isolates with computational comparison of a subset of seven meningococcal genome sequences. Principal Findings We found that lateral gene transfer of minimal mobile elements as well as prophages are major forces shaping meningococcal population structure. Extensive gene content comparison revealed novel associations of virulence with genetic elements besides the recently discovered meningococcal disease associated (MDA) island. In particular, we identified an association of virulence with a recently described canonical genomic island termed IHT-E and a differential distribution of genes encoding RTX toxin- and two-partner secretion systems among hyperinvasive and non-hyperinvasive lineages. By computationally screening also the core genome for signs of recombination, we provided evidence that about 40\% of the meningococcal core genes are affected by recombination primarily within metabolic genes as well as genes involved in DNA replication and repair. By comparison with the results of previous mCGH studies, our data indicated that genetic structuring as revealed by mCGH is stable over time and highly similar for isolates from different geographic origins. Conclusions Recombination comprising lateral transfer of entire genes as well as homologous intragenic recombination has a profound impact on meningococcal population structure and genome composition. Our data support the hypothesis that meningococcal virulence is polygenic in nature and that differences in metabolism might contribute to virulence.}, language = {en} } @phdthesis{Hemer2012, author = {Hemer, Sarah}, title = {Molecular characterization of evolutionarily conserved signaling systems of Echinococcus multilocularis and their utilization for the development of novel drugs against Echinococosis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-74007}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Alveolar echinococcosis (AE), a severe and life-threatening disease is caused by the small fox tapeworm Echinococcus multilocularis. Currently, the options of chemotherapeutic treatment are very limited and are based on benzimidazole compounds, which act merely parasitostatic in vivo and often display strong side effects. Therefore, new therapeutic drugs and targets are urgently needed. In the present work the role of two evolutionarily conserved signalling pathways in E. multilocularis, namely the insulin signalling cascade and Abl kinases, has been studied in regard to host-parasite interaction and the possible use in anti-AE chemotherapy.}, subject = {Fuchsbandwurm}, language = {en} } @article{MaidenFrosch2012, author = {Maiden, Martin C. J. and Frosch, Matthias}, title = {Can we, should we, eradicate the meningococcus?}, series = {Vaccine}, volume = {30}, journal = {Vaccine}, number = {Suppl. 2}, doi = {10.1016/j.vaccine.2011.12.068}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-125646}, pages = {B52-B56}, year = {2012}, abstract = {The eradication of infectious agents is an attractive means of disease control that, to date, has been achieved for only one human pathogen, the smallpox virus. The introduction of vaccines against Neisseria meningitidis into immunisation schedules, and particularly the conjugate polysaccharide vaccines which can interrupt transmission, raises the question of whether disease caused by this obligate human bacterium can be controlled, eliminated, or even eradicated. The limited number of meningococcal serogroups, lack of an animal reservoir, and importance of meningococcal disease are considerations in favour of eradication; however, the commensal nature of most infections, the high diversity of meningococcal populations, and the lack of comprehensive vaccines are all factors that suggest that this is not feasible. Indeed, any such attempt might be harmful by perturbing the human microbiome and its interaction with the immune system. On balance, the control and possible elimination of disease caused by particular disease-associated meningococcal genotypes is a more achievable and worthwhile goal.}, language = {en} } @article{BarthHerrmannTappeetal.2012, author = {Barth, Thomas F. E. and Herrmann, Tobias S. and Tappe, Dennis and Stark, Lorenz and Gr{\"u}ner, Beate and Buttenschoen, Klaus and Hillenbrand, Andreas and Juchems, Markus and Henne-Bruns, Doris and Kern, Petra and Seitz, Hanns M. and M{\"o}ller, Peter and Rausch, Robert L. and Kern, Peter and Deplazes, Peter}, title = {Sensitive and Specific Immunohistochemical Diagnosis of Human Alveolar Echinococcosis with the Monoclonal Antibody Em2G11}, series = {PLoS Neglected Tropical Diseases}, volume = {6}, journal = {PLoS Neglected Tropical Diseases}, number = {10}, doi = {10.1371/journal.pntd.0001877}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-135371}, pages = {e1877}, year = {2012}, abstract = {Background: Alveolar echinococcosis (AE) is caused by the metacestode stage of Echinococcus multilocularis. Differential diagnosis with cystic echinococcosis (CE) caused by E. granulosus and AE is challenging. We aimed at improving diagnosis of AE on paraffin sections of infected human tissue by immunohistochemical testing of a specific antibody. Methodology/Principal Findings: We have analysed 96 paraffin archived specimens, including 6 cutting needle biopsies and 3 fine needle aspirates, from patients with suspected AE or CE with the monoclonal antibody (mAb) Em2G11 specific for the Em2 antigen of E. multilocularis metacestodes. In human tissue, staining with mAb Em2G11 is highly specific for E. multilocularis metacestodes while no staining is detected in CE lesions. In addition, the antibody detects small particles of E. multilocularis (spems) of less than 1 mm outside the main lesion in necrotic tissue, liver sinusoids and lymphatic tissue most probably caused by shedding of parasitic material. The conventional histological diagnosis based on haematoxylin and eosin and PAS stainings were in accordance with the immunohistological diagnosis using mAb Em2G11 in 90 of 96 samples. In 6 samples conventional subtype diagnosis of echinococcosis had to be adjusted when revised by immunohistology with mAb Em2G11. Conclusions/Significance: Immunohistochemistry with the mAb Em2G11 is a new, highly specific and sensitive diagnostic tool for AE. The staining of small particles of E. multilocularis (spems) outside the main lesion including immunocompetent tissue, such as lymph nodes, suggests a systemic effect on the host.}, language = {en} } @article{HubertPawlikClausetal.2012, author = {Hubert, Kerstin and Pawlik, Marie-Christin and Claus, Heike and Jarva, Hanna and Meri, Seppo and Vogel, Ulrich}, title = {Opc Expression, LPS Immunotype Switch and Pilin Conversion Contribute to Serum Resistance of Unencapsulated Meningococci}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {9}, doi = {10.1371/journal.pone.0045132}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-135421}, pages = {e45132}, year = {2012}, abstract = {Neisseria meningitidis employs polysaccharides and outer membrane proteins to cope with human serum complement attack. To screen for factors influencing serum resistance, an assay was developed based on a colorimetric serum bactericidal assay. The screening used a genetically modified sequence type (ST)-41/44 clonal complex (cc) strain lacking LPS sialylation, polysaccharide capsule, the factor H binding protein (fHbp) and MutS, a protein of the DNA repair mechanism. After killing of >99.9\% of the bacterial cells by serum treatment, the colorimetric assay was used to screen 1000 colonies, of which 35 showed enhanced serum resistance. Three mutant classes were identified. In the first class of mutants, enhanced expression of Opc was identified. Opc expression was associated with vitronectin binding and reduced membrane attack complex deposition confirming recent observations. Lipopolysaccharide (LPS) immunotype switch from immunotype L3 to L8/L1 by lgtA and lgtC phase variation represented the second class. Isogenic mutant analysis demonstrated that in ST-41/44 cc strains the L8/L1 immunotype was more serum resistant than the L3 immunotype. Consecutive analysis revealed that the immunotypes L8 and L1 were frequently observed in ST-41/44 cc isolates from both carriage and disease. Immunotype switch to L8/L1 is therefore suggested to contribute to the adaptive capacity of this meningococcal lineage. The third mutant class displayed a pilE allelic exchange associated with enhanced autoaggregation. The mutation of the C terminal hypervariable region D of PilE included a residue previously associated with increased pilus bundle formation. We suggest that autoaggregation reduced the surface area accessible to serum complement and protected from killing. The study highlights the ability of meningococci to adapt to environmental stress by phase variation and intrachromosomal recombination affecting subcapsular antigens.}, language = {en} } @article{SchwerkPapandreouSchuhmannetal.2012, author = {Schwerk, Christian and Papandreou, Thalia and Schuhmann, Daniel and Nickol, Laura and Borkowski, Julia and Steinmann, Ulrike and Quednau, Natascha and Stump, Carolin and Weiss, Christel and Berger, J{\"u}rgen and Wolburg, Hartwig and Claus, Heike and Vogel, Ulrich and Ishikawa, Hiroshi and Tenenbaum, Tobias and Schroten, Horst}, title = {Polar Invasion and Translocation of Neisseria meningitidis and Streptococcus suis in a Novel Human Model of the Blood-Cerebrospinal Fluid Barrier}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {1}, doi = {10.1371/journal.pone.0030069}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131459}, pages = {e30069}, year = {2012}, abstract = {Acute bacterial meningitis is a life-threatening disease in humans. Discussed as entry sites for pathogens into the brain are the blood-brain and the blood-cerebrospinal fluid barrier (BCSFB). Although human brain microvascular endothelial cells (HBMEC) constitute a well established human in vitro model for the blood-brain barrier, until now no reliable human system presenting the BCSFB has been developed. Here, we describe for the first time a functional human BCSFB model based on human choroid plexus papilloma cells (HIBCPP), which display typical hallmarks of a BCSFB as the expression of junctional proteins and formation of tight junctions, a high electrical resistance and minimal levels of macromolecular flux when grown on transwell filters. Importantly, when challenged with the zoonotic pathogen Streptococcus suis or the human pathogenic bacterium Neisseria meningitidis the HIBCPP show polar bacterial invasion only from the physiologically relevant basolateral side. Meningococcal invasion is attenuated by the presence of a capsule and translocated N. meningitidis form microcolonies on the apical side of HIBCPP opposite of sites of entry. As a functionally relevant human model of the BCSFB the HIBCPP offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens.}, language = {en} } @article{MoremiMshanaKamugishaetal.2012, author = {Moremi, Nyambura and Mshana, Stephen E. and Kamugisha, Erasmus and Kataraihya, Johannes B. and Tappe, Dennis and Vogel, Ulrich and Lyamuya, Eligius F. and Claus, Heike}, title = {Predominance of methicillin resistant Staphylococcus aureus-ST88 and new ST1797 causing wound infection and abscesses}, series = {Journal of Infection in Developing Countries}, volume = {6}, journal = {Journal of Infection in Developing Countries}, number = {8}, doi = {10.3855/jidc.2093}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134746}, pages = {620-625}, year = {2012}, abstract = {Introduction: Although there has been a worldwide emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA), little is known about the molecular epidemiology of MRSA in Tanzania. Methodology: In this study, we characterized MRSA strains isolated from clinical specimens at the Bugando Medical Centre, Tanzania, between January and December 2008. Of 160 S. aureus isolates from 600 clinical specimens, 24 (15\%) were found to be MRSA. Besides molecular screening for the Panton Valentine leukocidin (PVL) genes by PCR, MRSA strains were further characterized by Multi-Locus Sequence Typing (MLST) and spa typing. Results: Despite considerable genetic diversity, the spa types t690 (29.1\%) and t7231 (41.6\%), as well as the sequence types (ST) 88 (54.2\%) and 1797 (29.1\%), were dominant among clinical isolates. The PVL genes were detected in 4 isolates; of these, 3 were found in ST 88 and one in ST1820. Resistance to erythromycin, clindamicin, gentamicin, tetracycline and co-trimoxazole was found in 45.8\%, 62.5\%, 41.6\%, 45.8\% and 50\% of the strains, respectively. Conclusion: We present the first thorough typing of MRSA at a Tanzanian hospital. Despite considerable genetic diversity, ST88 was dominant among clinical isolates at the Bugando Medical Centre. Active and standardized surveillance of nosocomial MRSA infection should be conducted in the future to analyse the infection and transmission rates and implement effective control measures.}, language = {en} } @article{NonoPletinckxLutzetal.2012, author = {Nono, Justin Komguep and Pletinckx, Katrien and Lutz, Manfred B. and Brehm, Klaus}, title = {Excretory/Secretory-Products of Echinococcus multilocularis Larvae Induce Apoptosis and Tolerogenic Properties in Dendritic Cells In Vitro}, series = {PLoS Neglected Tropical Diseases}, volume = {6}, journal = {PLoS Neglected Tropical Diseases}, number = {2}, doi = {10.1371/journal.pntd.0001516}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134280}, pages = {e1516}, year = {2012}, abstract = {Background: Alveolar echinococcosis, caused by Echinococcus multilocularis larvae, is a chronic disease associated with considerable modulation of the host immune response. Dendritic cells (DC) are key effectors in shaping the immune response and among the first cells encountered by the parasite during an infection. Although it is assumed that E. multilocularis, by excretory/secretory (E/S)-products, specifically affects DC to deviate immune responses, little information is available on the molecular nature of respective E/S-products and their mode of action. Methodology/Principal Findings: We established cultivation systems for exposing DC to live material from early (oncosphere), chronic (metacestode) and late (protoscolex) infectious stages. When co-incubated with Echinococcus primary cells, representing the invading oncosphere, or metacestode vesicles, a significant proportion of DC underwent apoptosis and the surviving DC failed to mature. In contrast, DC exposed to protoscoleces upregulated maturation markers and did not undergo apoptosis. After pre-incubation with primary cells and metacestode vesicles, DC showed a strongly impaired ability to be activated by the TLR ligand LPS, which was not observed in DC pre-treated with protoscolex E/S-products. While none of the larvae induced the secretion of pro-inflammatory IL-12p70, the production of immunosuppressive IL-10 was elevated in response to primary cell E/S-products. Finally, upon incubation with DC and naive T-cells, E/S-products from metacestode vesicles led to a significant expansion of Foxp3+ T cells in vitro. Conclusions: This is the first report on the induction of apoptosis in DC by cestode E/S-products. Our data indicate that the early infective stage of E. multilocularis is a strong inducer of tolerance in DC, which is most probably important for generating an immunosuppressive environment at an infection phase in which the parasite is highly vulnerable to host attacks. The induction of CD4+CD25+Foxp3+ T cells through metacestode E/S-products suggests that these cells fulfill an important role for parasite persistence during chronic echinococcosis.}, language = {en} } @phdthesis{Nono2012, author = {Nono, Justin}, title = {Immunomodulation through Excretory/Secretory Products of the parasitic Helminth Echinococcus multilocularis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85449}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Die Alveol{\"a}re Echinokokkose (AE) ist eine lebensbedrohliche Zoonose, die durch das Metazestoden-Larvenstadium des Fuchsbandwurms Echinococcus multilocularis ausgel{\"o}st wird. Nach Eintritt des Parasiten in den Zwischenwirt wird zun{\"a}chst eine potentiell anti-parasitische, Th1-dominierte Immunantwort ausgel{\"o}st, welche anschließend in der chronischen Phase graduell durch eine permissive, Th2-dominierte Antwort ersetzt wird. Als Ergebnis einer zugrunde liegenden Immunmodulation durch den Parasiten k{\"o}nnen Echinococcus-Larven f{\"u}r Jahre bis Jahrzehnte im Wirt persistieren und verhalten sich {\"a}hnlich einem perfekt transplantierten Organ. {\"U}ber die molekulare Basis der Immunmodulation durch den Parasiten ist derzeit wenig bekannt. In dieser Arbeit wurden geeignete Kultursysteme f{\"u}r verschiedene E. multilocularis Larvenstadien verwendet, um den Einfluss exkretorisch/sekretorischer Metaboliten (E/S-Produkte) auf Wirts-Immuneffektor-Zellen zu studieren. E/S-Produkte kultivierter Larven, die die fr{\"u}he (Prim{\"a}rzellen) und chronische (Metazestode) Phase der Infektion repr{\"a}sentieren induzierten Apoptose und tolerogene Eigenschaften in Dendritischen Zellen (DC) des Wirts, w{\"a}hrend solche von Kontroll-Larven (Protoskolizes) keine derartigen Effekte zeigten. Dies zeigt, dass die fr{\"u}hen infekti{\"o}sen Stadien von E. multilocularis in DC ein tolerierendes Milieu erzeugen, welches sehr wahrscheinlich die initiale Etablierung des Parasiten in einer Phase beg{\"u}nstigt, in der er h{\"o}chst sensitiv gegen{\"u}ber Wirtsangriffen ist. Interessanterweise f{\"o}rderten E/S-Produkte des Metazestoden in vitro die Konversion von CD4+ T-Zellen in Foxp3+, regulatorische T-Zellen (Treg) w{\"a}hrend E/S-Produkte von Prim{\"a}rzellen oder Protoskolizes dies nicht vermochten. Da Foxp3+ Tregs generell als immunosuppressorisch bekannt sind, deuten diese Daten an, dass der Metazestode aktiv eine Induktion von Tregs herbeif{\"u}hrt, um eine permissive Immunsuppression w{\"a}hrend einer Infektion zu erreichen. Eine substantielle Zunahme von Anzahl und Frequenz Foxp3+ Tregs konnte zudem in Peritoneal-Exsudaten von M{\"a}uuen nach intraperitonealer Injektion von Parasitengewebe gemessen werden, was anzeigt, dass eine Expansion von Foxp3+ Tregs auch w{\"a}hrend der in vivo Infektion von Bedeutung ist. Interessanterweise konnte in dieser Arbeit ein Activin-Orthologes des Parasiten, EmACT, identifiziert werden, weleches vom Metazestoden sekretiert wird und {\"a}hnlich wie humanes Activin in der Lage ist, eine TGF-β-abh{\"a}ngige Expansion von Tregs in vitro zu induzieren. Dies zeigt an, dass E. multilocularis evolutionsgeschichtlich konservierte Zytokine nutzt, um aktiv die Wirts-Immunantwort zu beeinflussen. Zusammenfassend deuten die gewonnenen Daten auf eine wichtige Rolle Foxp3+ Tregs, welche u.a. durch EmACT induziert werden, im immunologischen geschehen der AE hin. Ein weiterer Parasiten-Faktor, EmTIP, mit signifikanten Homologien zum T-cell Immunomodulatory Protein (TIP) des Menschen wurde in dieser Arbeit n{\"a}her charakterisiert. EmTIP konnte in der E/S-Fraktion von Prim{\"a}rzellen nachgewiesen werden und induzierte die Freisetzung von IFN-γ in CD4+ T-Helferzellen. Durch Zugabe von anti-EmTIP-Antik{\"o}rpern konnte zudem die Entwicklung des Parasiten zum Metazestoden in vitro gehemmt werden. EmTIP d{\"u}rfte daher einerseits bei der fr{\"u}hen Parasiten-Entwicklung im Zwischenwirt eine Rolle spielen und k{\"o}nnte im Zuge dessen auch die Auspr{\"a}gung der fr{\"u}hen, Th-1-dominierten Immunantwort w{\"a}hrend der AE beg{\"u}nstigen. Zusammenfassend wurden in dieser Arbeit zwei E. multilocularis E/S-Faktoren identifiziert, EmACT und EmTIP, die ein hohes immunmodulatorisches Potential besitzen. Die hier vorgestellten Daten liefern neue, fundamentale Einsichten in die molekularen Mechanismen der Parasiten-induzierten Immunmodulation bei der AE und sind hoch relevant f{\"u}r die Entwicklung anti-parasitischer Immuntherapien.}, subject = {Immunmodulation}, language = {en} } @article{SlaninaHeblingHaucketal.2012, author = {Slanina, Heiko and Hebling, Sabrina and Hauck, Christoph R. and Schubert-Unkmeir, Alexandra}, title = {Cell Invasion by Neisseria meningitidis Requires a Functional Interplay between the Focal Adhesion Kinase, Src and Cortactin}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75354}, year = {2012}, abstract = {Entry of Neisseria meningitidis (the meningococcus) into human brain microvascular endothelial cells (HBMEC) is mediated by fibronectin or vitronectin bound to the surface protein Opc forming a bridge to the respective integrins. This interaction leads to cytoskeletal rearrangement and uptake of meningococci. In this study, we determined that the focal adhesion kinase (FAK), which directly associates with integrins, is involved in integrin-mediated internalization of N. meningitidis in HBMEC. Inhibition of FAK activity by the specific FAK inhibitor PF 573882 reduced Opc-mediated invasion of HBMEC more than 90\%. Moreover, overexpression of FAK mutants that were either impaired in the kinase activity or were not capable of autophosphorylation or overexpression of the dominant-negative version of FAK (FRNK) blocked integrin-mediated internalization of N. meningitidis. Importantly, FAK-deficient fibroblasts were significantly less invaded by N. meningitidis. Furthermore, N. meningitidis induced tyrosine phosphorylation of several host proteins including the FAK/Src complex substrate cortactin. Inhibition of cortactin expression by siRNA silencing and mutation of critical amino acid residues within cortactin, that encompass Arp2/3 association and dynamin binding, significantly reduced meningococcal invasion into eukaryotic cells suggesting that both domains are critical for efficient uptake of N. meningitidis into eukaryotic cells. Together, these results indicate that N. meningitidis exploits the integrin signal pathway for its entry and that FAK mediates the transfer of signals from activated integrins to the cytoskeleton. A cooperative interplay between FAK, Src and cortactin then enables endocytosis of N. meningitidis into host cells.}, subject = {Medizin}, language = {en} } @article{GeyerChalmersMacKintoshetal.2013, author = {Geyer, Kathrin K. and Chalmers, Iain W. and MacKintosh, Neil and Hirst, Julie E. and Geoghegan, Rory and Badets, Mathieu and Brophy, Peter M. and Brehm, Klaus and Hoffmann, Karl F.}, title = {Cytosine methylation is a conserved epigenetic feature found throughout the phylum Platyhelminthes}, series = {BMC Genomics}, volume = {14}, journal = {BMC Genomics}, number = {462}, issn = {1471-2164}, doi = {10.1186/1471-2164-14-462}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121892}, year = {2013}, abstract = {Background: The phylum Platyhelminthes (flatworms) contains an important group of bilaterian organisms responsible for many debilitating and chronic infectious diseases of human and animal populations inhabiting the planet today. In addition to their biomedical and veterinary relevance, some platyhelminths are also frequently used models for understanding tissue regeneration and stem cell biology. Therefore, the molecular (genetic and epigenetic) characteristics that underlie trophic specialism, pathogenicity or developmental maturation are likely to be pivotal in our continued studies of this important metazoan group. Indeed, in contrast to earlier studies that failed to detect evidence of cytosine or adenine methylation in parasitic flatworm taxa, our laboratory has recently defined a critical role for cytosine methylation in Schistosoma mansoni oviposition, egg maturation and ovarian development. Thus, in order to identify whether this epigenetic modification features in other platyhelminth species or is a novelty of S. mansoni, we conducted a study simultaneously surveying for DNA methylation machinery components and DNA methylation marks throughout the phylum using both parasitic and non-parasitic representatives. Results: Firstly, using both S. mansoni DNA methyltransferase 2 (SmDNMT2) and methyl-CpG binding domain protein (SmMBD) as query sequences, we illustrate that essential DNA methylation machinery components are well conserved throughout the phylum. Secondly, using both molecular (methylation specific amplification polymorphism, MSAP) and immunological (enzyme-linked immunoabsorbent assay, ELISA) methodologies, we demonstrate that representative species (Echinococcus multilocularis, Protopolystoma xenopodis, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica and Polycelis nigra) within all four platyhelminth classes (Cestoda, Monogenea, Trematoda and 'Turbellaria') contain methylated cytosines within their genome compartments. Conclusions: Collectively, these findings provide the first direct evidence for a functionally conserved and enzymatically active DNA methylation system throughout the Platyhelminthes. Defining how this epigenetic feature shapes phenotypic diversity and development within the phylum represents an exciting new area of metazoan biology.}, language = {en} } @article{AtanasovBenkertThelenetal.2013, author = {Atanasov, Georgi and Benkert, Christoph and Thelen, Armin and Tappe, Dennis and Frosch, Matthias and Teichmann, Dieter and Barth, Thomas F. E. and Wittekind, Christian and Schubert, Stefan and Jonas, Sven}, title = {Alveolar echinococcosis-spreading disease challenging clinicians: A case report and literature review}, series = {World Journal of Gastroenterology}, volume = {19}, journal = {World Journal of Gastroenterology}, number = {26}, doi = {10.3748/wjg.v19.i26.4257}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131525}, pages = {4257-4261}, year = {2013}, abstract = {Human alveolar echinococcosis (AE) is a potentially deadly disease; recent studies have shown that the endemic area of Echinococcus multilocularis, its causative agent, is larger than previously known. This disease has low prevalence and remains underreported in Europe. Emerging clinical data show that diagnostic difficulties are still common. We report on a 76-year old patient suffering from AE lesions restricted to the left lobe of the liver who underwent a curative extended left hemihepatectomy. Prior to the resection a liver biopsy under the suspicion of an atypical malignancy was performed. After the intervention he developed a pseudoaneurysm of the hepatic artery that was successfully coiled. Surprisingly, during surgery, the macroscopic appearance of the tumour revealed a growth pattern that was rather typical for cystic echinococcosis (CE), i.e., a gross tumour composed of multiple large vesicles with several centimeters in diameter. In addition, there were neither extensive adhesions nor infiltrations of the neighboring pancreas and diaphragm as was expected from previous imaging results. The unexpected diagnosis of AE was confirmed by definite histopathology, specific polymerase chain reaction and serology results. This is a rare case of unusual macroscopic presentation of AE that posed immense diagnostic challenges and had an eventful course. To our knowledge this is the first case of an autochthonous infection in this particular geographic area of Germany, the federal state of Saxony. This report may provide new hints for an expanding area of risk for AE and emphasizes the risk of complications in the scope of diagnostic procedures and the limitations of modern radiological imaging.}, language = {en} } @article{HarrisonClausJiangetal.2013, author = {Harrison, Odile B. and Claus, Heike and Jiang, Ying and Bennett, Julia S. and Bratcher, Holly B. and Jolley, Keith A. and Corton, Craig and Care, Rory and Poolman, Jan T. and Zollinger, Wendell D. and Frasch, Carl E. and Stephens, David S. and Feavers, Ian and Frosch, Matthias and Parkhill, Julian and Vogel, Ulrich and Quail, Michael A. and Bentley, Stephen D. and Maiden, Martin C. J.}, title = {Description and Nomenclature of Neisseria meningitidis Capsule Locus}, series = {Emerging Infectious Diseases}, volume = {19}, journal = {Emerging Infectious Diseases}, number = {4}, doi = {10.3201/eid1904.111799}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131703}, pages = {566-573}, year = {2013}, abstract = {Pathogenic Neisseria meningitidis isolates contain a polysaccharide capsule that is the main virulence determinant for this bacterium. Thirteen capsular polysaccharides have been described, and nuclear magnetic resonance spectroscopy has enabled determination of the structure of capsular polysaccharides responsible for serogroup specificity. Molecular mechanisms involved in N. meningitidis capsule biosynthesis have also been identified, and genes involved in this process and in cell surface translocation are clustered at a single chromosomal locus termed cps. The use of multiple names for some of the genes involved in capsule synthesis, combined with the need for rapid diagnosis of serogroups commonly associated with invasive meningococcal disease, prompted a requirement for a consistent approach to the nomenclature of capsule genes. In this report, a comprehensive description of all N. meningitidis serogroups is provided, along with a proposed nomenclature, which was presented at the 2012 XVIIIth International Pathogenic Neisseria Conference.}, language = {en} } @article{MarcusVogelSchubertetal.2013, author = {Marcus, U. and Vogel, U. and Schubert, A. and Claus, H. and Baetzing-Feigenbaum, J. and Hellenbrand, W. and Wichmann, O.}, title = {A cluster of invasive meningococcal disease in young men who have sex with men in Berlin, October 2012 to May 2013}, series = {Eurosurveillance}, volume = {18}, journal = {Eurosurveillance}, number = {28}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131711}, pages = {6-8}, year = {2013}, abstract = {No abstract available.}, language = {en} } @article{TappeHaeuplerSchaeferetal.2013, author = {Tappe, Dennis and Haeupler, Alexandra and Sch{\"a}fer, Hansj{\"o}rg and Racz, Paul and Cramer, Jakob P. and Poppert, Sven}, title = {Armillifer armillatus Pentastomiasis in African Immigrant, Germany}, series = {Emerging Infectious Diseases}, volume = {19}, journal = {Emerging Infectious Diseases}, number = {3}, doi = {10.3201/eid1903.121508}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-129054}, pages = {507-508}, year = {2013}, abstract = {No abstract available.}, language = {en} } @article{FraunholzBernhardtSchuldesetal.2013, author = {Fraunholz, Martin and Bernhardt, J{\"o}rg and Schuldes, J{\"o}rg and Daniel, Rolf and Hecker, Michael and Sinh, Bhanu}, title = {Complete Genome Sequence of Staphylococcus aureus 6850, a Highly Cytotoxic and Clinically Virulent Methicillin-Sensitive Strain with Distant Relatedness to Prototype Strains}, series = {Genome Announcements}, volume = {1}, journal = {Genome Announcements}, number = {5}, doi = {10.1128/genomeA.00775-13}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-129294}, pages = {e00775-13}, year = {2013}, abstract = {Staphylococcus aureus is a frequent human commensal bacterium and pathogen. Here we report the complete genome sequence of strain 6850 (spa type t185; sequence type 50 [ST50]), a highly cytotoxic and clinically virulent methicillin-sensitive strain from a patient with complicated S. aureus bacteremia associated with osteomyelitis and septic arthritis.}, language = {en} } @article{BrehmKoziolKrohne2013, author = {Brehm, Klaus and Koziol, Uriel and Krohne, Georg}, title = {Anatomy and development of the larval nervous system in Echinococcus multilocularis}, series = {Frontiers in Zoology}, journal = {Frontiers in Zoology}, doi = {10.1186/1742-9994-10-24}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-96504}, year = {2013}, abstract = {Background The metacestode larva of Echinococcus multilocularis (Cestoda: Taeniidae) develops in the liver of intermediate hosts (typically rodents, or accidentally in humans) as a labyrinth of interconnected cysts that infiltrate the host tissue, causing the disease alveolar echinococcosis. Within the cysts, protoscoleces (the infective stage for the definitive canid host) arise by asexual multiplication. These consist of a scolex similar to that of the adult, invaginated within a small posterior body. Despite the importance of alveolar echinococcosis for human health, relatively little is known about the basic biology, anatomy and development of E. multilocularis larvae, particularly with regard to their nervous system. Results We describe the existence of a subtegumental nerve net in the metacestode cysts, which is immunoreactive for acetylated tubulin-α and contains small populations of nerve cells that are labeled by antibodies raised against several invertebrate neuropeptides. However, no evidence was found for the existence of cholinergic or serotoninergic elements in the cyst wall. Muscle fibers occur without any specific arrangement in the subtegumental layer, and accumulate during the invaginations of the cyst wall that form brood capsules, where protoscoleces develop. The nervous system of the protoscolex develops independently of that of the metacestode cyst, with an antero-posterior developmental gradient. The combination of antibodies against several nervous system markers resulted in a detailed description of the protoscolex nervous system, which is remarkably complex and already similar to that of the adult worm. Conclusions We provide evidence for the first time of the existence of a nervous system in the metacestode cyst wall, which is remarkable given the lack of motility of this larval stage, and the lack of serotoninergic and cholinergic elements. We propose that it could function as a neuroendocrine system, derived from the nervous system present in the bladder tissue of other taeniids. The detailed description of the development and anatomy of the protoscolex neuromuscular system is a necessary first step toward the understanding of the developmental mechanisms operating in these peculiar larval stages.}, language = {en} } @phdthesis{Pawlik2013, author = {Pawlik, Marie-Christin}, title = {Gene expression in the human pathogen Neisseria meningitidis: Adaptation to serum exposure and zinc limitation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-78758}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Neisseria meningitidis is a facultative human pathogen that occasionally shows strong resistance against serum complement exposure. Previously described factors that mediate meningococcal serum resistance are for example the capsule, LPS sialylation, and expression of the factor H binding protein. I aimed for identification of novel serum resistance factors, thereby following two approaches, i) the analysis of the impact of global regulators of gene expression on serum resistance; and ii) a comparative analysis of closely related strains differing in serum resistance. (i) Of six meningococcal global regulators of gene expression studied, only mutation of the zinc uptake regulator Zur reduced complement deposition on meningococci. Little was known about meningococcal Zur and regulatory processes in response to zinc. I therefore elucidated the yet unidentified meningococcal Zur regulon comparing the transcriptional response of the N. meningitidis strain MC58 under zinc-rich and zinc-deficient conditions using a common reference design of microarray analysis. The meningococcal Zur regulon comprises 17 genes, of which 15 genes were repressed and two genes were activated at high zinc condition. Amongst the Zur-repressed genes were genes involved in zinc uptake, tRNA modification, and ribosomal assembly. A 23 bp meningococcal consensus Zur binding motif (Zur box) with a conserved central palindrome was established (TGTTATDNHATAACA) and detected in the promoter region of all regulated transcriptional units (genes/operons). In vitro binding of meningococcal Zur to the Zur box of three selected genes was shown for the first time using EMSAs. Binding of meningococcal Zur to DNA depended specifically on zinc, and mutations in the palindromic sequence constrained Zur binding to the DNA motif. ii) Three closely related strains of ST-41/44 cc from invasive disease and carriage which differed in their resistance to serum complement exposure were analysed to identify novel mediators of serum resistance. I compared the strains' gene content by microarray analysis which revealed six genes being present in both carrier isolates, but absent in the invasive isolate. Four of them are part of two Islands of horizontally transferred DNA, i.e. IHT-B and -C. The working group furthermore applied a comprehensive screening assay, a transcriptome and a proteome analysis leading to identification of three target proteins. I contributed to establish the role of these three proteins in serum resistance: The adhesin Opc mediates serum resistance by binding of vitronectin, a negative regulator of the complement system; the hypothetical protein NMB0865 slightly contributes to serum resistance by a yet unknown mechanism; and NspA, recently identified to bind the negative complement regulator factor H, led to considerable reduced complement-mediated killing.}, subject = {Komplement }, language = {en} } @article{EliasHeuschmannSchmittetal.2013, author = {Elias, Johannes and Heuschmann, Peter U. and Schmitt, Corinna and Eckhardt, Frithjof and Boehm, Hartmut and Maier, Sebastian and Kolb-M{\"a}urer, Annette and Riedmiller, Hubertus and M{\"u}llges, Wolfgang and Weisser, Christoph and Wunder, Christian and Frosch, Matthias and Vogel, Ulrich}, title = {Prevalence dependent calibration of a predictive model for nasal carriage of methicillin-resistant Staphylococcus aureus}, series = {BMC Infectious Diseases}, journal = {BMC Infectious Diseases}, doi = {10.1186/1471-2334-13-111}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-96091}, year = {2013}, abstract = {Background Published models predicting nasal colonization with Methicillin-resistant Staphylococcus aureus among hospital admissions predominantly focus on separation of carriers from non-carriers and are frequently evaluated using measures of discrimination. In contrast, accurate estimation of carriage probability, which may inform decisions regarding treatment and infection control, is rarely assessed. Furthermore, no published models adjust for MRSA prevalence. Methods Using logistic regression, a scoring system (values from 0 to 200) predicting nasal carriage of MRSA was created using a derivation cohort of 3091 individuals admitted to a European tertiary referral center between July 2007 and March 2008. The expected positive predictive value of a rapid diagnostic test (GeneOhm, Becton \& Dickinson Co.) was modeled using non-linear regression according to score. Models were validated on a second cohort from the same hospital consisting of 2043 patients admitted between August 2008 and January 2012. Our suggested correction score for prevalence was proportional to the log-transformed odds ratio between cohorts. Calibration before and after correction, i.e. accurate classification into arbitrary strata, was assessed with the Hosmer-Lemeshow-Test. Results Treating culture as reference, the rapid diagnostic test had positive predictive values of 64.8\% and 54.0\% in derivation and internal validation corhorts with prevalences of 2.3\% and 1.7\%, respectively. In addition to low prevalence, low positive predictive values were due to high proportion (> 66\%) of mecA-negative Staphylococcus aureus among false positive results. Age, nursing home residence, admission through the medical emergency department, and ICD-10-GM admission diagnoses starting with "A" or "J" were associated with MRSA carriage and were thus included in the scoring system, which showed good calibration in predicting probability of carriage and the rapid diagnostic test's expected positive predictive value. Calibration for both probability of carriage and expected positive predictive value in the internal validation cohort was improved by applying the correction score. Conclusions Given a set of patient parameters, the presented models accurately predict a) probability of nasal carriage of MRSA and b) a rapid diagnostic test's expected positive predictive value. While the former can inform decisions regarding empiric antibiotic treatment and infection control, the latter can influence choice of screening method.}, language = {en} } @article{EliasFindlowBorrowetal.2013, author = {Elias, Johannes and Findlow, Jamie and Borrow, Ray and Tremmel, Angelika and Frosch, Matthias and Vogel, Ulrich}, title = {Persistence of antibodies in laboratory staff immunized with quadrivalent meningococcal polysaccharide vaccine}, series = {Journal of Occupational Medicine and Toxicology}, journal = {Journal of Occupational Medicine and Toxicology}, doi = {10.1186/1745-6673-8-4}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-95953}, year = {2013}, abstract = {Background Occupational exposure to live meningococci can potentially cause invasive meningococcal disease in laboratory staff. While, until recently, immunization with quadrivalent polysaccharide vaccine represented one cornerstone of protection, data on long-term persistence of antibodies in adults remain scarce. Methods We analyzed the relationship of antibody levels and time following quadrivalent polysaccharide vaccination (Mencevax® ACWY, GlaxoSmithKline) in a cross-sectional sample of 20 laboratory workers vaccinated at ages between 16.4 to 40.7 years from Germany. Sera were obtained 0.4 to 158.5 (median 35.3) months after vaccination. At the time of sampling, laboratory workers had been regularly exposed to meningococci for periods between 3.2 to 163.8 (median 41.2) months. Serum bactericidal assay (SBA) with rabbit complement and a microsphere-based flow analysis method were used to determine bactericidal titers and concentrations of IgG, respectively, against serogroups A, C, W135, and Y. Decay of antibodies was modeled using linear regression. Protective levels were defined as SBA titers ≥ 8. Results Half-lives of SBA titers against serogroups A, C, W135, and Y were estimated at 27.4, 21.9, 18.8, and 28.0 months, respectively. Average durations of protection were estimated at 183.9, 182.0, 114.6, and 216.4 months, respectively. Inter-individual variation was high; using lower margins of 95\% prediction intervals, minimal durations of protection against serogroups A, C, W135 and Y were estimated at 33.5, 24.6, 0.0, and 55.1 months, respectively. The proportion of staff with protective SBA titers against W135 (65.0\%) was significantly lower than proportions protected against A (95.0\%), C (94.7\%), and Y (95.0\%). Consistently, geometric mean titer (97.0) and geometric mean concentration of IgG (2.1 μg/ml) was lowest against serogroup W135. SBA titers in a subset of individuals with incomplete protection rose to ≥ 128 (≥ 8 fold) after reimmunization with a quadrivalent glycoconjugate vaccine. Conclusions The average duration of protection following immunization with a quadrivalent polysaccharide vaccine in adults was ≥ 115 months regardless of serogroup. A substantial proportion (approximately 23\% according to our decay model) of adult vaccinees may not retain protection against serogroup W135 for five years, the time suggested for reimmunization.}, language = {en} } @phdthesis{Pasquet2014, author = {Pasquet, Vivian}, title = {Characterization of thioredoxin and glutathione reductase activities of Mesocestoides vogae, a flatworm parasite useful as a laboratory model for the screening of drugs.}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-106759}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {Flatworm parasites (platyhelminths) cause serious infection diseases in humans, such as schistosomiasis and hydatid disease, mainly prevalent in developing countries. However, the current repertoire of drug armamentarium used to combat flatworm infections is limited. For instance, praziquantel is the only drug available for mass treatment of Schistosoma infections. In contrast to their hosts, flatworm parasites possess a distinct redox arrangement of redox pathways in which the selenoenzyme thioredoxin glutathione reductase (TGR) controls the overall redox homeostasis. Interference with this enzyme leads to parasite death. Hence, this key redox enzyme seems to be a new promising drug target against flatworm infections. Because most flatworms are difficult to cultivate in the laboratory (e.g. Echinococcus granulosus experimental infection in mice takes about 10 month to develop into cysts), this work was focused on Mesocestoides vogae (syn. corti), a non-human flatworm parasite which is an interesting laboratory model to study other flatworm infections: it is very rare in humans, can be easily manipulated both in vivo and in vitro and grows extremely fast in mice. With the aim to assess TGR inhibitors as possible drugs to treat flatworm infections, the thioredoxin and glutathione pathways of M.vogae were studied. Here, the objectives were to study whether the biochemical pathways that maintain the redox homeostasis in M. vogae conform to the general biochemical scenario proposed for other platyhelminth parasites. Here, it was proven that M. vogae extracts possess both thioredoxin and glutathione reductase activities. The thioredoxin and glutathione reductase activities were partially purified from total extracts by a combination of ammonium sulfate precipitation, anion exchange and hydroxyapatite chromatography. Both activities co-purified in all steps which strongly indicates the existence of TGR rather than a single TR and GR. Furthermore partially purified activities could be inhibited by the organogold compound auranofin, a known TGR inhibitor. Moreover, the glutathione reductase activity displays hysteresis (a peculiar kinetic behavior) at high concentrations of oxidised glutathione, a feature typical of flatworm TGRs, but not of conventional GR. Although M. vogae activities could not be purified to homogeneity, the overall results strongly indicate that this flatworm possesses TGR and lacks conventional GR and TR. Furthermore the thiadiazole WPQ75 and the N-oxide VL16E (a furoxan derivate) were identified as inhibitors of TGR activity of M.vogae at a 10 µM concentration. These inhibitors were able to kill M.vogae larval worms in vitro as well as in experimental infection in mice. Due to the existence of TGR activity in M.vogae, the possibility to inhibit this activity with recently discovered inhibitors of flatworm TGR and the successes achieved by testing these inhibitors both in vitro and in vivo, it is strongly evident that M. vogae would be an excellent model to assess TGR inhibitors in flatworm infections.}, subject = {Thioredoxin}, language = {en} } @article{BrehmKomguepNonoLutz2014, author = {Brehm, Klaus and Komguep Nono, Justin and Lutz, Manfred B.}, title = {EmTIP, a T-Cell Immunomodulatory Protein Secreted by the Tapeworm Echinococcus multilocularis Is Important}, doi = {10.1371/journal.pntd.0002632}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-111407}, year = {2014}, abstract = {Background Alveolar echinococcosis (AE), caused by the metacestode of the tapeworm Echinococcus multilocularis, is a lethal zoonosis associated with host immunomodulation. T helper cells are instrumental to control the disease in the host. Whereas Th1 cells can restrict parasite proliferation, Th2 immune responses are associated with parasite proliferation. Although the early phase of host colonization by E. multilocularis is dominated by a potentially parasitocidal Th1 immune response, the molecular basis of this response is unknown. Principal Findings We describe EmTIP, an E. multilocularis homologue of the human T-cell immunomodulatory protein, TIP. By immunohistochemistry we show EmTIP localization to the intercellular space within parasite larvae. Immunoprecipitation and Western blot experiments revealed the presence of EmTIP in the excretory/secretory (E/S) products of parasite primary cell cultures, representing the early developing metacestode, but not in those of mature metacestode vesicles. Using an in vitro T-cell stimulation assay, we found that primary cell E/S products promoted interferon (IFN)-γ release by murine CD4+ T-cells, whereas metacestode E/S products did not. IFN-γ release by T-cells exposed to parasite products was abrogated by an anti-EmTIP antibody. When recombinantly expressed, EmTIP promoted IFN-γ release by CD4+ T-cells in vitro. After incubation with anti-EmTIP antibody, primary cells showed an impaired ability to proliferate and to form metacestode vesicles in vitro. Conclusions We provide for the first time a possible explanation for the early Th1 response observed during E. multilocularis infections. Our data indicate that parasite primary cells release a T-cell immunomodulatory protein, EmTIP, capable of promoting IFN-γ release by CD4+ T-cells, which is probably driving or supporting the onset of the early Th1 response during AE. The impairment of primary cell proliferation and the inhibition of metacestode vesicle formation by anti-EmTIP antibodies suggest that this factor fulfills an important role in early E. multilocularis development within the intermediate host.}, language = {en} } @article{DeogratiasMushiPaternoetal.2014, author = {Deogratias, Anna-Pendo and Mushi, Martha F. and Paterno, Laurent and Tappe, Dennis and Seni, Jeremiah and Kabymera, Rogatus and Kidenya, Benson R. and Mshana, Stephen E.}, title = {Prevalence and determinants of Campylobacter infection among under five children with acute watery diarrhea in Mwanza, North Tanzania}, series = {Archives of Public Health}, volume = {72}, journal = {Archives of Public Health}, number = {17}, doi = {10.1186/2049-3258-72-17}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-120749}, year = {2014}, abstract = {BACKGROUND: Campylobacteriosis, a zoonotic bacterial disease observed world-wide, is becoming the most commonly recognized cause of bacterial gastroenteritis in humans. This study was done to determine the prevalence and determinants of Campylobacter infection among under-fives with acute watery diarrhea in Mwanza City, Tanzania. METHOD: This cross-sectional hospital-based study was conducted at Bugando Medical Centre (BMC) and Sekou Toure Hospital in Mwanza City. All inpatients and outpatients under-fives who met the inclusion criteria from October 2012 to April 2013 were enrolled in the study. Demographic and clinical data were obtained using standardized data collection tools. Stool samples were collected for gram staining and culture for Campylobacter spp. on Preston selective agar media. In addition, blood slides for malaria and HIV tests were done to all patients. RESULTS: A total of 300 children were enrolled with a median age of 12 [interquartile range, 8-19] months. Of these, 169 (56.5\%) were from BMC and 131 (43.7\%) from Sekou-Toure hospital. One hundred and seventy (56.7\%) of the participating children were male. Of 300 under-fives with acute watery diarrhea, 29 patients (9.7\%) were found to have Campylobacter infection. A significant higher number of children with Campylobacter infection were found in Sekou Toure hospital compared to BMC [16.0\% (21/29) versus 4.7\% (8/29), p = 0.002)]. Age above 2 years was independently found to predict campylobacter infection (OR: 2.9, 95\% CI 1.1-7.7, p = 0.0037). Of 30 patients with a positive blood slide for Plasmodium falciparum, 20.0\% were also positive for Campylobacter infection (OR: 3.9, 95\% CI 1.2-10.1, p = 0.021). CONCLUSION: Campylobacter infection shows a comparatively low prevalence in under-fives with acute watery diarrhea in Mwanza city and is independently associated with positive slides for malaria and an age above 2 years. Further studies are needed to type the most prevalent Campylobacter species and to determine their antibiotic susceptibility pattern.}, language = {en} } @article{BrehonyTrotterRamsayetal.2014, author = {Brehony, Carina and Trotter, Caronline L. and Ramsay, Mary E. and Chandra, Manosree and Jolley, Keith A. and van der Ende, Arie and Carion, Fran{\c{c}}oise and Berthelsen, Lene and Hoffmann, Steen and Harðard{\´o}ttir, Hj{\"o}rd{\´i}s and Vazques, Julio A. and Murphy, Karen and Toropainen, Maija and Cani{\c{c}}a, Manuela and Ferreira, Eugenia and Diggle, Mathew and Edwards, Giles F. and Taha, Muhamed-Kheir and Stefanelli, Paola and Kriz, Paula and Gray, Steve J. and Fox, Andrew J. and Jacobsson, Susanne and Claus, Heike and Vogel, Ulrich and Tzanakaki, Georgina and Heuberger, Sigrid and Caugant, Dominique A. and Frosch, Matthias and Maiden, Martin C. J.}, title = {Implications of Differential Age Distribution of Disease-Associated Meningococcal Lineages for Vaccine Development}, series = {Clinical and Vaccine Immunology : CVI}, volume = {21}, journal = {Clinical and Vaccine Immunology : CVI}, number = {6}, doi = {10.1128/cvi.00133-14}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-120808}, pages = {847-53}, year = {2014}, abstract = {New vaccines targeting meningococci expressing serogroup B polysaccharide have been developed, with some being licensed in Europe. Coverage depends on the distribution of disease-associated genotypes, which may vary by age. It is well established that a small number of hyperinvasive lineages account for most disease, and these lineages are associated with particular antigens, including vaccine candidates. A collection of 4,048 representative meningococcal disease isolates from 18 European countries, collected over a 3-year period, were characterized by multilocus sequence typing (MLST). Age data were available for 3,147 isolates. The proportions of hyperinvasive lineages, identified as particular clonal complexes (ccs) by MLST, differed among age groups. Subjects <1 year of age experienced lower risk of sequence type 11 (ST-11) cc, ST-32 cc, and ST-269 cc disease and higher risk of disease due to unassigned STs, 1- to 4-year-olds experienced lower risk of ST-11 cc and ST-32 cc disease, 5- to 14-year-olds were less likely to experience ST-11 cc and ST-269 cc disease, and ≥25-year-olds were more likely to experience disease due to less common ccs and unassigned STs. Younger and older subjects were vulnerable to a more diverse set of genotypes, indicating the more clonal nature of genotypes affecting adolescents and young adults. Knowledge of temporal and spatial diversity and the dynamics of meningococcal populations is essential for disease control by vaccines, as coverage is lineage specific. The nonrandom age distribution of hyperinvasive lineages has consequences for the design and implementation of vaccines, as different variants, or perhaps targets, may be required for different age groups.}, language = {en} } @article{LichteneggerBinaRoieretal.2014, author = {Lichtenegger, Sabine and Bina, Isabelle and Roier, Sandro and Bauernfeind, Stilla and Keidel, Kristina and Schild, Stefan and Anthony, Mark and Reidl, Joachim}, title = {Characterization of lactate utilization and its implication on the physiology of Haemophilus influenzae}, series = {International Journal of Medical Microbiology}, volume = {304}, journal = {International Journal of Medical Microbiology}, number = {3-4}, doi = {10.1016/j.ijmm.2014.02.010}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121335}, pages = {490-8}, year = {2014}, abstract = {Haemophilus influenzae is a Gram-negative bacillus and a frequent commensal of the human nasopharynx. Earlier work demonstrated that in H. influenzae type b, l-lactate metabolism is associated with serum resistance and in vivo survival of the organism. To further gain insight into lactate utilization of the non-typeable (NTHi) isolate 2019 and laboratory prototype strain Rd KW20, deletion mutants of the l-lactate dehydrogenase (lctD) and permease (lctP) were generated and characterized. It is shown, that the apparent KM of l-lactate uptake is 20.1μM as determined for strain Rd KW20. Comparison of the COPD isolate NTHi 2019-R with the corresponding lctP knockout strain for survival in human serum revealed no lactate dependent serum resistance. In contrast, we observed a 4-fold attenuation of the mutant strain in a murine model of nasopharyngeal colonization. Characterization of lctP transcriptional control shows that the lactate utilization system in H. influenzae is not an inductor inducible system. Rather negative feedback regulation was observed in the presence of l-lactate and this is dependent on the ArcAB regulatory system. Additionally, for 2019 it was found that lactate may have signaling function leading to increased cell growth in late log phase under conditions where no l-lactate is metabolized. This effect seems to be ArcA independent and was not observed in strain Rd KW20. We conclude that l-lactate is an important carbon-source and may act as host specific signal substrate which fine tunes the globally acting ArcAB regulon and may additionally affect a yet unknown signaling system and thus may contribute to enhanced in vivo survival.}, language = {en} } @article{BrehmKoziol2014, author = {Brehm, Klaus and Koziol, Uriel}, title = {On the importance of targeting parasite stem cells in anti-echinococcosis drug development}, series = {Parasite}, volume = {21}, journal = {Parasite}, issn = {1252-607X}, doi = {10.1051/parasite/2014070}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-118030}, pages = {72}, year = {2014}, abstract = {The life-threatening diseases alveolar and cystic echinococcoses are caused by larvae of the tapeworms Echinococcus multilocularis and E. granulosus, respectively. In both cases, intermediate hosts, such as humans, are infected by oral uptake of oncosphere larvae, followed by asexual multiplication and almost unrestricted growth of the metacestode within host organs. Besides surgery, echinococcosis treatment relies on benzimidazole-based chemotherapy, directed against parasite beta-tubulin. However, since beta-tubulins are highly similar between cestodes and humans, benzimidazoles can only be applied at parasitostatic doses and are associated with adverse side effects. Mostly aiming at identifying alternative drug targets, the nuclear genome sequences of E. multilocularis and E. granulosus have recently been characterized, revealing a large number of druggable targets that are expressed by the metacestode. Furthermore, recent cell biological investigations have demonstrated that E. multilocularis employs pluripotent stem cells, called germinative cells, which are the only parasite cells capable of proliferation and which give rise to all differentiated cells. Hence, the germinative cells are the crucial cell type mediating proliferation of E. multilocularis, and most likely also E. granulosus, within host organs and should also be responsible for parasite recurrence upon discontinuation of chemotherapy. Interestingly, recent investigations have also indicated that germinative cells might be less sensitive to chemotherapy because they express a beta-tubulin isoform with limited affinity to benzimidazoles. In this article, we briefly review the recent findings concerning Echinococcus genomics and stem cell research and propose that future research into anti-echinococcosis drugs should also focus on the parasite's stem cell population.}, language = {en} } @article{SchoenKischkiesEliasetal.2014, author = {Schoen, Christoph and Kischkies, Laura and Elias, Johannes and Ampattu, Biju Joseph}, title = {Metabolism and virulence in Neisseria meningitidis}, doi = {10.3389/fcimb.2014.00114}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-113118}, year = {2014}, abstract = {A longstanding question in infection biology addresses the genetic basis for invasive behavior in commensal pathogens. A prime example for such a pathogen is Neisseria meningitidis. On the one hand it is a harmless commensal bacterium exquisitely adapted to humans, and on the other hand it sometimes behaves like a ferocious pathogen causing potentially lethal disease such as sepsis and acute bacterial meningitis. Despite the lack of a classical repertoire of virulence genes in N. meningitidis separating commensal from invasive strains, molecular epidemiology suggests that carriage and invasive strains belong to genetically distinct populations. In recent years, it has become increasingly clear that metabolic adaptation enables meningococci to exploit host resources, supporting the concept of nutritional virulence as a crucial determinant of invasive capability. Here, we discuss the contribution of core metabolic pathways in the context of colonization and invasion with special emphasis on results from genome-wide surveys. The metabolism of lactate, the oxidative stress response, and, in particular, glutathione metabolism as well as the denitrification pathway provide examples of how meningococcal metabolism is intimately linked to pathogenesis. We further discuss evidence from genome-wide approaches regarding potential metabolic differences between strains from hyperinvasive and carriage lineages and present new data assessing in vitro growth differences of strains from these two populations. We hypothesize that strains from carriage and hyperinvasive lineages differ in the expression of regulatory genes involved particularly in stress responses and amino acid metabolism under infection conditions.}, language = {en} } @article{SchubertKoziolCailliauetal.2014, author = {Schubert, Andreas and Koziol, Uriel and Cailliau, Katia and Vanderstraete, Mathieu and Dissous, Colette and Brehm, Klaus}, title = {Targeting Echinococcus multilocularis Stem Cells by Inhibition of the Polo-Like Kinase EmPlk1}, doi = {10.1371/journal.pntd.0002870}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-112806}, year = {2014}, abstract = {Background Alveolar echinococcosis (AE) is a life-threatening disease caused by larvae of the fox-tapeworm Echinococcus multilocularis. Crucial to AE pathology is continuous infiltrative growth of the parasite's metacestode stage, which is driven by a population of somatic stem cells, called germinative cells. Current anti-AE chemotherapy using benzimidazoles is ineffective in eliminating the germinative cell population, thus leading to remission of parasite growth upon therapy discontinuation. Methodology/Principal findings We herein describe the characterization of EmPlk1, encoded by the gene emplk1, which displays significant homologies to members of the Plk1 sub-family of Polo-like kinases that regulate mitosis in eukaryotic cells. We demonstrate germinative cell-specific expression of emplk1 by RT-PCR, transcriptomics, and in situ hybridization. We also show that EmPlk1 can induce germinal vesicle breakdown when heterologously expressed in Xenopus oocytes, indicating that it is an active kinase. This activity was significantly suppressed in presence of BI 2536, a Plk1 inhibitor that has been tested in clinical trials against cancer. Addition of BI 2536 at concentrations as low as 20 nM significantly blocked the formation of metacestode vesicles from cultivated Echinococcus germinative cells. Furthermore, low concentrations of BI 2536 eliminated the germinative cell population from mature metacestode vesicles in vitro, yielding parasite tissue that was no longer capable of proliferation. Conclusions/Significance We conclude that BI 2536 effectively inactivates E. multilocularis germinative cells in parasite larvae in vitro by direct inhibition of EmPlk1, thus inducing mitotic arrest and germinative cell killing. Since germinative cells are decisive for parasite proliferation and metastasis formation within the host, BI 2536 and related compounds are very promising compounds to complement benzimidazoles in AE chemotherapy. Author Summary The lethal disease AE is characterized by continuous and infiltrative growth of the metacestode larva of the tapeworm E. multilocularis within host organs. This cancer-like progression is exclusively driven by a population of parasite stem cells (germinative cells) that have to be eliminated for an effective cure of the disease. Current treatment options, using benzimidazoles, are parasitostatic only, and thus obviously not effective in germinative cell killing. We herein describe a novel, druggable parasite enzyme, EmPlk1, that specifically regulates germinative cell proliferation. We show that a compound, BI 2536, originally designed to inhibit the human ortholog of EmPlk1, can also inhibit the parasite protein at low doses. Furthermore, low doses of BI 2536 eliminated germinative cells from Echinococcus larvae in vitro and prevented parasite growth and development. We propose that BI 2536 and related compounds are promising drugs to complement current benzimidazole treatment for achieving parasite killing.}, language = {en} } @article{SchubertUnkmeirSchneiderSchauliesGulbinsetal.2014, author = {Schubert-Unkmeir, Alexandra and Schneider-Schaulies, Sibylle and Gulbins, Erich and Hebling, Sabrina and Simonis, Alexander}, title = {Differential Activation of Acid Sphingomyelinase and Ceramide Release Determines Invasiveness of Neisseria meningitidis into Brain Endothelial Cells}, doi = {10.1371/journal.ppat.1004160}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-113031}, year = {2014}, abstract = {The interaction with brain endothelial cells is central to the pathogenicity of Neisseria meningitidis infections. Here, we show that N. meningitidis causes transient activation of acid sphingomyelinase (ASM) followed by ceramide release in brain endothelial cells. In response to N. meningitidis infection, ASM and ceramide are displayed at the outer leaflet of the cell membrane and condense into large membrane platforms which also concentrate the ErbB2 receptor. The outer membrane protein Opc and phosphatidylcholine-specific phospholipase C that is activated upon binding of the pathogen to heparan sulfate proteoglycans, are required for N. meningitidis-mediated ASM activation. Pharmacologic or genetic ablation of ASM abrogated meningococcal internalization without affecting bacterial adherence. In accordance, the restricted invasiveness of a defined set of pathogenic isolates of the ST-11/ST-8 clonal complex into brain endothelial cells directly correlated with their restricted ability to induce ASM and ceramide release. In conclusion, ASM activation and ceramide release are essential for internalization of Opc-expressing meningococci into brain endothelial cells, and this segregates with invasiveness of N. meningitidis strains. Author Summary Neisseria meningitidis, an obligate human pathogen, is a causative agent of septicemia and meningitis worldwide. Meningococcal infection manifests in a variety of forms, including meningitis, meningococcemia with meningitis or meningococcemia without obvious meningitis. The interaction of N. meningitidis with human cells lining the blood vessels of the blood-cerebrospinal fluid barrier is a prerequisite for the development of meningitis. As a major pathogenicity factor, the meningococcal outer membrane protein Opc enhances bacterial entry into brain endothelial cells, however, mechanisms underlying trapping of receptors and signaling molecules following this interaction remained elusive. We now show that Opc-expressing meningococci activate acid sphingomyelinase (ASM) in brain endothelial cells, which hydrolyses sphingomyelin to cause ceramide release and formation of extended ceramide-enriched membrane platforms wherein ErbB2, an important receptor involved in bacterial uptake, clusters. Mechanistically, ASM activation relied on binding of N. meningitidis to its attachment receptor, HSPG, followed by activation of PC-PLC. Meningococcal isolates of the ST-11 clonal complex, which are reported to be more likely to cause severe sepsis, but rarely meningitis, barely invaded brain endothelial cells and revealed a highly restricted ability to induce ASM and ceramide release. Thus, our results unravel a differential activation of the ASM/ceramide system by the species N. meningitidis determining its invasiveness into brain endothelial cells.}, language = {en} } @article{BrehmKoziolRauschendorferetal.2014, author = {Brehm, Klaus and Koziol, Uriel and Rauschendorfer, Theresa and Rodr{\´i}guez, Luis Zanon and Krohne, Georg}, title = {The unique stem cell system of the immortal larva of the human parasite Echinococcus multilocularis}, doi = {10.1186/2041-9139-5-10}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-110315}, year = {2014}, abstract = {Background It is believed that in tapeworms a separate population of undifferentiated cells, the germinative cells, is the only source of cell proliferation throughout the life cycle (similar to the neoblasts of free living flatworms). In Echinococcus multilocularis, the metacestode larval stage has a unique development, growing continuously like a mass of vesicles that infiltrate the tissues of the intermediate host, generating multiple protoscoleces by asexual budding. This unique proliferation potential indicates the existence of stem cells that are totipotent and have the ability for extensive self-renewal. Results We show that only the germinative cells proliferate in the larval vesicles and in primary cell cultures that undergo complete vesicle regeneration, by using a combination of morphological criteria and by developing molecular markers of differentiated cell types. The germinative cells are homogeneous in morphology but heterogeneous at the molecular level, since only sub-populations express homologs of the post-transcriptional regulators nanos and argonaute. Important differences are observed between the expression patterns of selected neoblast marker genes of other flatworms and the E. multilocularis germinative cells, including widespread expression in E. multilocularis of some genes that are neoblast-specific in planarians. Hydroxyurea treatment results in the depletion of germinative cells in larval vesicles, and after recovery following hydroxyurea treatment, surviving proliferating cells grow as patches that suggest extensive self-renewal potential for individual germinative cells. Conclusions In E. multilocularis metacestodes, the germinative cells are the only proliferating cells, presumably driving the continuous growth of the larval vesicles. However, the existence of sub-populations of the germinative cells is strongly supported by our data. Although the germinative cells are very similar to the neoblasts of other flatworms in function and in undifferentiated morphology, their unique gene expression pattern and the evolutionary loss of conserved stem cells regulators suggest that important differences in their physiology exist, which could be related to the unique biology of E. multilocularis larvae.}, language = {en} } @article{BrehmHemerKonradetal.2014, author = {Brehm, Klaus and Hemer, Sarah and Konrad, Christian and Spiliotis, Markus and Koziol, Uriel and Schaack, Dominik and F{\"o}rster, Sabine and Gelmedin, Verena and Stadelmann, Britta and Dandekar, Thomas and Hemphill, Andrew}, title = {Host insulin stimulates Echinococcus multilocularis insulin signalling pathways and larval development}, doi = {10.1186/1741-7007-12-5}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-110357}, year = {2014}, abstract = {Background The metacestode of the tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a lethal zoonosis. Infections are initiated through establishment of parasite larvae within the intermediate host's liver, where high concentrations of insulin are present, followed by tumour-like growth of the metacestode in host organs. The molecular mechanisms determining the organ tropism of E. multilocularis or the influences of host hormones on parasite proliferation are poorly understood. Results Using in vitro cultivation systems for parasite larvae we show that physiological concentrations (10 nM) of human insulin significantly stimulate the formation of metacestode larvae from parasite stem cells and promote asexual growth of the metacestode. Addition of human insulin to parasite larvae led to increased glucose uptake and enhanced phosphorylation of Echinococcus insulin signalling components, including an insulin receptor-like kinase, EmIR1, for which we demonstrate predominant expression in the parasite's glycogen storage cells. We also characterized a second insulin receptor family member, EmIR2, and demonstrated interaction of its ligand binding domain with human insulin in the yeast two-hybrid system. Addition of an insulin receptor inhibitor resulted in metacestode killing, prevented metacestode development from parasite stem cells, and impaired the activation of insulin signalling pathways through host insulin. Conclusions Our data indicate that host insulin acts as a stimulant for parasite development within the host liver and that E. multilocularis senses the host hormone through an evolutionarily conserved insulin signalling pathway. Hormonal host-parasite cross-communication, facilitated by the relatively close phylogenetic relationship between E. multilocularis and its mammalian hosts, thus appears to be important in the pathology of alveolar echinococcosis. This contributes to a closer understanding of organ tropism and parasite persistence in larval cestode infections. Furthermore, our data show that Echinococcus insulin signalling pathways are promising targets for the development of novel drugs.}, language = {en} } @article{StijnisDijkmansBartetal.2015, author = {Stijnis, Kees and Dijkmans, Anneke C. and Bart, Aldert and Brosens, Lodewijk A. A. and Muntau, Birgit and Schoen, Christoph and Barth, Thomas F. and van Gulik, Thomas and van Gool, Tom and Grobusch, Martin P. and Tappe, Dennis}, title = {Echinococcus vogeli in Immigrant from Suriname to the Netherlands}, series = {Emerging Infectious Diseases}, volume = {21}, journal = {Emerging Infectious Diseases}, number = {3}, doi = {10.3201/eid2103.141205}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143953}, pages = {528-530}, year = {2015}, language = {en} } @article{KoziolRadioSmircichetal.2015, author = {Koziol, Uriel and Radio, Santiago and Smircich, Pablo and Zarowiecki, Magdalena and Fern{\´a}ndez, Cecilia and Brehm, Klaus}, title = {A novel terminal-repeat retrotransposon in miniature (TRIM) is massively expressed in Echinococcus multilocularis stem cells}, series = {Genome Biology and Evolution}, volume = {7}, journal = {Genome Biology and Evolution}, number = {8}, doi = {10.1093/gbe/evv126}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-148306}, pages = {2136-2153}, year = {2015}, abstract = {Taeniid cestodes (including the human parasites Echinococcus spp. and Taenia solium) have very few mobile genetic elements (MGEs) in their genome, despite lacking a canonical PIWI pathway. The MGEs of these parasites are virtually unexplored, and nothing is known about their expression and silencing. In this work, we report the discovery of a novel family of small nonautonomous long terminal repeat retrotransposons (also known as terminal-repeat retrotransposons in miniature, TRIMs) which we have named ta-TRIM (taeniid TRIM). ta-TRIMs are only the second family of TRIM elements discovered in animals, and are likely the result of convergent reductive evolution in different taxonomic groups. These elements originated at the base of the taeniid tree and have expanded during taeniid diversification, including after the divergence of closely related species such as Echinococcus multilocularis and Echinococcus granulosus. They are massively expressed in larval stages, from a small proportion of full-length copies and from isolated terminal repeats that show transcriptional read-through into downstream regions, generating novel noncoding RNAs and transcriptional fusions to coding genes. In E. multilocularis, ta-TRIMs are specifically expressed in the germinative cells (the somatic stem cells) during asexual reproduction of metacestode larvae. This would provide a developmental mechanism for insertion of ta-TRIMs into cells that will eventually generate the adult germ line. Future studies of active and inactive ta-TRIM elements could give the first clues on MGE silencing mechanisms in cestodes.}, language = {en} } @phdthesis{Herz2015, author = {Herz, Michaela}, title = {Molecular characterization of the serotonin and cAMP-signalling pathways in Echinococcus}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-139249}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {Alveolar and cystic echinococcosis, caused by Echinococcus multilocularis and Echinococcus granulosus respectively, are severe zoonotic diseases with limited treatment options. The sole curative treatment is the surgical removal of the complete parasite material. Due to late diagnosis, chemotherapeutic treatment often is the only treatment option. Treatment is based on benzimidazoles, which merely act parasitostatic and often display strong side effects. Therefore, new therapeutic drugs are urgently needed. Evolutionarily conserved signalling pathways are known to be involved in hostparasite cross-communication, parasite development and survival. Moreover, they represent potential targets for chemotherapeutic drugs. In this context the roles of the serotonin- and cAMP-signalling pathways in Echinococcus were studied. Genes encoding serotonin receptors, a serotonin transporter and enzymes involved in serotonin biosynthesis could be identified in the E. multilocularis and E. granulosus genomes indicating that these parasites are capable of synthesizing and perceiving serotonin signals. Also the influence of exogenous serotonin on parasite development was studied. Serotonin significantly increased metacestode vesicle formation from primary cells and re-differentiation of protoscoleces. Inhibition of serotonin transport with citalopram significantly reduced metacestode vesicle formation from primary cells and caused death of protoscoleces and metacestodes. Furthermore, it could be shown that serotonin increased phosphorylation of protein kinase A substrates. Taken together, these results show that serotonin and serotonin transport are essential for Echinococcus development and survival. Consequently, components of the serotonin pathway represent potential drug targets. In this work the cAMP-signalling pathway was researched with focus on G-protein coupled receptors and adenylate cyclases. 76 G-protein coupled receptors, including members of all major families were identified in the E. multilocularis genome. Four genes homologous to adenylate cyclase IX were identified in the E. multilocularis genome and three in the E. granulosus genome. While glucagon caused no significant effects, the adenylate cyclase activator forskolin and the adenylate cyclase inhibitor 2', 5' didesoxyadenosine influenced metacestode vesicle formation from primary cells, re-differentiation of protoscoleces and survival of metacestodes. It was further shown that forskolin increases phosphorylation of protein kinase A substrates, indicating that forskolin activates the cAMP-pathway also in cestodes. These results indicate that the cAMP signalling pathway plays an important role in Echinococcus development and survival. To complement this work, the influence of different media and additives on E. granulosus protoscoleces was investigated. Anaerobic conditions and the presence of FBS prolonged protoscolex survival while different media influenced protoscolex activation and development. Taken together, this work provided important insights into developmental processes in Echinococcus and potential drug targets for echinococcosis chemotherapy.}, subject = {Serotonin}, language = {en} } @article{VendelovadeLimaLorenzattoetal.2016, author = {Vendelova, Emilia and de Lima, Jeferson Camargo and Lorenzatto, Karina Rodrigues and Monteiro, Karina Mariante and Mueller, Thomas and Veepaschit, Jyotishman and Grimm, Clemens and Brehm, Klaus and Hrčkov{\´a}, Gabriela and Lutz, Manfred B. and Ferreira, Henrique B. and Nono, Justin Komguep}, title = {Proteomic Analysis of Excretory-Secretory Products of Mesocestoides corti Metacestodes Reveals Potential Suppressors of Dendritic Cell Functions}, series = {PLoS Neglected Tropical Diseases}, volume = {10}, journal = {PLoS Neglected Tropical Diseases}, number = {10}, doi = {10.1371/journal.pntd.0005061}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-166742}, pages = {e0005061}, year = {2016}, abstract = {Accumulating evidences have assigned a central role to parasite-derived proteins in immunomodulation. Here, we report on the proteomic identification and characterization of immunomodulatory excretory-secretory (ES) products from the metacestode larva (tetrathyridium) of the tapeworm Mesocestoides corti (syn. M. vogae). We demonstrate that ES products but not larval homogenates inhibit the stimuli-driven release of the pro-inflammatory, Th1-inducing cytokine IL-12p70 by murine bone marrow-derived dendritic cells (BMDCs). Within the ES fraction, we biochemically narrowed down the immunosuppressive activity to glycoproteins since active components were lipid-free, but sensitive to heat- and carbohydrate-treatment. Finally, using bioassay-guided chromatographic analyses assisted by comparative proteomics of active and inactive fractions of the ES products, we defined a comprehensive list of candidate proteins released by M. corti tetrathyridia as potential suppressors of DC functions. Our study provides a comprehensive library of somatic and ES products and highlight some candidate parasite factors that might drive the subversion of DC functions to facilitate the persistence of M. corti tetrathyridia in their hosts.}, language = {en} } @article{WalterCollenburgJaptoketal.2016, author = {Walter, T. and Collenburg, L. and Japtok, L. and Kleuser, B. and Schneider-Schaulies, S. and M{\"u}ller, N. and Becam, J. and Schubert-Unkmeir, A. and Kong, J. N. and Bieberich, E. and Seibel, J.}, title = {Incorporation and visualization of azido-functionalized N-oleoyl serinol in Jurkat cells, mouse brain astrocytes, 3T3 fibroblasts and human brain microvascular endothelial cells}, series = {Chemical Communications}, volume = {52}, journal = {Chemical Communications}, number = {55}, doi = {10.1039/c6cc02879a}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-191263}, pages = {8612-8614}, year = {2016}, abstract = {The synthesis and biological evaluation of azido-N-oleoyl serinol is reported. It mimicks biofunctional lipid ceramides and has shown to be capable of click reactions for cell membrane imaging in Jurkat and human brain microvascular endothelial cells.}, language = {en} } @article{KoziolJareroOlsonetal.2016, author = {Koziol, Uriel and Jarero, Francesca and Olson, Peter D. and Brehm, Klaus}, title = {Comparative analysis of Wnt expression identifies a highly conserved developmental transition in flatworms}, series = {BMC Biology}, volume = {14}, journal = {BMC Biology}, number = {10}, doi = {10.1186/s12915-016-0233-x}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146566}, year = {2016}, abstract = {Background Early developmental patterns of flatworms are extremely diverse and difficult to compare between distant groups. In parasitic flatworms, such as tapeworms, this is confounded by highly derived life cycles involving indirect development, and even the true orientation of the tapeworm antero-posterior (AP) axis has been a matter of controversy. In planarians, and metazoans generally, the AP axis is specified by the canonical Wnt pathway, and we hypothesized that it could also underpin axial formation during larval metamorphosis in tapeworms. Results By comparative gene expression analysis of Wnt components and conserved AP markers in the tapeworms Echinococcus multilocularis and Hymenolepis microstoma, we found remarkable similarities between the early stages of larval metamorphosis in tapeworms and late embryonic and adult development in planarians. We demonstrate posterior expression of specific Wnt factors during larval metamorphosis and show that scolex formation is preceded by localized expression of Wnt inhibitors. In the highly derived larval form of E. multilocularis, which proliferates asexually within the mammalian host, we found ubiquitous expression of posterior Wnt factors combined with localized expression of Wnt inhibitors that correlates with the asexual budding of scoleces. As in planarians, muscle cells are shown to be a source of secreted Wnt ligands, providing an explanation for the retention of a muscle layer in the immotile E. multilocularis larva. Conclusions The strong conservation of gene expression between larval metamorphosis in tapeworms and late embryonic development in planarians suggests, for the first time, a homologous developmental period across this diverse phylum. We postulate these to represent the phylotypic stages of these flatworm groups. Our results support the classical notion that the scolex is the true anterior end of tapeworms. Furthermore, the up-regulation of Wnt inhibitors during the specification of multiple anterior poles suggests a mechanism for the unique asexual reproduction of E. multilocularis larvae.}, language = {en} } @article{TahaClausLappannetal.2016, author = {Taha, Muhamed-Kheir and Claus, Heike and Lappann, Martin and Veyrier, Fr{\´e}d{\´e}ric J. and Otto, Andreas and Becher, D{\"o}rte and Deghmane, Ala-Eddine and Frosch, Matthias and Hellenbrand, Wiebke and Hong, Eva and du Ch{\^a}telet, Isabelle Parent and Prior, Karola and Harmsen, Dag and Vogel, Ulrich}, title = {Evolutionary Events Associated with an Outbreak of Meningococcal Disease in Men Who Have Sex with Men}, series = {PLoS ONE}, volume = {11}, journal = {PLoS ONE}, number = {5}, doi = {10.1371/journal.pone.0154047}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179870}, year = {2016}, abstract = {Meningococci spread via respiratory droplets, whereas the closely related gonococci are transmitted sexually. Several outbreaks of invasive meningococcal disease have been reported in Europe and the United States among men who have sex with men (MSM). We recently identified an outbreak of serogroup C meningococcal disease among MSM in Germany and France. In this study, genomic and proteomic techniques were used to analyze the outbreak isolates. In addition, genetically identical urethritis isolates were recovered from France and Germany and included in the analysis. Genome sequencing revealed that the isolates from the outbreak among MSM and from urethritis cases belonged to a clade within clonal complex 11. Proteome analysis showed they expressed nitrite reductase, enabling anaerobic growth as previously described for gonococci. Invasive isolates from MSM, but not urethritis isolates, further expressed functional human factor H binding protein associated with enhanced survival in a newly developed transgenic mouse model expressing human factor H, a complement regulatory protein. In conclusion, our data suggest that urethritis and outbreak isolates followed a joint adaptation route including adaption to the urogenital tract.}, language = {en} } @phdthesis{HagmanngebKischkies2016, author = {Hagmann [geb. Kischkies], Laura Violetta}, title = {Stringent response regulation and its impact on ex vivo survival in the commensal pathogen \(Neisseria\) \(meningitidis\)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144352}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {Neisseria meningitidis is a commensal bacterium which sometimes causes serious disease in humans. Recent studies in numerous human pathogenic bacteria have shown that the stringent response contributes to bacterial virulence. Therefore, this study analyzed the regulation of the stringent response in meningococci and in particular of RelA as well as its contribution to ex vivo fitness in a strain- and condition- dependent manner by using the carriage strain α522 and the hyperinvasive strain MC58 in different in vitro and ex vivo conditions. Growth experiments revealed that both wild-type strains were almost indistinguishable in their ex vivo phenotypes. However, quantitative real time PCR (qRT-PCR) found differences in the gene expression of relA between both strains. Furthermore, in contrast to the MC58 RelA mutant strain α522 deficient in RelA was unable to survive in human whole blood, although both strains showed the same ex vivo phenotypes in saliva and cerebrospinal fluid. Moreover, strain α522 was depended on a short non-coding AT-rich repeat element (ATRrelA) in the promoter region of relA to survive in human blood. Furthermore, cell culture experiments with human epithelial cells revealed that in both strains the deletion of relA resulted in a significantly decreased invasion rate while not significantly affecting adhesion. In order to better understand the conditional lethality of the relA deletion, computational and experimental analyses were carried out to unravel differences in amino acid biosynthetic pathways between both strains. Whereas strain MC58 is able to synthesize all 20 amino acids, strain α522 has an auxotrophy for cysteine and glutamine. In addition, the in vitro growth experiments found that RelA is required for growth in the absence of external amino acids in both strains. Furthermore, the mutant strain MC58 harboring an ATRrelA in its relA promoter region showed improved growth in minimal medium supplemented with L-cysteine and/or L-glutamine compared to the wild-type strain. Contrary, in strain α522 no differences between the wild-type and the ATRrelA deletion mutant were observed. Together this indicates that ATRrelA interferes with the complex regulatory interplay between the stringent response pathway and L-cysteine as well as L-glutamine metabolism. It further suggests that meningococcal virulence is linked to relA in a strain- and condition- depended manner. In conclusion, this work highlighted the role of the stringent response and of non-coding regulatory elements for bacterial virulence and indicates that virulence might be related to the way how meningococci accomplish growth within the host environments.}, subject = {Neisseria meningitidis}, language = {en} } @article{KochHellenbrandSchinketal.2016, author = {Koch, J. and Hellenbrand, W. and Schink, S. and Wichmann, O. and Carganico, A. and Drewes, J. and Kruspe, M. and Suckau, M. and Claus, H. and Marcus, U.}, title = {Evaluation of a temporary vaccination recommendation in response to an outbreak of invasive meningococcal serogroup C disease in men who have sex with men in Berlin, 2013-2014}, series = {Eurosurveillance}, volume = {21}, journal = {Eurosurveillance}, number = {5}, doi = {10.2807/1560-7917.ES.2016.21.5.30122}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165070}, pages = {pii=30122}, year = {2016}, abstract = {Meningococcal serogroup C (MenC) vaccination of men who have sex with men (MSM) was temporarily recommended to control an outbreak of invasive MenC disease among MSM in Berlin in 2012-2013. Vaccination was offered to HIV-infected MSM free of charge; others had to request reimbursement or pay out of pocket. We aimed to assess (i) awareness and acceptance of this recommendation through an online survey of MSM, (ii) implementation through a survey of primary care physicians and analysis of vaccine prescriptions, and (iii) impact through analysis of notified cases. Among online survey respondents, 60\% were aware of the recommendation. Of these, 39\% had obtained vaccination (70\% of HIV-infected, 13\% of HIV-negative/non-tested MSM). Awareness of recommendation and vaccination were positively associated with HIV infection, primary care physicians' awareness of respondents' sexual orientation, and exposure to multiple information sources. Most (26/30) physicians informed clients about the recommendation. Physicians considered concerns regarding reimbursement, vaccine safety and lack of perceived disease risk as primary barriers. After the recommendation, no further outbreak-related cases occurred. To reach and motivate target groups, communication of a new outbreak-related vaccination recommendation should address potential concerns through as many information channels as possible and direct reimbursement of costs should be enabled.}, language = {en} } @article{DoranFuldeGratzetal.2016, author = {Doran, Kelly S. and Fulde, Marcus and Gratz, Nina and Kim, Brandon J. and Nau, Roland and Prasadarao, Nemani and Schubert-Unkmeir, Alexandra and Tuomanen, Elaine I. and Valentin-Weigand, Peter}, title = {Host-pathogen interactions in bacterial meningitis}, series = {Acta Neuropathologica}, volume = {131}, journal = {Acta Neuropathologica}, number = {2}, doi = {10.1007/s00401-015-1531-z}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-191034}, pages = {185-209}, year = {2016}, abstract = {Bacterial meningitis is a devastating disease occurring worldwide with up to half of the survivors left with permanent neurological sequelae. Due to intrinsic properties of the meningeal pathogens and the host responses they induce, infection can cause relatively specific lesions and clinical syndromes that result from interference with the function of the affected nervous system tissue. Pathogenesis is based on complex host-pathogen interactions, some of which are specific for certain bacteria, whereas others are shared among different pathogens. In this review, we summarize the recent progress made in understanding the molecular and cellular events involved in these interactions. We focus on selected major pathogens, Streptococcus pneumonia, S. agalactiae (Group B Streptococcus), Neisseria meningitidis, and Escherichia coli K1, and also include a neglected zoonotic pathogen, Streptococcus suis. These neuroinvasive pathogens represent common themes of host-pathogen interactions, such as colonization and invasion of mucosal barriers, survival in the blood stream, entry into the central nervous system by translocation of the blood-brain and blood-cerebrospinal fluid barrier, and induction of meningeal inflammation, affecting pia mater, the arachnoid and subarachnoid spaces.}, language = {en} } @article{StockPetrašMelteretal.2016, author = {Stock, Nina Katharina and Petr{\´a}š, Petr and Melter, Oto and Kapounov{\´a}, Gabriela and Vopalkov{\´a}, Petra and Kubele, Jan and Vaniš, V{\´a}clav and Tkadlec, Jan and Buk{\´a}čkov{\´a}, Eva and Machov{\´a}, Ivana and Jindr{\´a}k, Vlastimil}, title = {Importance of Multifaceted Approaches in Infection Control: A Practical Experience from an Outbreak Investigation}, series = {PLoS ONE}, volume = {11}, journal = {PLoS ONE}, number = {6}, doi = {10.1371/journal.pone.0157981}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-166891}, pages = {e0157981}, year = {2016}, abstract = {Background This study presents the results of a multidisciplinary, nosocomial MRSA outbreak investigation in an 8-bed medical intensive care unit (ICU). The identification of seven MRSA positive patients in the beginning of 2014 led to the closure of the ward for several weeks. A multidisciplinary, retrospective investigation was initiated in order to identify the reason and the source for the outbreak, describe MRSA transmission in the department and identify limitations in infection control. Methods The investigation comprised an epidemiological description of MRSA cases from 2012 to 2014 and a characterization of MRSA isolates, including phage-, spa- and PFGE-typing. Additionally, MRSA screening was performed from the hospital staff and the environment. To identify the reason for the outbreak, work-related, psychological and behavioral factors were investigated by impartial audits and staff interviews. Results Thirty-one MRSA cases were registered during the study period, and 36 isolates were investigated. Molecular typing determined the outbreak strain (phage type 54/812, PFGE type A4, spa type t003) and identified the probable index case. Nasal carriage in one employee and a high environmental contamination with the outbreak strain was documented. Important gaps in nursing procedures and general management were identified. Elevated stress levels and communication problems preceded the outbreak. Compliance with hand hygiene and isolation procedures was evaluated as appropriate. Conclusion This study demonstrates the complexity of controlling hospital-associated infections. The combined use of different typing methods is beneficial for outbreak investigations. Psychological, behavioral and other work-related factors have an important impact on the spread of nosocomial pathogens. These factors should be addressed and integrated in routine infection control practice.}, language = {en} } @article{MoremiClausMshana2016, author = {Moremi, Nyambura and Claus, Heike and Mshana, Stephen E.}, title = {Antimicrobial resistance pattern: a report of microbiological cultures at a tertiary hospital in Tanzania}, series = {BMC Infectious Diseases}, volume = {16}, journal = {BMC Infectious Diseases}, number = {756}, doi = {10.1186/s12879-016-2082-1}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161185}, year = {2016}, abstract = {Background Antimicrobial resistance has been declared by the World Health Organization as a threat to the public health. The aim of this study was to analyze antimicrobial resistance patterns of the common pathogens occurring at the Bugando Medical Centre (BMC), Mwanza, Tanzania to provide data for antimicrobial stewardship programmes. Methods A total of 3330 microbiological culture results scripts representing non-repetitive specimens reported between June 2013 and May 2015 were retrieved and analyzed for pathogens and their susceptibility patterns using STATA-11 software. Results Out of 3330 specimens, 439 (13.2\%) had positive culture. Staphylococcus aureus (n = 100; 22.8\%), Klebsiella pneumoniae (n = 65; 14.8\%) and Escherichia coli (n = 41; 9.3\%) were the most frequently isolated bacteria. Of 78 Staphylococcus aureus tested, 27 (34.6\%) were found to be methicillin resistant Staphylococcus aureus (MRSA). Rates of resistance of Klebsiella pneumoniae and Escherichia coli isolates to third generation cephalosporins were 38.5\% (25/65) and 29.3\% (12/41) respectively. Staphylococcus aureus and Klesbiella pneumoniae were commonly isolated from bloodstream infections while Escherichia coli and Pseudomonas aeruginosa were the predominant isolates from urinary tract and wounds infections respectively. Of 23 Salmonella species isolated, 22 (95\%) were recovered from the blood. Nine of the 23 Salmonella species isolates (39\%) were found to be resistant to third generation cephalosporins. The resistance rate of gram-negative bacteria to third generation cephalosporins increased from 26.5\% in 2014 to 57.9\% in 2015 (p = 0.004) while the rate of MRSA decreased from 41.2\% in 2013 to 9.5\% in 2015 (p = 0.016). Multidrug-resistant gram-negative isolates were commonly isolated from Intensive Care Units and it was noted that, the majority of invasive infections were due to gram-negative bacteria. Conclusion There is an increase in proportion of gram-negative isolates resistant to third generation cephalosporins. The diversity of potential pathogens resistant to commonly prescribed antibiotics underscores the importance of sustained and standardized antimicrobial resistance surveillance and antibiotic stewardship programmes in developing countries.}, language = {en} } @article{HellenbrandClausSchinketal.2016, author = {Hellenbrand, Wiebke and Claus, Heike and Schink, Susanne and Marcus, Ulrich and Wichmann, Ole and Vogel, Ulrich}, title = {Risk of Invasive Meningococcal Disease in Men Who Have Sex with Men: Lessons Learned from an Outbreak in Germany, 2012-2013}, series = {PLoS ONE}, volume = {11}, journal = {PLoS ONE}, number = {8}, doi = {10.1371/journal.pone.0160126}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-166842}, pages = {e0160126}, year = {2016}, abstract = {Background We undertook investigations in response to an invasive meningococcal disease (IMD) outbreak in men who have sex with men (MSM) in Berlin 2012-2013 to better understand meningococcal transmission and IMD risk in MSM. Methods We retrospectively searched for further IMD cases in MSM in Germany through local health departments and undertook exploratory interviews. We performed antigen sequence typing, characterized fHbp and aniA genes of strains with the outbreak finetype and reviewed epidemiologically or spatiotemporally linked cases from 2002-2014. Results Among the 148 IMD-cases notified from 01.01.2012-30.09.2013 in 18-59 year-old men we identified 13 MSM in 6 federal states: 11 serogroup C (MenC, all finetype C:P1.5-1,10-8:F3-6), 2 MenB. Interviews with 7 MSM revealed frequent meeting of multiple partners online or via mobile apps and illicit drug use as potential risk factors. MenC incidence was 13-fold higher in MSM than non-MSM. MenC isolates from 9/11 MSM had a novel fHbp allele 766. All C:P1.5-1,10-8:F3-6 strains from MSM versus 16/23 from non-MSM had intact aniA genes (p = 0.04). Although definitive evidence for transmission among MSM in epidemiological or spatiotemporal clusters in 2002-2014 was lacking, clusters were more frequent in men aged 20-49 years. Molecular analysis of C:P1.5-1,10-8:F3-6 strains revealed cases with intact aniA since 2007, mainly associated with fHbp361, fHbp766 and fHbp813, all involving one or more MSM. Conclusions MenC incidence was elevated in MSM during the study period. Multiple casual sexual contacts and illicit drug use were common in affected MSM. In all strains from MSM we detected an intact aniA gene coding for a nitrite reductase, which permits survival in microanaerobic environments and could play a role in meningococcal transmission in MSM through urogenital colonization. Furthermore, meningococcal transmission among MSM may be sustained over large areas and thus require modified spatiotemporal scanning algorithms for timely detection and control.}, language = {en} } @article{KohlmorgenEliasSchoen2017, author = {Kohlmorgen, Britta and Elias, Johannes and Schoen, Christoph}, title = {Improved performance of the artus Mycobacterium tuberculosis RG PCR kit in a low incidence setting: a retrospective monocentric study}, series = {Scientific Reports}, volume = {7}, journal = {Scientific Reports}, number = {14127}, doi = {10.1038/s41598-017-14367-z}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-159248}, year = {2017}, abstract = {Tuberculosis (TB) and the spread of Mycobacterium tuberculosis complex (MTBC) strains resistant against rifampin (RIF) and isoniazid (INH) pose a serious threat to global health. However, rapid and reliable MTBC detection along with RIF/INH susceptibility testing are challenging in low prevalence countries due to the higher rate of false positives. Here, we provide the first performance data for the artus MTBC PCR assay in a low prevalence setting. We analyze 1323 respiratory and 311 non-respiratory samples with the artus MTBC PCR assay as well as by mycobacterial culture and microscopy. We propose retesting of specimens in duplicate and consideration of a determined cycle-threshold value cut-off greater than 34, as this significantly increases accuracy, specificity, and negative predictive value without affecting sensitivity. Furthermore, we tested fourteen MTBC positive samples with the GenoType MTBDRplus test and demonstrate that using an identical DNA extraction protocol for both assays does not impair downstream genotypic testing for RIF and INH susceptibility. In conclusion, our procedure optimizes the use of the artus MTB assay with workload efficient methods in a low incidence setting. Combining the modified artus MTB with the GenoType MTBDRplus assays allows rapid and accurate detection of MTBC and RIF/INH resistance.}, language = {en} } @article{AmpattuHagmannLiangetal.2017, author = {Ampattu, Biju Joseph and Hagmann, Laura and Liang, Chunguang and Dittrich, Marcus and Schl{\"u}ter, Andreas and Blom, Jochen and Krol, Elizaveta and Goesmann, Alexander and Becker, Anke and Dandekar, Thomas and M{\"u}ller, Tobias and Schoen, Christoph}, title = {Transcriptomic buffering of cryptic genetic variation contributes to meningococcal virulence}, series = {BMC Genomics}, volume = {18}, journal = {BMC Genomics}, number = {282}, doi = {10.1186/s12864-017-3616-7}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-157534}, year = {2017}, abstract = {Background: Commensal bacteria like Neisseria meningitidis sometimes cause serious disease. However, genomic comparison of hyperinvasive and apathogenic lineages did not reveal unambiguous hints towards indispensable virulence factors. Here, in a systems biological approach we compared gene expression of the invasive strain MC58 and the carriage strain α522 under different ex vivo conditions mimicking commensal and virulence compartments to assess the strain-specific impact of gene regulation on meningococcal virulence. Results: Despite indistinguishable ex vivo phenotypes, both strains differed in the expression of over 500 genes under infection mimicking conditions. These differences comprised in particular metabolic and information processing genes as well as genes known to be involved in host-damage such as the nitrite reductase and numerous LOS biosynthesis genes. A model based analysis of the transcriptomic differences in human blood suggested ensuing metabolic flux differences in energy, glutamine and cysteine metabolic pathways along with differences in the activation of the stringent response in both strains. In support of the computational findings, experimental analyses revealed differences in cysteine and glutamine auxotrophy in both strains as well as a strain and condition dependent essentiality of the (p)ppGpp synthetase gene relA and of a short non-coding AT-rich repeat element in its promoter region. Conclusions: Our data suggest that meningococcal virulence is linked to transcriptional buffering of cryptic genetic variation in metabolic genes including global stress responses. They further highlight the role of regulatory elements for bacterial virulence and the limitations of model strain approaches when studying such genetically diverse species as N. meningitidis.}, language = {en} } @article{KlughammerDittrichBlometal.2017, author = {Klughammer, Johanna and Dittrich, Marcus and Blom, Jochen and Mitesser, Vera and Vogel, Ulrich and Frosch, Matthias and Goesmann, Alexander and M{\"u}ller, Tobias and Schoen, Christoph}, title = {Comparative genome sequencing reveals within-host genetic changes in Neisseria meningitidis during invasive disease}, series = {PLoS ONE}, volume = {12}, journal = {PLoS ONE}, number = {1}, doi = {10.1371/journal.pone.0169892}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-159547}, pages = {e0169892}, year = {2017}, abstract = {Some members of the physiological human microbiome occasionally cause life-threatening disease even in immunocompetent individuals. A prime example of such a commensal pathogen is Neisseria meningitidis, which normally resides in the human nasopharynx but is also a leading cause of sepsis and epidemic meningitis. Using N. meningitidis as model organism, we tested the hypothesis that virulence of commensal pathogens is a consequence of within host evolution and selection of invasive variants due to mutations at contingency genes, a mechanism called phase variation. In line with the hypothesis that phase variation evolved as an adaptation to colonize diverse hosts, computational comparisons of all 27 to date completely sequenced and annotated meningococcal genomes retrieved from public databases showed that contingency genes are indeed enriched for genes involved in host interactions. To assess within-host genetic changes in meningococci, we further used ultra-deep whole-genome sequencing of throat-blood strain pairs isolated from four patients suffering from invasive meningococcal disease. We detected up to three mutations per strain pair, affecting predominantly contingency genes involved in type IV pilus biogenesis. However, there was not a single (set) of mutation(s) that could invariably be found in all four pairs of strains. Phenotypic assays further showed that these genetic changes were generally not associated with increased serum resistance, higher fitness in human blood ex vivo or differences in the interaction with human epithelial and endothelial cells in vitro. In conclusion, we hypothesize that virulence of meningococci results from accidental emergence of invasive variants during carriage and without within host evolution of invasive phenotypes during disease progression in vivo.}, language = {en} } @article{MoremiClausVogeletal.2017, author = {Moremi, Nyambura and Claus, Heike and Vogel, Ulrich and Mshana, Stephen E.}, title = {Surveillance of surgical site infections by Pseudomonas aeruginosa and strain characterization in Tanzanian hospitals does not provide proof for a role of hospital water plumbing systems in transmission}, series = {Antimicrobial Resistance and Infection Control}, volume = {6}, journal = {Antimicrobial Resistance and Infection Control}, number = {56}, doi = {10.1186/s13756-017-0216-x}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-158168}, year = {2017}, abstract = {Background The role of hospital water systems in the development of Pseudomonas aeruginosa (P. aeruginosa) surgical site infections (SSIs) in low-income countries is barely studied. This study characterized P. aeruginosa isolates from patients and water in order to establish possible epidemiological links. Methods: Between December 2014 and September 2015, rectal and wound swabs, and water samples were collected in the frame of active surveillance for SSIs in the two Tanzanian hospitals. Typing of P. aeruginosa was done by multi-locus sequence typing. Results: Of 930 enrolled patients, 536 were followed up, of whom 78 (14.6\%, 95\% CI; 11.6-17.5) developed SSIs. P. aeruginosa was found in eight (14\%) of 57 investigated wounds. Of the 43 water sampling points, 29 were positive for P. aeruginosa. However, epidemiological links to wound infections were not confirmed. The P. aeruginosa carriage rate on admission was 0.9\% (8/930). Of the 363 patients re-screened upon discharge, four (1.1\%) possibly acquired P. aeruginosa during hospitalization. Wound infections of the three of the eight P. aeruginosa SSIs were caused by a strain of the same sequence type (ST) as the one from intestinal carriage. Isolates from patients were more resistant to antibiotics than water isolates. Conclusions: The P. aeruginosa SSI rate was low. There was no evidence for transmission from tap water. Not all P. aeruginosa SSI were proven to be endogenous, pointing to other routes of transmission.}, language = {en} } @article{DickKraussHillenkampetal.2017, author = {Dick, Julia and Krauß, Patrizia and Hillenkamp, Jost and Kohlmorgen, Britta and Schoen, Christoph}, title = {Postoperative Tropheryma whipplei endophthalmitis - a case report highlighting the additive value of molecular testing}, series = {JMM Case Reports}, volume = {4}, journal = {JMM Case Reports}, doi = {10.1099/jmmcr.0.005124}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-158823}, pages = {e005124}, year = {2017}, abstract = {Introduction. Tropheryma whipplei is the causative agent of Whipple's disease. Gastrointestinal and lymphatic tissues are affected in the majority of cases, resulting in diarrhoea, malabsorption and fever. Here, we report a rare case of ocular manifestation in a patient lacking the typical Whipple symptoms. Case presentation. A 74-year-old Caucasian female presented with blurred vision in the right eye over a period of 1-2 months, accompanied by stinging pain and conjunctival hyperaemia for the last 2 days. Upon admission, visual acuity was hand motion in the affected eye. Ophthalmological examination showed typical signs of intraocular inflammation. Diagnostic and therapeutic pars plana vitrectomy including vitreous biopsy and intravitreal instillation of vancomycin and amikacin was performed within hours of initial presentation. Both microscopic analysis and microbial cultures of the vitreous biopsy remained negative for bacteria and fungi. The postoperative antibiotic regime included intravenous administration of ceftriaxone in combination with topical tobramycin and ofloxacin. Due to the empirical therapy the inflammation ceased and the patient was discharged after 5 days with cefpodoxime orally and local antibiotic and steroidal therapy. Meanwhile, the vitreous body had undergone testing by PCR for the eubacterial 16S rRNA gene, which was found to be positive. Analysis of the PCR product revealed a specific sequence of T. whipplei. Conclusion. In our patient, endophthalmitis was the first and only symptom of Morbus Whipple, while most patients with Whipple's disease suffer from severe gastrointestinal symptoms. 16S rDNA PCR should be considered for any intraocular infection when microscopy and standard culture methods remain negative.}, language = {en} } @article{MoremiClausVogeletal.2017, author = {Moremi, Nyambura and Claus, Heike and Vogel, Ulrich and Mshana, Stephen E.}, title = {Faecal carriage of CTX-M extended-spectrum beta-lactamase-producing Enterobacteriaceae among street children dwelling in Mwanza city, Tanzania}, series = {PLoS ONE}, volume = {12}, journal = {PLoS ONE}, number = {9}, doi = {10.1371/journal.pone.0184592}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170331}, pages = {e0184592}, year = {2017}, abstract = {Background Data on ESBL carriage of healthy people including children are scarce especially in developing countries. We analyzed the prevalence and genotypes of ESBL-producing Enterobacteriaceae (EPE) in Tanzanian street children with rare contact to healthcare facilities but significant interactions with the environment, animals and other people. Methodology/ Principle findings Between April and July 2015, stool samples of 107 street children, who live in urban Mwanza were analyzed for EPE. Intestinal carriage of EPE was found in 34 (31.8\%, 95\% CI; 22.7-40.3) children. Of the 36 isolates from 34 children, 30 (83.3\%) were Escherichia coli (E. coli) and six Klebsiella pneumoniae (K. pneumoniae). Out of 36 isolates, 36 (100\%), 35 (97\%), 25 (69\%) and 16 (44\%) were resistant to tetracycline, trimethoprim-sulfamethoxazole, ciprofloxacin and gentamicin, respectively. Beta-lactamase genes and the multilocus sequence types of E. coli and K. pneumoniae were characterized. ESBL gene bla\(_{CTX-M-15}\) was detected in 75\% (27/36) of ESBL isolates. Sequence types (STs) 131, 10, 448 and 617 were the most prevalent in E. coli. Use of local herbs (OR: 3.5, 95\% CI: 1.51-8.08, P = 0.003) and spending day and night on streets (OR: 3.6, 95\% CI: 1.44-8.97, P = 0.005) were independent predictors of ESBL carriage. Conclusions/ Significance We observed a high prevalence of bla\(_{CTX-M-15}\) in EPE collected from street children in Tanzania. Detection of E. coli STs 131, 10, 38 and 648, which have been observed worldwide in animals and people, highlights the need for multidisciplinary approaches to understand the epidemiology and drivers of antimicrobial resistance in low-income countries.}, language = {en} } @article{BecamWalterBurgertetal.2017, author = {Becam, J{\´e}r{\^o}me and Walter, Tim and Burgert, Anne and Schlegel, Jan and Sauer, Markus and Seibel, J{\"u}rgen and Schubert-Unkmeir, Alexandra}, title = {Antibacterial activity of ceramide and ceramide analogs against pathogenic Neisseria}, series = {Scientific Reports}, volume = {7}, journal = {Scientific Reports}, doi = {10.1038/s41598-017-18071-w}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-159367}, pages = {17627}, year = {2017}, abstract = {Certain fatty acids and sphingoid bases found at mucosal surfaces are known to have antibacterial activity and are thought to play a more direct role in innate immunity against bacterial infections. Herein, we analysed the antibacterial activity of sphingolipids, including the sphingoid base sphingosine as well as short-chain C\(_{6}\) and long-chain C\(_{16}\)-ceramides and azido-functionalized ceramide analogs against pathogenic Neisseriae. Determination of the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) demonstrated that short-chain ceramides and a ω-azido-functionalized C\(_{6}\)-ceramide were active against Neisseria meningitidis and N. gonorrhoeae, whereas they were inactive against Escherichia coli and Staphylococcus aureus. Kinetic assays showed that killing of N. meningitidis occurred within 2 h with ω-azido-C\(_{6}\)-ceramide at 1 X the MIC. Of note, at a bactericidal concentration, ω-azido-C\(_{6}\)-ceramide had no significant toxic effect on host cells. Moreover, lipid uptake and localization was studied by flow cytometry and confocal laser scanning microscopy (CLSM) and revealed a rapid uptake by bacteria within 5 min. CLSM and super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy demonstrated homogeneous distribution of ceramide analogs in the bacterial membrane. Taken together, these data demonstrate the potent bactericidal activity of sphingosine and synthetic short-chain ceramide analogs against pathogenic Neisseriae.}, language = {en} } @article{WagenerLoiko2017, author = {Wagener, Johannes and Loiko, Veronika}, title = {Recent insights into the paradoxical effect of echinocandins}, series = {Journal of Fungi}, volume = {4}, journal = {Journal of Fungi}, number = {1}, issn = {2309-608X}, doi = {10.3390/jof4010005}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-197960}, pages = {5}, year = {2017}, abstract = {Echinocandin antifungals represent one of the most important drug classes for the treatment of invasive fungal infections. The mode of action of the echinocandins relies on inhibition of the β-1,3-glucan synthase, an enzyme essentially required for the synthesis of the major fungal cell wall carbohydrate β-1,3-glucan. Depending on the species, echinocandins may exert fungicidal or fungistatic activity. Apparently independent of this differential activity, a surprising in vitro phenomenon called the "paradoxical effect" can be observed. The paradoxical effect is characterized by the ability of certain fungal isolates to reconstitute growth in the presence of higher echinocandin concentrations, while being fully susceptible at lower concentrations. The nature of the paradoxical effect is not fully understood and has been the focus of multiple studies in the last two decades. Here we concisely review the current literature and propose an updated model for the paradoxical effect, taking into account recent advances in the field.}, language = {en} } @article{GlaserSilwedelFehrholzetal.2017, author = {Glaser, Kirsten and Silwedel, Christine and Fehrholz, Markus and Waaga-Gasser, Ana M. and Henrich, Birgit and Claus, Heike and Speer, Christian P.}, title = {Ureaplasma Species Differentially Modulate Pro- and Anti-Inflammatory Cytokine Responses in Newborn and Adult Human Monocytes Pushing the State Toward Pro-Inflammation}, series = {Frontiers in Cellular and Infection Microbiology}, volume = {7}, journal = {Frontiers in Cellular and Infection Microbiology}, number = {484}, doi = {10.3389/fcimb.2017.00484}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-169958}, year = {2017}, abstract = {Background: Ureaplasma species have been associated with chorioamnionitis and preterm birth and have been implicated in the pathogenesis of neonatal short and long-term morbidity. However, being mostly commensal bacteria, controversy remains on the pro-inflammatory capacity of Ureaplasma. Discussions are ongoing on the incidence and impact of prenatal, perinatal, and postnatal infection. The present study addressed the impact of Ureaplasma isolates on monocyte-driven inflammation. Methods: Cord blood monocytes of term neonates and adult monocytes, either native or LPS-primed, were cultured with Ureaplasma urealyticum (U. urealyticum) serovar 8 (Uu8) and Ureaplasma parvum serovar 3 (Up3). Using qRT-PCR, cytokine flow cytometry, and multi-analyte immunoassay, we assessed mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-8, IL-12p40, IL-10, and IL-1 receptor antagonist (IL-1ra) as well as Toll-like receptor (TLR) 2 and TLR4. Results: Uu8 and Up3 induced mRNA expression and protein release of TNF-α, IL-1β and IL-8 in term neonatal and adult monocytes (p < 0.01 and p < 0.05). Intracellular protein expression of TNF-α, IL-1β and IL-8 in Ureaplasma-stimulated cells paralleled those results. Ureaplasma-induced cytokine levels did not significantly differ from LPS-mediated levels except for lower intracellular IL-1β in adult monocytes (Uu8: p < 0.05). Remarkably, ureaplasmas did not induce IL-12p40 response and promoted lower amounts of anti-inflammatory IL-10 and IL-1ra than LPS, provoking a cytokine imbalance more in favor of pro-inflammation (IL-1β/IL-10, IL-8/IL-10 and IL-8/IL-1ra: p < 0.01, vs. LPS). In contrast to LPS, both isolates induced TLR2 mRNA in neonatal and adult cells (p < 0.001 and p < 0.05) and suppressed TLR4 mRNA in adult monocytes (p < 0.05). Upon co-stimulation, Uu8 and Up3 inhibited LPS-induced intracellular IL-1β (p < 0.001 and p < 0.05) and IL-8 in adult monocytes (p < 0.01), while LPS-induced neonatal cytokines were maintained or aggravated (p < 0.05). Conclusion: Our data demonstrate a considerable pro-inflammatory capacity of Ureaplasma isolates in human monocytes. Stimulating pro-inflammatory cytokine responses while hardly inducing immunomodulatory and anti-inflammatory cytokines, ureaplasmas might push monocyte immune responses toward pro-inflammation. Inhibition of LPS-induced cytokines in adult monocytes in contrast to sustained inflammation in term neonatal monocytes indicates a differential modulation of host immune responses to a second stimulus. Modification of TLR2 and TLR4 expression may shape host susceptibility to inflammation.}, language = {en} } @article{HeidrichBauriedlBarquistetal.2017, author = {Heidrich, Nadja and Bauriedl, Saskia and Barquist, Lars and Li, Lei and Schoen, Christoph and Vogel, J{\"o}rg}, title = {The primary transcriptome of Neisseria meningitidis and its interaction with the RNA chaperone Hfq}, series = {Nucleic Acids Research}, volume = {45}, journal = {Nucleic Acids Research}, number = {10}, doi = {10.1093/nar/gkx168}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170828}, pages = {6147-6167}, year = {2017}, abstract = {Neisseria meningitidis is a human commensal that can also cause life-threatening meningitis and septicemia. Despite growing evidence for RNA-based regulation in meningococci, their transcriptome structure and output of regulatory small RNAs (sRNAs) are incompletely understood. Using dRNA-seq, we have mapped at single-nucleotide resolution the primary transcriptome of N. meningitidis strain 8013. Annotation of 1625 transcriptional start sites defines transcription units for most protein-coding genes but also reveals a paucity of classical σ70-type promoters, suggesting the existence of activators that compensate for the lack of -35 consensus sequences in N. meningitidis. The transcriptome maps also reveal 65 candidate sRNAs, a third of which were validated by northern blot analysis. Immunoprecipitation with the RNA chaperone Hfq drafts an unexpectedly large post-transcriptional regulatory network in this organism, comprising 23 sRNAs and hundreds of potential mRNA targets. Based on this data, using a newly developed gfp reporter system we validate an Hfq-dependent mRNA repression of the putative colonization factor PrpB by the two trans-acting sRNAs RcoF1/2. Our genome-wide RNA compendium will allow for a better understanding of meningococcal transcriptome organization and riboregulation with implications for colonization of the human nasopharynx.}, language = {en} } @article{HerrmannMuenstermannStrobeletal.2018, author = {Herrmann, Johannes and Muenstermann, Marcel and Strobel, Lea and Schubert-Unkmeir, Alexandra and Woodruff, Trent M. and Gray-Owen, Scott D. and Klos, Andreas and Johswich, Kay O.}, title = {Complement C5a receptor 1 exacerbates the pathophysiology of N. meningitidis sepsis and is a potential target for disease treatment}, series = {mBio}, volume = {9}, journal = {mBio}, number = {1}, doi = {10.1128/mBio.01755-17}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175792}, pages = {e01755-17}, year = {2018}, abstract = {Sepsis caused by Neisseria meningitidis (meningococcus) is a rapidly progressing, life-threatening disease. Because its initial symptoms are rather unspecific, medical attention is often sought too late, i.e., when the systemic inflammatory response is already unleashed. This in turn limits the success of antibiotic treatment. The complement system is generally accepted as the most important innate immune determinant against invasive meningococcal disease since it protects the host through the bactericidal membrane attack complex. However, complement activation concomitantly liberates the C5a peptide, and it remains unclear whether this potent anaphylatoxin contributes to protection and/or drives the rapidly progressing immunopathogenesis associated with meningococcal disease. Here, we dissected the specific contribution of C5a receptor 1 (C5aR1), the canonical receptor for C5a, using a mouse model of meningococcal sepsis. Mice lacking C3 or C5 displayed susceptibility that was enhanced by >1,000-fold or 100-fold, respectively, consistent with the contribution of these components to protection. In clear contrast, C5ar1\(^{-/-}\) mice resisted invasive meningococcal infection and cleared N. meningitidis more rapidly than wild-type (WT) animals. This favorable outcome stemmed from an ameliorated inflammatory cytokine response to N. meningitidis in C5ar1\(^{-/-}\) mice in both in vivo and ex vivo whole-blood infections. In addition, inhibition of C5aR1 signaling without interference with the complement bactericidal activity reduced the inflammatory response also in human whole blood. Enticingly, pharmacologic C5aR1 blockade enhanced mouse survival and lowered meningococcal burden even when the treatment was administered after sepsis induction. Together, our findings demonstrate that C5aR1 drives the pathophysiology associated with meningococcal sepsis and provides a promising target for adjunctive therapy. Importance: The devastating consequences of N. meningitidis sepsis arise due to the rapidly arising and self-propagating inflammatory response that mobilizes antibacterial defenses but also drives the immunopathology associated with meningococcemia. The complement cascade provides innate broad-spectrum protection against infection by directly damaging the envelope of pathogenic microbes through the membrane attack complex and triggers an inflammatory response via the C5a peptide and its receptor C5aR1 aimed at mobilizing cellular effectors of immunity. Here, we consider the potential of separating the bactericidal activities of the complement cascade from its immune activating function to improve outcome of N. meningitidis sepsis. Our findings demonstrate that the specific genetic or pharmacological disruption of C5aR1 rapidly ameliorates disease by suppressing the pathogenic inflammatory response and, surprisingly, allows faster clearance of the bacterial infection. This outcome provides a clear demonstration of the therapeutic benefit of the use of C5aR1-specific inhibitors to improve the outcome of invasive meningococcal disease.}, language = {en} } @article{SilwedelSpeerHaarmannetal.2018, author = {Silwedel, Christine and Speer, Christian P. and Haarmann, Axel and Fehrholz, Markus and Claus, Heike and Buttmann, Mathias and Glaser, Kirsten}, title = {Novel insights into neuroinflammation: bacterial lipopolysaccharide, tumor necrosis factor α, and Ureaplasma species differentially modulate atypical chemokine receptor 3 responses in human brain microvascular endothelial cells}, series = {Journal of Neuroinflammation}, volume = {15}, journal = {Journal of Neuroinflammation}, number = {156}, doi = {10.1186/s12974-018-1170-0}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175952}, year = {2018}, abstract = {Background: Atypical chemokine receptor 3 (ACKR3, synonym CXCR7) is increasingly considered relevant in neuroinflammatory conditions, in which its upregulation contributes to compromised endothelial barrier function and may ultimately allow inflammatory brain injury. While an impact of ACKR3 has been recognized in several neurological autoimmune diseases, neuroinflammation may also result from infectious agents, including Ureaplasma species (spp.). Although commonly regarded as commensals of the adult urogenital tract, Ureaplasma spp. may cause invasive infections in immunocompromised adults as well as in neonates and appear to be relevant pathogens in neonatal meningitis. Nonetheless, clinical and in vitro data on Ureaplasma-induced inflammation are scarce. Methods: We established a cell culture model of Ureaplasma meningitis, aiming to analyze ACKR3 variances as a possible pathomechanism in Ureaplasma-associated neuroinflammation. Non-immortalized human brain microvascular endothelial cells (HBMEC) were exposed to bacterial lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α), and native as well as LPS-primed HBMEC were cultured with Ureaplasma urealyticum serovar 8 (Uu8) and U. parvum serovar 3 (Up3). ACKR3 responses were assessed via qRT-PCR, RNA sequencing, flow cytometry, and immunocytochemistry. Results: LPS, TNF-α, and Ureaplasma spp. influenced ACKR3 expression in HBMEC. LPS and TNF-α significantly induced ACKR3 mRNA expression (p < 0.001, vs. control), whereas Ureaplasma spp. enhanced ACKR3 protein expression in HBMEC (p < 0.01, vs. broth control). Co-stimulation with LPS and either Ureaplasma isolate intensified ACKR3 responses (p < 0.05, vs. LPS). Furthermore, stimulation wielded a differential influence on the receptor's ligands. Conclusions: We introduce an in vitro model of Ureaplasma meningitis. We are able to demonstrate a pro-inflammatory capacity of Ureaplasma spp. in native and, even more so, in LPS-primed HBMEC, underlining their clinical relevance particularly in a setting of co-infection. Furthermore, our data may indicate a novel role for ACKR3, with an impact not limited to auto-inflammatory diseases, but extending to infection-related neuroinflammation as well. AKCR3-induced blood-brain barrier breakdown might constitute a potential common pathomechanism.}, language = {en} } @article{StrobelJohswich2018, author = {Strobel, Lea and Johswich, Kay O.}, title = {Anticoagulants impact on innate immune responses and bacterial survival in whole blood models of Neisseria meningitidis infection}, series = {Scientific Reports}, volume = {8}, journal = {Scientific Reports}, number = {10225}, doi = {10.1038/s41598-018-28583-8}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176226}, year = {2018}, abstract = {Neisseria meningitidis (meningococcus) causes invasive diseases such as meningitis or septicaemia. Ex vivo infection of human whole blood is a valuable tool to study meningococcal virulence factors and the host innate immune responses. In order to consider effects of cellular mediators, the coagulation cascade must be inhibited to avoid clotting. There is considerable variation in the anticoagulants used among studies of N. meningitidis whole blood infections, featuring citrate, heparin or derivatives of hirudin, a polypeptide from leech saliva. Here, we compare the influence of these three different anticoagulants, and additionally Mg/EGTA, on host innate immune responses as well as on viability of N. meningitidis strains isolated from healthy carriers and disease cases, reflecting different sequence types and capsule phenotypes. We found that the anticoagulants significantly impact on cellular responses and, strain-dependently, also on bacterial survival. Hirudin does not inhibit complement and is therefore superior over the other anticoagulants; indeed hirudin-plasma most closely reflects the characteristics of serum during N. meningitidis infection. We further demonstrate the impact of heparin on complement activation on N. meningitidis and its consequences on meningococcal survival in immune sera, which appears to be independent of the heparin binding antigens Opc and NHBA.}, language = {en} } @article{RufBrantlWagener2018, author = {Ruf, Dominik and Brantl, Victor and Wagener, Johannes}, title = {Mitochondrial Fragmentation in \(Aspergillus\) \(fumigatus\) as Early Marker of Granulocyte Killing Activity}, series = {Frontiers in Cellular and Infection Microbiology}, volume = {8}, journal = {Frontiers in Cellular and Infection Microbiology}, number = {128}, doi = {10.3389/fcimb.2018.00128}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-227133}, year = {2018}, abstract = {The host's defense against invasive mold infections relies on diverse antimicrobial activities of innate immune cells. However, studying these mechanisms in vitro is complicated by the filamentous nature of such pathogens that typically form long, branched, multinucleated and compartmentalized hyphae. Here we describe a novel method that allows for the visualization and quantification of the antifungal killing activity exerted by human granulocytes against hyphae of the opportunistic pathogen Aspergillus fumigatus. The approach relies on the distinct impact of fungal cell death on the morphology of mitochondria that were visualized with green fluorescent protein (GFP). We show that oxidative stress induces complete fragmentation of the tubular mitochondrial network which correlates with cell death of affected hyphae. Live cell microscopy revealed a similar and non-reversible disruption of the mitochondrial morphology followed by fading of fluorescence in Aspergillus hyphae that were killed by human granulocytes. Quantitative microscopic analysis of fixed samples was subsequently used to estimate the antifungal activity. By utilizing this assay, we demonstrate that lipopolysaccharides as well as human serum significantly increase the killing efficacy of the granulocytes. Our results demonstrate that evaluation of the mitochondrial morphology can be utilized to assess the fungicidal activity of granulocytes against A. fumigatus hyphae.}, language = {en} } @article{PrausseLehnertTimmeetal.2018, author = {Prauße, Maria T. E. and Lehnert, Teresa and Timme, Sandra and H{\"u}nniger, Kerstin and Leonhardt, Ines and Kurzai, Oliver and Figge, Marc Thilo}, title = {Predictive Virtual Infection Modeling of Fungal Immune Evasion in Human Whole Blood}, series = {Frontiers in Immunology}, volume = {9}, journal = {Frontiers in Immunology}, number = {560}, issn = {1664-3224}, doi = {10.3389/fimmu.2018.00560}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-197493}, year = {2018}, abstract = {Bloodstream infections by the human-pathogenic fungi Candida albicans and Candida glabrata increasingly occur in hospitalized patients and are associated with high mortality rates. The early immune response against these fungi in human blood comprises a concerted action of humoral and cellular components of the innate immune system. Upon entering the blood, the majority of fungal cells will be eliminated by innate immune cells, i.e., neutrophils and monocytes. However, recent studies identified a population of fungal cells that can evade the immune response and thereby may disseminate and cause organ dissemination, which is frequently observed during candidemia. In this study, we investigate the so far unresolved mechanism of fungal immune evasion in human whole blood by testing hypotheses with the help of mathematical modeling. We use a previously established state-based virtual infection model for whole-blood infection with C. albicans to quantify the immune response and identified the fungal immune-evasion mechanism. While this process was assumed to be spontaneous in the previous model, we now hypothesize that the immune-evasion process is mediated by host factors and incorporate such a mechanism in the model. In particular, we propose, based on previous studies that the fungal immune-evasion mechanism could possibly arise through modification of the fungal surface by as of yet unknown proteins that are assumed to be secreted by activated neutrophils. To validate or reject any of the immune-evasion mechanisms, we compared the simulation of both immune-evasion models for different infection scenarios, i.e., infection of whole blood with either C. albicans or C. glabrata under non-neutropenic and neutropenic conditions. We found that under non-neutropenic conditions, both immune-evasion models fit the experimental data from whole-blood infection with C. albicans and C. glabrata. However, differences between the immune-evasion models could be observed for the infection outcome under neutropenic conditions with respect to the distribution of fungal cells across the immune cells. Based on these predictions, we suggested specific experimental studies that might allow for the validation or rejection of the proposed immune-evasion mechanism.}, language = {en} } @article{SchlegelPetersDooseetal.2019, author = {Schlegel, Jan and Peters, Simon and Doose, S{\"o}ren and Schubert-Unkmeir, Alexandra and Sauer, Markus}, title = {Super-resolution microscopy reveals local accumulation of plasma membrane gangliosides at Neisseria meningitidis Invasion Sites}, series = {Frontiers in Cell and Developmental Biology}, volume = {7}, journal = {Frontiers in Cell and Developmental Biology}, number = {194}, doi = {10.3389/fcell.2019.00194}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201639}, year = {2019}, abstract = {Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the β2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection.}, language = {en} } @article{SilwedelHaarmannFehrholzetal.2019, author = {Silwedel, Christine and Haarmann, Axel and Fehrholz, Markus and Claus, Heike and Speer, Christian P. and Glaser, Kirsten}, title = {More than just inflammation: Ureaplasma species induce apoptosis in human brain microvascular endothelial cells}, series = {Journal of Neuroinflammation}, volume = {16}, journal = {Journal of Neuroinflammation}, doi = {10.1186/s12974-019-1413-8}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-200711}, pages = {38}, year = {2019}, abstract = {Background Ureaplasma species (spp.) are commonly regarded as low-virulent commensals but may cause invasive diseases in immunocompromised adults and in neonates, including neonatal meningitis. The interactions of Ureaplasma spp. with host defense mechanisms are poorly understood. This study addressed Ureaplasma-driven cell death, concentrating on apoptosis as well as inflammatory cell death. Methods Human brain microvascular endothelial cells (HBMEC) were exposed to Ureaplasma (U.) urealyticum serovar 8 (Uu8) and U. parvum serovar 3 (Up3). Resulting numbers of dead cells as well as mRNA levels and enzyme activity of key agents in programmed cell death were assessed by flow cytometry, RNA sequencing, and qRT-PCR, respectively. xCELLigence data were used for real-time monitoring of changes in cell adhesion properties. Results Both Ureaplasma isolates induced cell death (p < 0.05, vs. broth). Furthermore, Ureaplasma spp. enhanced mRNA levels for genes in apoptosis, including caspase 3 (Up3 p < 0.05, vs. broth), caspase 7 (p < 0.01), and caspase 9 (Up3 p < 0.01). Caspase 3 activity was increased upon Uu8 exposure (p < 0.01). Vice versa, Ureaplasma isolates downregulated mRNA levels for proteins involved in inflammatory cell death, namely caspase 1 (Uu8 p < 0.01, Up3 p < 0.001), caspase 4 (Uu8 p < 0.05, Up3 p < 0.01), NOD-like receptor pyrin domain-containing 3 (Uu8 p < 0.05), and receptor-interacting protein kinase 3 (p < 0.05). Conclusions By inducing apoptosis in HBMEC as main constituents of the blood-brain barrier, Ureaplasma spp. may provoke barrier breakdown. Simultaneous suppression of inflammatory cell death may additionally attenuate host defense strategies. Ultimate consequence could be invasive and long-term CNS infections by Ureaplasma spp.}, language = {en} } @article{WaltherWagnerKurzai2019, author = {Walther, Grit and Wagner, Lysett and Kurzai, Oliver}, title = {Updates on the taxonomy of Mucorales with an emphasis on clinically important taxa}, series = {Journal of Fungi}, volume = {5}, journal = {Journal of Fungi}, number = {4}, issn = {2309-608X}, doi = {10.3390/jof5040106}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-193081}, year = {2019}, abstract = {Fungi of the order Mucorales colonize all kinds of wet, organic materials and represent a permanent part of the human environment. They are economically important as fermenting agents of soybean products and producers of enzymes, but also as plant parasites and spoilage organisms. Several taxa cause life-threatening infections, predominantly in patients with impaired immunity. The order Mucorales has now been assigned to the phylum Mucoromycota and is comprised of 261 species in 55 genera. Of these accepted species, 38 have been reported to cause infections in humans, as a clinical entity known as mucormycosis. Due to molecular phylogenetic studies, the taxonomy of the order has changed widely during the last years. Characteristics such as homothallism, the shape of the suspensors, or the formation of sporangiola are shown to be not taxonomically relevant. Several genera including Absidia, Backusella, Circinella, Mucor, and Rhizomucor have been amended and their revisions are summarized in this review. Medically important species that have been affected by recent changes include Lichtheimia corymbifera, Mucor circinelloides, and Rhizopus microsporus. The species concept of Rhizopus arrhizus (syn. R. oryzae) is still a matter of debate. Currently, species identification of the Mucorales is best performed by sequencing of the internal transcribed spacer (ITS) region. Ecologically, the Mucorales represent a diverse group but for the majority of taxa, the ecological role and the geographic distribution remain unknown. Understanding the biology of these opportunistic fungal pathogens is a prerequisite for the prevention of infections, and, consequently, studies on the ecology of the Mucorales are urgently needed.}, language = {en} } @article{GomesWestermannSauerweinetal.2019, author = {Gomes, Sara F. Martins and Westermann, Alexander J. and Sauerwein, Till and Hertlein, Tobias and F{\"o}rstner, Konrad U. and Ohlsen, Knut and Metzger, Marco and Shusta, Eric V. and Kim, Brandon J. and Appelt-Menzel, Antje and Schubert-Unkmeir, Alexandra}, title = {Induced pluripotent stem cell-derived brain endothelial cells as a cellular model to study Neisseria meningitidis infection}, series = {Frontiers in Microbiology}, volume = {10}, journal = {Frontiers in Microbiology}, number = {1181}, doi = {10.3389/fmicb.2019.01181}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201562}, year = {2019}, abstract = {Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs.}, language = {en} } @article{MuenstermannStrobelKlosetal.2019, author = {Muenstermann, Marcel and Strobel, Lea and Klos, Andreas and Wetsel, Rick A. and Woodruff, Trent M. and K{\"o}hl, J{\"o}rg and Johswich, Kay O.}, title = {Distinct roles of the anaphylatoxin receptors C3aR, C5aR1 and C5aR2 in experimental meningococcal infections}, series = {Virulence}, volume = {10}, journal = {Virulence}, number = {1}, doi = {10.1080/21505594.2019.1640035}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-200496}, pages = {677-694}, year = {2019}, abstract = {The complement system is pivotal in the defense against invasive disease caused by Neisseria meningitidis (Nme, meningococcus), particularly via the membrane attack complex. Complement activation liberates the anaphylatoxins C3a and C5a, which activate three distinct G-protein coupled receptors, C3aR, C5aR1 and C5aR2 (anaphylatoxin receptors, ATRs). We recently discovered that C5aR1 exacerbates the course of the disease, revealing a downside of complement in Nme sepsis. Here, we compared the roles of all three ATRs during mouse nasal colonization, intraperitoneal infection and human whole blood infection with Nme. Deficiency of complement or ATRs did not alter nasal colonization, but significantly affected invasive disease: Compared to WT mice, the disease was aggravated in C3ar\(^{-/-}\) mice, whereas C5ar1\(^{-/-}\) and C5ar2\(^{-/-}\) mice showed increased resistance to meningococcal sepsis. Surprisingly, deletion of either of the ATRs resulted in lower cytokine/chemokine responses, irrespective of the different susceptibilities of the mice. This was similar in ex vivo human whole blood infection using ATR inhibitors. Neutrophil responses to Nme were reduced in C5ar1\(^{-/-}\) mouse blood. Upon stimulation with C5a plus Nme, mouse macrophages displayed reduced phosphorylation of ERK1/2, when C5aR1 or C5aR2 were ablated or inhibited, suggesting that both C5a-receptors prime an initial macrophage response to Nme. Finally, in vivo blockade of C5aR1 alone (PMX205) or along with C5aR2 (A8\(^{Δ71-73}\)) resulted in ameliorated disease, whereas neither antagonizing C3aR (SB290157) nor its activation with a "super-agonist" peptide (WWGKKYRASKLGLAR) demonstrated a benefit. Thus, C5aR1 and C5aR2 augment disease pathology and are interesting targets for treatment, whereas C3aR is protective in experimental meningococcal sepsis.}, language = {en} } @article{HerzBrehm2019, author = {Herz, Michaela and Brehm, Klaus}, title = {Evidence for densovirus integrations into tapeworm genomes}, series = {Parasites \& Vectors}, volume = {12}, journal = {Parasites \& Vectors}, doi = {10.1186/s13071-019-3820-1}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-202478}, pages = {560}, year = {2019}, abstract = {Background Tapeworms lack a canonical piRNA-pathway, raising the question of how they can silence existing mobile genetic elements (MGE). Investigation towards the underlying mechanisms requires information on tapeworm transposons which is, however, presently scarce. Methods The presence of densovirus-related sequences in tapeworm genomes was studied by bioinformatic approaches. Available RNA-Seq datasets were mapped against the Echinococcus multilocularis genome to calculate expression levels of densovirus-related genes. Transcription of densovirus loci was further analyzed by sequencing and RT-qPCR. Results We herein provide evidence for the presence of densovirus-related elements in a variety of tapeworm genomes. In the high-quality genome of E. multilocularis we identified more than 20 individual densovirus integration loci which contain the information for non-structural and structural virus proteins. The majority of densovirus loci are present as head-to-tail concatemers in isolated repeat containing regions of the genome. In some cases, unique densovirus loci have integrated close to histone gene clusters. We show that some of the densovirus loci of E. multilocularis are actively transcribed, whereas the majority are transcriptionally silent. RT-qPCR data further indicate that densovirus expression mainly occurs in the E. multilocularis stem cell population, which probably forms the germline of this organism. Sequences similar to the non-structural densovirus genes present in E. multilocularis were also identified in the genomes of E. canadensis, E. granulosus, Hydatigera taeniaeformis, Hymenolepis diminuta, Hymenolepis microstoma, Hymenolepis nana, Taenia asiatica, Taenia multiceps, Taenia saginata and Taenia solium. Conclusions Our data indicate that densovirus integration has occurred in many tapeworm species. This is the first report on widespread integration of DNA viruses into cestode genomes. Since only few densovirus integration sites were transcriptionally active in E. multilocularis, our data are relevant for future studies into gene silencing mechanisms in tapeworms. Furthermore, they indicate that densovirus-based vectors might be suitable tools for genetic manipulation of cestodes.}, language = {en} } @article{KimMcDonaghDengetal.2019, author = {Kim, Brandon J. and McDonagh, Maura A. and Deng, Liwen and Gastfriend, Benjamin D. and Schubert-Unkmeir, Alexandra and Doran, Kelly S. and Shusta, Eric V.}, title = {Streptococcus agalactiae disrupts P-glycoprotein function in brain endothelial cells}, series = {Fluids and Barriers of the CNS}, volume = {16}, journal = {Fluids and Barriers of the CNS}, doi = {10.1186/s12987-019-0146-5}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201895}, pages = {26}, year = {2019}, abstract = {Bacterial meningitis is a serious life threatening infection of the CNS. To cause meningitis, blood-borne bacteria need to interact with and penetrate brain endothelial cells (BECs) that comprise the blood-brain barrier. BECs help maintain brain homeostasis and they possess an array of efflux transporters, such as P-glycoprotein (P-gp), that function to efflux potentially harmful compounds from the CNS back into the circulation. Oftentimes, efflux also serves to limit the brain uptake of therapeutic drugs, representing a major hurdle for CNS drug delivery. During meningitis, BEC barrier integrity is compromised; however, little is known about efflux transport perturbations during infection. Thus, understanding the impact of bacterial infection on P-gp function would be important for potential routes of therapeutic intervention. To this end, the meningeal bacterial pathogen, Streptococcus agalactiae, was found to inhibit P-gp activity in human induced pluripotent stem cell-derived BECs, and live bacteria were required for the observed inhibition. This observation was correlated to decreased P-gp expression both in vitro and during infection in vivo using a mouse model of bacterial meningitis. Given the impact of bacterial interactions on P-gp function, it will be important to incorporate these findings into analyses of drug delivery paradigms for bacterial infections of the CNS.}, language = {en} } @article{KimShustaDoran2019, author = {Kim, Brandon J. and Shusta, Eric V. and Doran, Kelly S.}, title = {Past and current perspectives in modeling bacteria and blood-brain barrier interactions}, series = {Frontiers in Microbiology}, volume = {10}, journal = {Frontiers in Microbiology}, number = {1336}, doi = {10.3389/fmicb.2019.01336}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201766}, year = {2019}, abstract = {The central nervous system (CNS) barriers are highly specialized cellular barriers that promote brain homeostasis while restricting pathogen and toxin entry. The primary cellular constituent regulating pathogen entry in most of these brain barriers is the brain endothelial cell (BEC) that exhibits properties that allow for tight regulation of CNS entry. Bacterial meningoencephalitis is a serious infection of the CNS and occurs when bacteria can cross specialized brain barriers and cause inflammation. Models have been developed to understand the bacterial - BEC interaction that lead to pathogen crossing into the CNS, however, these have been met with challenges due to these highly specialized BEC phenotypes. This perspective provides a brief overview and outlook of the in vivo and in vitro models currently being used to study bacterial brain penetration, and opinion on improved models for the future.}, language = {en} } @article{ClausHubertBecheretal.2019, author = {Claus, Heike and Hubert, Kerstin and Becher, D{\"o}rte and Otto, Andreas and Pawlik, Marie-Christin and Lappann, Ines and Strobel, Lea and Vogel, Ulrich and Johswich, Kay}, title = {A homopolymeric adenosine tract in the promoter region of nspA influences factor H-mediated serum resistance in Neisseria meningitidis}, series = {Scientific Reports}, volume = {9}, journal = {Scientific Reports}, doi = {10.1038/s41598-019-39231-0}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-200956}, pages = {2736}, year = {2019}, abstract = {Although usually asymptomatically colonizing the human nasopharynx, the Gram-negative bacterium Neisseria meningitidis (meningococcus) can spread to the blood stream and cause invasive disease. For survival in blood, N. meningitidis evades the complement system by expression of a polysaccharide capsule and surface proteins sequestering the complement regulator factor H (fH). Meningococcal strains belonging to the sequence type (ST-) 41/44 clonal complex (cc41/44) cause a major proportion of serogroup B meningococcal disease worldwide, but they are also common in asymptomatic carriers. Proteome analysis comparing cc41/44 isolates from invasive disease versus carriage revealed differential expression levels of the outer membrane protein NspA, which binds fH. Deletion of nspA reduced serum resistance and NspA expression correlated with fH sequestration. Expression levels of NspA depended on the length of a homopolymeric tract in the nspA promoter: A 5-adenosine tract dictated low NspA expression, whereas a 6-adenosine motif guided high NspA expression. Screening German cc41/44 strain collections revealed the 6-adenosine motif in 39\% of disease isolates, but only in 3.4\% of carriage isolates. Thus, high NspA expression is associated with disease, but not strictly required. The 6-adenosine nspA promoter is most common to the cc41/44, but is also found in other hypervirulent clonal complexes.}, language = {en} } @article{SilwedelSpeerHaarmannetal.2019, author = {Silwedel, Christine and Speer, Christian P. and Haarmann, Axel and Fehrholz, Markus and Claus, Heike and Schlegel, Nicolas and Glaser, Kirsten}, title = {Ureaplasma species modulate cytokine and chemokine responses in human brain microvascular endothelial cells}, series = {International Journal of Molecular Science}, volume = {20}, journal = {International Journal of Molecular Science}, number = {14}, issn = {1422-0067}, doi = {10.3390/ijms20143583}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201848}, year = {2019}, abstract = {Ureaplasma species are common colonizers of the adult genitourinary tract and often considered as low-virulence commensals. Intraamniotic Ureaplasma infections, however, facilitate chorioamnionitis and preterm birth, and cases of Ureaplasma-induced neonatal sepsis, pneumonia, and meningitis raise a growing awareness of their clinical relevance. In vitro studies are scarce but demonstrate distinct Ureaplasma-driven impacts on immune mechanisms. The current study addressed cytokine and chemokine responses upon exposure of native or lipopolysaccharide (LPS) co-stimulated human brain microvascular endothelial cells (HBMEC) to Ureaplasma urealyticum or U. parvum, using qRT-PCR, RNA sequencing, multi-analyte immunoassay, and flow cytometry. Ureaplasma exposure in native HBMEC reduced monocyte chemoattractant protein (MCP)-3 mRNA expression (p < 0.01, vs. broth). In co-stimulated HBMEC, Ureaplasma spp. attenuated LPS-evoked mRNA responses for C-X-C chemokine ligand 5, MCP-1, and MCP-3 (p < 0.05, vs. LPS) and mitigated LPS-driven interleukin (IL)-1α protein secretion, as well as IL-8 mRNA and protein responses (p < 0.05). Furthermore, Ureaplasma isolates increased C-X-C chemokine receptor 4 mRNA levels in native and LPS co-stimulated HBMEC (p < 0.05). The presented results may imply immunomodulatory capacities of Ureaplasma spp. which may ultimately promote chronic colonization and long-term neuroinflammation.}, language = {en} } @article{SpringerWaltherRickertsetal.2019, author = {Springer, Jan and Walther, Grit and Rickerts, Volker and Hamprecht, Axel and Willinger, Birgit and Teschner, Daniel and Einsele, Hermann and Kurzai, Oliver and Loeffler, Juergen}, title = {Detection of Fusarium Species in Clinical Specimens by Probe-Based Real-Time PCR}, series = {Journal of Fungi}, volume = {5}, journal = {Journal of Fungi}, number = {4}, issn = {2309-608X}, doi = {10.3390/jof5040105}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-193111}, pages = {105}, year = {2019}, abstract = {The mold Fusarium is a ubiquitous fungus causing plant, animal and human infections. In humans, Fusarium spp. are the major cause of eye infections in patients wearing contact lenses or after local trauma. Systemic infections by Fusarium spp. mainly occur in immunosuppressed patients and can disseminate throughout the human body. Due to high levels of resistance to antifungals a fast identification of the causative agent is an urgent need. By using a probe-based real-time PCR assay specific for the genus Fusarium we analysed several different clinical specimens detecting Fusarium spp. commonly found in clinical samples in Germany. Also, a large collection of lung fluid samples of haematological patients was analysed (n = 243). In these, two samples (0.8\%) were reproducibly positive, but only one could be confirmed by sequencing. For this case of probable invasive fungal disease (IFD) culture was positive for Fusarium species. Here we describe a rapid, probe-based real-time PCR assay to specifically detect DNA from a broad range of Fusarium species and its application to clinically relevant specimens.}, language = {en} } @article{DrayssClausHubertetal.2019, author = {Drayß, Maria and Claus, Heike and Hubert, Kerstin and Thiel, Katrin and Berger, Anja and Sing, Andreas and van der Linden, Mark and Vogel, Ulrich and L{\^a}m, Thi{\^e}n-Tr{\´i}}, title = {Asymptomatic carriage of Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, Group A Streptococcus and Staphylococcus aureus among adults aged 65 years and older}, series = {PLoS ONE}, volume = {14}, journal = {PLoS ONE}, number = {2}, doi = {10.1371/journal.pone.0212052}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201042}, pages = {e0212052}, year = {2019}, abstract = {Objective The aim of this study was to determine the prevalence of Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, group A Streptococcus (GAS), and Staphylococcus aureus in asymptomatic elderly people and to unravel risk factors leading to colonization. Methods A multi-centre cross-sectional study was conducted including 677 asymptomatic adults aged 65 years or more, living at home or in nursing homes. Study areas were Greater Aachen (North-Rhine-Westphalia) and Wuerzburg (Bavaria), both regions with medium to high population density. Nasal and oropharyngeal swabs as well as questionnaires were collected from October 2012 to May 2013. Statistical analysis included multiple logistic regression models. Results The carriage rate was 1.9\% ([95\%CI: 1.0-3.3\%]; 13/677) for H. influenzae, 0.3\% ([95\%CI: 0-1.1\%]; 2/677) for N. meningitidis and 0\% ([95\% CI: 0-0.5\%]; 0/677) for S. pneumoniae and GAS. Staphylococcus aureus was harboured by 28.5\% of the individuals ([95\% CI: 25.1-32.1\%]; 193/677) and 0.7\% ([95\% CI: 0.2-1.7\%]; 5/677) were positive for methicillin-resistant S. aureus. Among elderly community-dwellers colonization with S. aureus was significantly associated with higher educational level (adjusted OR: 1.905 [95\% CI: 1.248-2.908]; p = 0.003). Among nursing home residents colonization was associated with being married (adjusted OR: 3.367 [1.502-7.546]; p = 0.003). Conclusion The prevalence of N. meningitidis, H. influenzae, S. pneumoniae and GAS was low among older people in Germany. The S. aureus rate was expectedly high, while MRSA was found in less than 1\% of the individuals.}, language = {en} } @article{LuberLutzAbeleHornetal.2019, author = {Luber, Verena and Lutz, Mathias and Abele-Horn, Marianne and Einsele, Hermann and Grigoleit, G{\"o}tz Ulrich and Mielke, Stephan}, title = {Excretion of Ascaris lumbricoides following reduced-intensity allogeneic hematopoietic stem cell transplantation and consecutive treatment with mebendazole}, series = {Transplant Infectious Disease}, volume = {22}, journal = {Transplant Infectious Disease}, number = {1}, issn = {1399-3062}, doi = {10.1111/tid.13224}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-219608}, pages = {1-4}, year = {2019}, abstract = {Here, we present the unique case of a 51-year-old German patient with multiple myeloma excreting Ascaris lumbricoides in his stool five weeks after allogeneic hematopoietic stem cell transplantation. Stool analysis remained negative for the presence of eggs, and there was no eosinophilia in the peripheral blood at any time around stem cell transplantation. The patient was commenced on a three-day treatment with mebendazole, which was well tolerated. No serious interactions with the concomitant post-transplant medication or negative effects on the hematopoiesis were observed, and the myeloma still is in complete remission. To our knowledge, this is the first report on excretion of A lumbricoides in the context of allogeneic stem cell transplantation. The case is remarkable with view to the fact that the parasite has supposedly survived all courses of myeloma treatment including autologous and allogeneic conditioning. Parasitosis with A lumbricoides has a worldwide prevalence of about a billion and is extremely rare in northern Europe. Possibly the patient got infected during a trip to Egypt years before multiple myeloma was diagnosed.}, language = {en} } @article{AbimannanSumathiKrishnarajasekharetal.2019, author = {Abimannan, Nagarajan and Sumathi, G. and Krishnarajasekhar, O. R. and Sinha, Bhanu and Krishnan, Padma}, title = {Clonal Clusters and Virulence Factors of Methicillin-Resistant \(Staphylococcus\) \(Aureus\): Evidence for Community-Acquired Methicillin-Resistant \(Staphylococcus\) \(Aureus\) Infiltration into Hospital Settings in Chennai, South India}, series = {Indian Journal of Medical Microbiology}, volume = {37}, journal = {Indian Journal of Medical Microbiology}, number = {3}, doi = {10.4103/ijmm.IJMM_18_271}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-226963}, pages = {326-336}, year = {2019}, abstract = {Background and Objective: Staphylococcus aureus is one of the major pathogens of nosocomial infections as wells as community-acquired (CA) infections worldwide. So far, large-scale comprehensive molecular and epidemiological characterisation of S. aureus from very diverse settings has not been carried out in India. The objective of this study is to evaluate the molecular, epidemiological and virulence characteristics of S. aureus in both community and hospital settings in Chennai, southern India. Methods: S. aureus isolates were obtained from four different groups (a) healthy individuals from closed community settings, (b) inpatients from hospitals, (c) outpatients from hospitals, representing isolates of hospital-community interface and (d) HIV-infected patients to define isolates associated with the immunocompromised. Antibiotic susceptibility testing, multiplex polymerase chain reactions for detection of virulence and resistance determinants, molecular typing including Staphylococcal cassette chromosome mec (SCCmec) and agr typing, were carried out. Sequencing-based typing was done using spa and multilocus sequence typing (MLST) methods. Clonal complexes (CC) of hospital and CA methicillin-resistant S. aureus (MRSA) were identified and compared for virulence and resistance. Results and Conclusion: A total of 769 isolates of S. aureus isolates were studied. The prevalence of MRSA was found to be 7.17\%, 81.67\%, 58.33\% and 22.85\% for groups a, b, c and d, respectively. Of the four SCCmec types (I, III, IV and V) detected, SCCmec V was found to be predominant. Panton-Valentine leucocidin toxin genes were detected among MRSA isolates harbouring SCCmec IV and V. A total of 78 spa types were detected, t657 being the most prevalent. 13 MLST types belonging to 9 CC were detected. CC1 (ST-772, ST-1) and CC8 (ST238, ST368 and ST1208) were found to be predominant among MRSA. CA-MRSA isolates with SCCmec IV and V were isolated from all study groups including hospitalised patients and were found to be similar by molecular tools. This shows that CA MRSA has probably infiltrated into the hospital settings.}, language = {en} } @article{MoremiClausVogeletal.2019, author = {Moremi, Nyambura and Claus, Heike and Vogel, Ulrich and Mshana, Stephen E.}, title = {The role of patients and healthcare workers Staphylococcus aureus nasal colonization in occurrence of surgical site infection among patients admitted in two centers in Tanzania}, series = {Antimicrobial Resistance \& Infection Control}, volume = {8}, journal = {Antimicrobial Resistance \& Infection Control}, doi = {10.1186/s13756-019-0554-y}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-224185}, year = {2019}, abstract = {Background Colonization with Staphylococcus aureus has been identified as a risk for subsequent occurrence of infection. This study investigated the relationship between S. aureus colonization of patients and healthcare workers (HCWs), and subsequent surgical site infections (SSI). Methods Between December 2014 and September 2015, a total of 930 patients and 143 HCWs were enrolled from the Bugando Medical Centre and Sekou Toure hospital in Mwanza, Tanzania. On admission and discharge nasal swabs, with an additional of wound swab for those who developed SSI were collected from patients whereas HCWs were swabbed once. Identification and antimicrobial susceptibility testing were done by VITEK-MS and VITEK-2, respectively. Detection of Panton Valentine leukocidin (PVL) and mecA genes was done by PCR. S. aureus isolates were further characterized by spa typing and Multi-Locus Sequence Typing (MLST). Results Among 930 patients screened for S. aureus on admission, 129 (13.9\%) were positive of which 5.4\% (7/129) were methicillin-resistant S. aureus (MRSA). Amongst 363 patients rescreened on discharge, 301 patients had been tested negative on admission of whom 29 (9.6\%) turned positive after their hospital stay. Three (10.3\%) of the 29 acquired S. aureus were MRSA. Inducible Clindamycin resistance occurred more often among acquired S. aureus isolates than among isolates from admission [34.5\% (10/29) vs. 17.1\% (22/129), P = 0.018]. S. aureus contributed to 21.1\% (n = 12) of the 57 cases of investigated SSIs among 536 patients followed. Seven out of eight S. aureus carriage/infection pairs had the same spa and sequence types. The previously reported dominant PVL-positive ST88 MRSA strain with spa type t690 was detected in patients and HCW. Conclusion A significant proportion of patients acquired S. aureus during hospitalization. The finding of more than 90\% of S. aureus SSI to be of endogenous source underscores the need of improving infection prevention and control measures including screening and decolonization of high risk patients.}, language = {en} } @article{AlZabenMedyukhinaDietrichetal.2019, author = {Al-Zaben, Naim and Medyukhina, Anna and Dietrich, Stefanie and Marolda, Alessandra and H{\"u}nniger, Kerstin and Kurzai, Oliver and Figge, Marc Thilo}, title = {Automated tracking of label-free cells with enhanced recognition of whole tracks}, series = {Scientific Reports}, volume = {9}, journal = {Scientific Reports}, doi = {10.1038/s41598-019-39725-x}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-221093}, year = {2019}, abstract = {Migration and interactions of immune cells are routinely studied by time-lapse microscopy of in vitro migration and confrontation assays. To objectively quantify the dynamic behavior of cells, software tools for automated cell tracking can be applied. However, many existing tracking algorithms recognize only rather short fragments of a whole cell track and rely on cell staining to enhance cell segmentation. While our previously developed segmentation approach enables tracking of label-free cells, it still suffers from frequently recognizing only short track fragments. In this study, we identify sources of track fragmentation and provide solutions to obtain longer cell tracks. This is achieved by improving the detection of low-contrast cells and by optimizing the value of the gap size parameter, which defines the number of missing cell positions between track fragments that is accepted for still connecting them into one track. We find that the enhanced track recognition increases the average length of cell tracks up to 2.2-fold. Recognizing cell tracks as a whole will enable studying and quantifying more complex patterns of cell behavior, e.g. switches in migration mode or dependence of the phagocytosis efficiency on the number and type of preceding interactions. Such quantitative analyses will improve our understanding of how immune cells interact and function in health and disease.}, language = {en} } @article{LorsonRuoppNadernezhadetal.2020, author = {Lorson, Thomas and Ruopp, Matthias and Nadernezhad, Ali and Eiber, Julia and Vogel, Ulrich and Jungst, Tomasz and L{\"u}hmann, Tessa}, title = {Sterilization Methods and Their Influence on Physicochemical Properties and Bioprinting of Alginate as a Bioink Component}, series = {ACS Omega}, volume = {5}, journal = {ACS Omega}, number = {12}, doi = {10.1021/acsomega.9b04096}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-229460}, pages = {6481-6486}, year = {2020}, abstract = {Bioprinting has emerged as a valuable threedimensional (3D) biomanufacturing method to fabricate complex hierarchical cell-containing constructs. Spanning from basic research to clinical translation, sterile starting materials are crucial. In this study, we present pharmacopeia compendial sterilization methods for the commonly used bioink component alginate. Autoclaving (sterilization in saturated steam) and sterile filtration followed by lyophilization as well as the pharmacopeia non-compendial method, ultraviolet (UV)-irradiation for disinfection, were assessed. The impact of the sterilization methods and their effects on physicochemical and rheological properties, bioprinting outcome, and sterilization efficiency of alginate were detailed. Only sterile filtration followed by lyophilization as the sterilization method retained alginate's physicochemical properties and bioprinting behavior while resulting in a sterile outcome. This set of methods provides a blueprint for the analysis of sterilization effects on the rheological and physicochemical pattern of bioink components and is easily adjustable for other polymers used in the field of biofabrication in the future.}, language = {en} } @article{ApsemidouFuellerIdelevichetal.2020, author = {Apsemidou, Athanasia and F{\"u}ller, Miriam Antonie and Idelevich, Evgeny A. and Kurzai, Oliver and Tragiannidis, Athanasios and Groll, Andreas H.}, title = {Candida lusitaniae breakthrough fungemia in an immuno-compromised adolescent: case report and review of the literature}, series = {Journal of Fungi}, volume = {6}, journal = {Journal of Fungi}, number = {4}, issn = {2309-608X}, doi = {10.3390/jof6040380}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-220125}, year = {2020}, abstract = {Candida lusitaniae is a rare cause of candidemia that is known for its unique capability to rapidly acquire resistance to amphotericin B. We report the case of an adolescent with grade IV graft-vs.-host disease after hematopoietic cell transplantation who developed catheter-associated C. lusitaniae candidemia while on therapeutic doses of liposomal amphotericin B. We review the epidemiology of C. lusitaniae bloodstream infections in adult and pediatric patients, the development of resistance, and its role in breakthrough candidemia. Appropriate species identification, in vitro susceptibility testing, and source control are pivotal to optimal management of C. lusitaniae candidemia. Initial antifungal therapy may consist of an echinocandin and be guided by in vitro susceptibility and clinical response.}, language = {en} } @article{DichtlForsterOrmannsetal.2020, author = {Dichtl, Karl and Forster, Johannes and Ormanns, Steffen and Horns, Heidi and Suerbaum, Sebastian and Seybold, Ulrich and Wagener, Johannes}, title = {Comparison of β-D-Glucan and galactomannan in serum for detection of invasive aspergillosis: retrospective analysis with focus on early diagnosis}, series = {Journal of Fungi}, volume = {6}, journal = {Journal of Fungi}, number = {4}, issn = {2309-608X}, doi = {10.3390/jof6040253}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-216298}, year = {2020}, abstract = {The early diagnosis of invasive aspergillosis (IA) relies mainly on computed tomography imaging and testing for fungal biomarkers such as galactomannan (GM). We compared an established ELISA for the detection of GM with a turbidimetric assay for detection of the panfungal biomarker β-D-glucan (BDG) for early diagnosis of IA. A total of 226 serum specimens from 47 proven and seven probable IA cases were analysed. Sensitivity was calculated for samples obtained closest to the day of IA-diagnosis (d0). Additional analyses were performed by including samples obtained during the presumed course of disease. Most IA cases involved the respiratory system (63\%), and Aspergillus fumigatus was the most frequently isolated species (59\%). For proven cases, sensitivity of BDG/GM analysis was 57\%/40\%. Including all samples dating from -6 to +1 weeks from d0 increased sensitivities to 74\%/51\%. Sensitivity of BDG testing was as high as or higher than GM testing for all subgroups and time intervals analysed. BDG testing was less specific (90-93\%) than GM testing (99-100\%). Combining BDG and GM testing resulted in sensitivity/specificity of 70\%/91\%. Often, BDG testing was positive before GM testing. Our study backs the use of BDG for diagnosis of suspected IA. We suggest combining BDG and GM to improve the overall sensitivity.}, language = {en} } @article{BauriedlGerovacHeidrichetal.2020, author = {Bauriedl, Saskia and Gerovac, Milan and Heidrich, Nadja and Bischler, Thorsten and Barquist, Lars and Vogel, J{\"o}rg and Schoen, Christoph}, title = {The minimal meningococcal ProQ protein has an intrinsic capacity for structure-based global RNA recognition}, series = {Nature Communications}, volume = {11}, journal = {Nature Communications}, doi = {10.1038/s41467-020-16650-6}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-230040}, year = {2020}, abstract = {FinO-domain proteins are a widespread family of bacterial RNA-binding proteins with regulatory functions. Their target spectrum ranges from a single RNA pair, in the case of plasmid-encoded FinO, to global RNA regulons, as with enterobacterial ProQ. To assess whether the FinO domain itself is intrinsically selective or promiscuous, we determine in vivo targets of Neisseria meningitidis, which consists of solely a FinO domain. UV-CLIP-seq identifies associations with 16 small non-coding sRNAs and 166 mRNAs. Meningococcal ProQ predominantly binds to highly structured regions and generally acts to stabilize its RNA targets. Loss of ProQ alters transcript levels of >250 genes, demonstrating that this minimal ProQ protein impacts gene expression globally. Phenotypic analyses indicate that ProQ promotes oxidative stress resistance and DNA damage repair. We conclude that FinO domain proteins recognize some abundant type of RNA shape and evolve RNA binding selectivity through acquisition of additional regions that constrain target recognition. FinO-domain proteins are bacterial RNA-binding proteins with a wide range of target specificities. Here, the authors employ UV CLIP-seq and show that minimal ProQ protein of Neisseria meningitidis binds to various small non-coding RNAs and mRNAs involved in virulence.}, language = {en} } @phdthesis{Bauriedl2020, author = {Bauriedl, Saskia Corinna}, title = {The influence of riboregulation on fitness and virulence in Neisseria meningitidis}, doi = {10.25972/OPUS-19297}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192978}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Neisseria meningitidis (N. meningitidis) is a human commensal that occasionally causes life-threatening infections such as bacterial meningitis and septicemia. Despite experi-mental evidence that the expression of small non-coding RNAs (sRNAs) as well as the RNA chaperone Hfq affect meningococcal physiology, the impact of RNA-based regula-tion (riboregulation) on fitness and virulence in N. meningitidis is only poorly understood. Therefore, this study addressed these issues using a combination of high-throughput tech-nologies. A differential RNA-sequencing (dRNA-seq) approach was applied to produce a single-nucleotide resolution map of the primary transcriptome of N. meningitidis strain 8013. The dRNA-seq analysis predicted 1,625 transcriptional start sites including 65 putative sRNAs, of which 20 were further validated by northern blot analysis. By Hfq RNA im-munopreci-pitation sequencing a large Hfq-centered post-transcriptional regulatory net-work comprising 23 sRNAs and 401 potential mRNA targets was identified. Rifampicin stability assays demonstrated that Hfq binding confers enhanced stability on its associat-ed sRNAs. Based on these data, the interactions of two paralogous sRNAs and their cog-nate target mRNA prpB were validated in vivo as well as in vitro. Both sRNAs directly repress prpB encoding a methylisocitrate lyse which was previously shown to be involved in meningococcal colonization of the human nasopharynx. Besides the well-described RNA chaperone Hfq, FinO-domain proteins have recently been recognized as a widespread family of RNA-binding proteins (RBPs) with regulatory roles in diverse bacteria. They display an intriguing bandwidth of target sites, ranging from a single RNA pair as recognized by plasmid-encoded FinO to the global RNA regu-lons of enterobacterial ProQ proteins. To better understand the intrinsic targeting mode of this RBP family, in vivo targets of the minimal ProQ protein of N. meningitidis were de-termined. In vivo UV crosslinking with RNA deep sequencing (UV-CLIP) identified as-sociations of ProQ with 16 sRNAs and 166 mRNAs encoding a variety of biological functions and thus revealed ProQ as another global RBP in meningococci. It could be shown that meningococcal ProQ predominantly binds to highly structured RNA regions including DNA uptake sequences (DUS) and rho-independent transcription terminators and stabilizes many of its RNA targets as proved by rifampicin stability experiments. As expected from the large suite of ProQ-bound RNAs, proQ deletion globally affects both gene and protein expression in N. meningitidis, changing the expression levels of at least 244 mRNAs and 80 proteins. Phenotypic analyses suggested that ProQ promotes oxida-tive stress tolerance and UV damage repair capacity, both of which are required for full virulence of N. meningitidis. Together, this work uncovers the co-existence of two major post-transcriptional regulons, one governed by ProQ, the other by Hfq, in N. meningitidis. It further highlights the role of these distinct RBPs and its associated sRNAs to bacterial virulence and indicates that riboregulation is likely to contribute to the way how meningococci adapt to different host niches.}, subject = {Neisseria meningitidis}, language = {en} } @article{WurmbScholtesKolibayetal.2020, author = {Wurmb, Thomas and Scholtes, Katja and Kolibay, Felix and Schorscher, Nora and Ertl, Georg and Ernestus, Ralf-Ingo and Vogel, Ulrich and Franke, Axel and Kowalzik, Barbara}, title = {Hospital preparedness for mass critical care during SARS-CoV-2 pandemic}, series = {Critical Care}, volume = {24}, journal = {Critical Care}, doi = {10.1186/s13054-020-03104-0}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-230201}, year = {2020}, abstract = {Mass critical care caused by the severe acute respiratory syndrome corona virus 2 pandemic poses an extreme challenge to hospitals. The primary goal of hospital disaster preparedness and response is to maintain conventional or contingency care for as long as possible. Crisis care must be delayed as long as possible by appropriate measures. Increasing the intensive care unit (ICU) capacities is essential. In order to adjust surge capacity, the reduction of planned, elective patient care is an adequate response. However, this involves numerous problems that must be solved with a sense of proportion. This paper summarises preparedness and response measures recommended to acute care hospitals.}, language = {en} } @article{GoetzPanzerTrinksetal.2020, author = {G{\"o}tz, Ralph and Panzer, Sabine and Trinks, Nora and Eilts, Janna and Wagener, Johannes and Turr{\`a}, David and Di Pietro, Antonio and Sauer, Markus and Terpitz, Ulrich}, title = {Expansion Microscopy for Cell Biology Analysis in Fungi}, series = {Frontiers in Microbiology}, volume = {11}, journal = {Frontiers in Microbiology}, issn = {1664-302X}, doi = {10.3389/fmicb.2020.00574}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-202569}, year = {2020}, abstract = {Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes.}, language = {en} } @misc{TortMitrevaBrehmetal.2020, author = {Tort, Jose F. and Mitreva, Makedonka and Brehm, Klaus R. and Rinaldi, Gabriel}, title = {Editorial: Novel Frontiers in Helminth Genomics}, series = {Frontiers in Genetics}, volume = {11}, journal = {Frontiers in Genetics}, number = {791}, issn = {1664-8021}, doi = {10.3389/fgene.2020.00791}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-210209}, year = {2020}, abstract = {No abstract available.}, language = {en} } @article{SturmGeisselMartinetal.2020, author = {Sturm, Laura and Geißel, Bernadette and Martin, Ronny and Wagener, Johannes}, title = {Differentially Regulated Transcription Factors and ABC Transporters in a Mitochondrial Dynamics Mutant Can Alter Azole Susceptibility of Aspergillus fumigatus}, series = {Frontiers in Microbiology}, volume = {11}, journal = {Frontiers in Microbiology}, issn = {1664-302X}, doi = {10.3389/fmicb.2020.01017}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204874}, year = {2020}, abstract = {Azole resistance of the fungal pathogen Aspergillus fumigatus is an emerging problem. To identify novel mechanisms that could mediate azole resistance in A. fumigatus, we analyzed the transcriptome of a mitochondrial fission/fusion mutant that exhibits increased azole tolerance. Approximately 12\% of the annotated genes are differentially regulated in this strain. This comprises upregulation of Cyp51A, the azole target structure, upregulation of ATP-binding cassette (ABC) superfamily and major facilitator superfamily (MFS) transporters and differential regulation of transcription factors. To study their impact on azole tolerance, conditional mutants were constructed of seven ABC transporters and 17 transcription factors. Under repressed conditions, growth rates and azole susceptibility of the mutants were similar to wild type. Under induced conditions, several transcription factor mutants showed growth phenotypes. In addition, four ABC transporter mutants and seven transcription factor mutants exhibited altered azole susceptibility. However, deletion of individual identified ABC transporters and transcription factors did not affect the increased azole tolerance of the fission/fusion mutant. Our results revealed the ability of multiple ABC transporters and transcription factors to modulate the azole susceptibility of A. fumigatus and support a model where mitochondrial dysfunctions trigger a drug resistance network that mediates azole tolerance of this mold.}, language = {en} } @article{SchlesingerWeissbrichWedekinketal.2020, author = {Schlesinger, Tobias and Weißbrich, Benedikt and Wedekink, Florian and Notz, Quirin and Herrmann, Johannes and Krone, Manuel and Sitter, Magdalena and Schmid, Benedikt and Kredel, Markus and Stumpner, Jan and D{\"o}lken, Lars and Wischhusen, J{\"o}rg and Kranke, Peter and Meybohm, Patrick and Lotz, Christpher}, title = {Biodistribution and serologic response in SARS-CoV-2 induced ARDS: A cohort study}, series = {PLoS One}, volume = {15, 2020}, journal = {PLoS One}, number = {11}, doi = {10.1371/journal.pone.0242917}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-231348}, year = {2020}, abstract = {Background The viral load and tissue distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain important questions. The current study investigated SARS-CoV-2 viral load, biodistribution and anti-SARS-CoV-2 antibody formation in patients suffering from severe corona virus disease 2019 (COVID-19) induced acute respiratory distress syndrome (ARDS). Methods This is a retrospective single-center study in 23 patients with COVID-19-induced ARDS. Data were collected within routine intensive care. SARS-CoV-2 viral load was assessed via reverse transcription quantitative polymerase chain reaction (RT-qPCR). Overall, 478 virology samples were taken. Anti-SARS-CoV-2-Spike-receptor binding domain (RBD) antibody detection of blood samples was performed with an enzyme-linked immunosorbent assay. Results Most patients (91\%) suffered from severe ARDS during ICU treatment with a 30-day mortality of 30\%. None of the patients received antiviral treatment. Tracheal aspirates tested positive for SARS-CoV-2 in 100\% of the cases, oropharyngeal swabs only in 77\%. Blood samples were positive in 26\% of the patients. No difference of viral load was found in tracheal or blood samples with regard to 30-day survival or disease severity. SARS-CoV-2 was never found in dialysate. Serologic testing revealed significantly lower concentrations of SARS-CoV-2 neutralizing IgM and IgA antibodies in survivors compared to non-survivors (p = 0.009). Conclusions COVID-19 induced ARDS is accompanied by a high viral load of SARS-CoV-2 in tracheal aspirates, which remained detectable in the majority throughout intensive care treatment. Remarkably, SARS-CoV-2 RNA was never detected in dialysate even in patients with RNAemia. Viral load or the buildup of neutralizing antibodies was not associated with 30-day survival or disease severity.}, language = {en} } @article{FlemmingHankirErnestusetal.2020, author = {Flemming, S. and Hankir, M. and Ernestus, R.-I. and Seyfried, F. and Germer, C.-T. and Meybohm, P. and Wurmb, T. and Vogel, U. and Wiegering, A.}, title = {Surgery in times of COVID-19 — recommendations for hospital and patient management}, series = {Langenbeck's Archives of Surgery}, volume = {405}, journal = {Langenbeck's Archives of Surgery}, issn = {1435-2443}, doi = {10.1007/s00423-020-01888-x}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-231766}, pages = {359-364}, year = {2020}, abstract = {Background The novel coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), has escalated rapidly to a global pandemic stretching healthcare systems worldwide to their limits. Surgeonshave had to immediately react to this unprecedented clinical challenge by systematically repurposing surgical wards. Purpose To provide a detailed set of guidelines developed in a surgical ward at University Hospital Wuerzburg to safelyaccommodate the exponentially rising cases of SARS-CoV-2 infected patients without compromising the care of emergencysurgery and oncological patients or jeopardizing the well-being of hospital staff. Conclusions The dynamic prioritization of SARS-CoV-2 infected and surgical patient groups is key to preserving life whilemaintaining high surgical standards. Strictly segregating patient groups in emergency rooms, non-intensive care wards andoperating areas prevents viral spread while adequately training and carefully selecting hospital staff allow them to confidentlyand successfully undertake their respective clinical duties.}, language = {en} } @article{WeissSchlegelTerpitzetal.2020, author = {Weiss, Esther and Schlegel, Jan and Terpitz, Ulrich and Weber, Michael and Linde, J{\"o}rg and Schmitt, Anna-Lena and H{\"u}nniger, Kerstin and Marischen, Lothar and Gamon, Florian and Bauer, Joachim and L{\"o}ffler, Claudia and Kurzai, Oliver and Morton, Charles Oliver and Sauer, Markus and Einsele, Hermann and Loeffler, Juergen}, title = {Reconstituting NK Cells After Allogeneic Stem Cell Transplantation Show Impaired Response to the Fungal Pathogen Aspergillus fumigatus}, series = {Frontiers in Immunology}, volume = {11}, journal = {Frontiers in Immunology}, issn = {1664-3224}, doi = {10.3389/fimmu.2020.02117}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-212581}, year = {2020}, abstract = {Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1β, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56\(^{bright}\)CD16\(^{dim}\) cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.}, language = {en} }