TY - JOUR A1 - Wippel, Carolin A1 - Maurer, Jana A1 - Fortsch, Christina A1 - Hupp, Sabrina A1 - Bohl, Alexandra A1 - Ma, Jiangtao A1 - Mitchell, Timothy J. A1 - Bunkowski, Stephanie A1 - Brück, Wolfgang A1 - Nau, Roland A1 - Iliev, Asparouh I. T1 - Bacterial Cytolysin during Meningitis Disrupts the Regulation of Glutamate in the Brain, Leading to Synaptic Damage JF - PLoS Pathogens N2 - Abstract Streptococcus pneumoniae (pneumococcal) meningitis is a common bacterial infection of the brain. The cholesterol-dependent cytolysin pneumolysin represents a key factor, determining the neuropathogenic potential of the pneumococci. Here, we demonstrate selective synaptic loss within the superficial layers of the frontal neocortex of post-mortem brain samples from individuals with pneumococcal meningitis. A similar effect was observed in mice with pneumococcal meningitis only when the bacteria expressed the pore-forming cholesterol-dependent cytolysin pneumolysin. Exposure of acute mouse brain slices to only pore-competent pneumolysin at disease-relevant, non-lytic concentrations caused permanent dendritic swelling, dendritic spine elimination and synaptic loss. The NMDA glutamate receptor antagonists MK801 and D-AP5 reduced this pathology. Pneumolysin increased glutamate levels within the mouse brain slices. In mouse astrocytes, pneumolysin initiated the release of glutamate in a calcium-dependent manner. We propose that pneumolysin plays a significant synapto- and dendritotoxic role in pneumococcal meningitis by initiating glutamate release from astrocytes, leading to subsequent glutamate-dependent synaptic damage. We outline for the first time the occurrence of synaptic pathology in pneumococcal meningitis and demonstrate that a bacterial cytolysin can dysregulate the control of glutamate in the brain, inducing excitotoxic damage. Author Summary Bacterial meningitis is one of the most devastating brain diseases. Among the bacteria that cause meningitis, Streptococcus pneumoniae is the most common. Meningitis predominantly affects children, especially in the Third World, and most of them do not survive. Those that do survive often suffer permanent brain damage and hearing problems. The exact morphological substrates of brain damage in Streptococcus pneumoniae meningitis remain largely unknown. In our experiments, we found that the brain cortex of patients with meningitis demonstrated a loss of synapses (the contact points among neurons, responsible for the processes of learning and memory), and we identified the major pneumococcal neurotoxin pneumolysin as a sufficient cause of this loss. The effect was not direct but was mediated by the brain neurotransmitter glutamate, which was released upon toxin binding by one of the non-neuronal cell types of the brain – the astrocytes. Pneumolysin initiated calcium influx in astrocytes and subsequent glutamate release. Glutamate damaged the synapses via NMDA-receptors – a mechanism similar to the damage occurring in brain ischemia. Thus, we show that synaptic loss is present in pneumococcal meningitis, and we identify the toxic bacterial protein pneumolysin as the major factor in this process. These findings alter our understanding of bacterial meningitis and establish new therapeutic strategies for this fatal disease. KW - synapses KW - brain damage KW - astrocytes KW - neuronal dendrites KW - meningitis KW - glutamate KW - bacterial meningitis KW - neocortex Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130462 VL - 9 IS - 6 ER - TY - JOUR A1 - Weibel, Stephanie A1 - Basse-Luesebrink, Thomas Christian A1 - Hess, Michael A1 - Hofmann, Elisabeth A1 - Seubert, Carolin A1 - Langbein-Laugwitz, Johanna A1 - Gentschev, Ivaylo A1 - Sturm, Volker Jörg Friedrich A1 - Ye, Yuxiang A1 - Kampf, Thomas A1 - Jakob, Peter Michael A1 - Szalay, Aladar A. T1 - Imaging of Intratumoral Inflammation during Oncolytic Virotherapy of Tumors by \(^{19}\)F-Magnetic Resonance Imaging (MRI) JF - PLoS ONE N2 - Background Oncolytic virotherapy of tumors is an up-coming, promising therapeutic modality of cancer therapy. Unfortunately, non-invasive techniques to evaluate the inflammatory host response to treatment are rare. Here, we evaluate \(^{19}\)F magnetic resonance imaging (MRI) which enables the non-invasive visualization of inflammatory processes in pathological conditions by the use of perfluorocarbon nanoemulsions (PFC) for monitoring of oncolytic virotherapy. Methodology/Principal Findings The Vaccinia virus strain GLV-1h68 was used as an oncolytic agent for the treatment of different tumor models. Systemic application of PFC emulsions followed by \(^1H\)/\(^{19}\)F MRI of mock-infected and GLV-1h68-infected tumor-bearing mice revealed a significant accumulation of the \(^{19}\)F signal in the tumor rim of virus-treated mice. Histological examination of tumors confirmed a similar spatial distribution of the \(^{19}\)F signal hot spots and \(CD68^+\)-macrophages. Thereby, the \(CD68^+\)-macrophages encapsulate the GFP-positive viral infection foci. In multiple tumor models, we specifically visualized early inflammatory cell recruitment in Vaccinia virus colonized tumors. Furthermore, we documented that the \(^{19}\)F signal correlated with the extent of viral spreading within tumors. Conclusions/Significance These results suggest \(^{19}\)F MRI as a non-invasive methodology to document the tumor-associated host immune response as well as the extent of intratumoral viral replication. Thus, \(^{19}\)F MRI represents a new platform to non-invasively investigate the role of the host immune response for therapeutic outcome of oncolytic virotherapy and individual patient response. KW - inflammation KW - fluorescence microscopy KW - oncolytic viruses KW - fluorescence imaging KW - macrophages KW - magnetic resonance imaging KW - histology KW - in vivo imaging Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130311 VL - 8 IS - 3 ER - TY - JOUR A1 - Pfeiffer, Verena A1 - Götz, Rudolf A1 - Xiang, Chaomei A1 - Camarero, Guadelupe A1 - Braun, Attila A1 - Zhang, Yina A1 - Blum, Robert A1 - Heinsen, Helmut A1 - Nieswandt, Bernhard A1 - Rapp, Ulf R. T1 - Ablation of BRaf Impairs Neuronal Differentiation in the Postnatal Hippocampus and Cerebellum JF - PLoS ONE N2 - This study focuses on the role of the kinase BRaf in postnatal brain development. Mice expressing truncated, non-functional BRaf in neural stem cell-derived brain tissue demonstrate alterations in the cerebellum, with decreased sizes and fuzzy borders of the glomeruli in the granule cell layer. In addition we observed reduced numbers and misplaced ectopic Purkinje cells that showed an altered structure of their dendritic arborizations in the hippocampus, while the overall cornus ammonis architecture appeared to be unchanged. In male mice lacking BRaf in the hippocampus the size of the granule cell layer was normal at postnatal day 12 (P12) but diminished at P21, as compared to control littermates. This defect was caused by a reduced ability of dentate gyrus progenitor cells to differentiate into NeuN positive granule cell neurons. In vitro cell culture of P0/P1 hippocampal cells revealed that BRaf deficient cells were impaired in their ability to form microtubule-associated protein 2 positive neurons. Together with the alterations in behaviour, such as autoaggression and loss of balance fitness, these observations indicate that in the absence of BRaf all neuronal cellular structures develop, but neuronal circuits in the cerebellum and hippocampus are partially disturbed besides impaired neuronal generation in both structures. KW - granule cells KW - hippocampus KW - neurons KW - neuronal dendrites KW - embryos KW - dentate gyrus KW - neuronal differentiation KW - cerebellum Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130304 VL - 8 IS - 3 ER - TY - JOUR A1 - Beitzinger, Christoph A1 - Bronnhuber, Annika A1 - Duscha, Kerstin A1 - Riedl, Zsuzsanna A1 - Huber-Lang, Markus A1 - Benz, Roland A1 - Hajos, György A1 - Barth, Holger T1 - Designed Azolopyridinium Salts Block Protective Antigen Pores In Vitro and Protect Cells from Anthrax Toxin JF - PLoS ONE N2 - Background Several intracellular acting bacterial protein toxins of the AB-type, which are known to enter cells by endocytosis, are shown to produce channels. This holds true for protective antigen (PA), the binding component of the tripartite anthrax-toxin of Bacillus anthracis. Evidence has been presented that translocation of the enzymatic components of anthrax-toxin across the endosomal membrane of target cells and channel formation by the heptameric/octameric \(PA_{63}\) binding/translocation component are related phenomena. Chloroquine and some 4-aminoquinolones, known as potent drugs against Plasmodium falciparium infection of humans, block efficiently the \(PA_{63}\)-channel in a dose dependent way. Methodology/Principal Findings Here we demonstrate that related positively charged heterocyclic azolopyridinium salts block the \(PA_{63}\)-channel in the µM range, when both, inhibitor and \(PA_{63}\) are added to the same side of the membrane, the cis-side, which corresponds to the lumen of acidified endosomal vesicles of target cells. Noise-analysis allowed the study of the kinetics of the plug formation by the heterocycles. In vivo experiments using J774A.1 macrophages demonstrated that the inhibitors of \(PA_{63}\)-channel function also efficiently block intoxication of the cells by the combination lethal factor and \(PA_{63}\) in the same concentration range as they block the channels in vitro. Conclusions/Significance These results strongly argue in favor of a transport of lethal factor through the \(PA_{63}\)-channel and suggest that the heterocycles used in this study could represent attractive candidates for development of novel therapeutic strategies against anthrax. KW - intoxication KW - chloroquine KW - toxins KW - anthrax KW - cell membranes KW - lipid bilayer KW - macrophages KW - membrane potential Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130097 VL - 8 IS - 6 ER - TY - JOUR A1 - Abdali, Narges A1 - Barth, Enrico A1 - Norouzy, Amir A1 - Schulz, Robert A1 - Nau, Werner M. A1 - Kleinekathofer, Ulrich A1 - Tauch, Andreas A1 - Benz, Roland T1 - Corynebacterium jeikeium jk0268 Constitutes for the 40 Amino Acid Long PorACj, Which Forms a Homooligomeric and Anion- Selective Cell Wall Channel JF - PLoS ONE N2 - Corynebacterium jeikeium, a resident of human skin, is often associated with multidrug resistant nosocomial infections in immunodepressed patients. C. jeikeium K411 belongs to mycolic acid-containing actinomycetes, the mycolata and contains a channel-forming protein as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent treated cell walls and in extracts of whole cells using organic solvents. A gene coding for a 40 amino acid long polypeptide possibly responsible for the pore-forming activity was identified in the known genome of C. jeikeium by its similar chromosomal localization to known porH and porA genes of other Corynebacterium strains. The gene jk0268 was expressed in a porin deficient Corynebacterium glutamicum strain. For purification temporarily histidine-tailed or with a GST-tag at the N-terminus, the homogeneous protein caused channel-forming activity with an average conductance of 1.25 nS in 1M KCl identical to the channels formed by the detergent extracts. Zero-current membrane potential measurements of the voltage dependent channel implied selectivity for anions. This preference is according to single-channel analysis caused by some excess of cationic charges located in the channel lumen formed by oligomeric alpha-helical wheels. The channel has a suggested diameter of 1.4 nm as judged from the permeability of different sized hydrated anions using the Renkin correction factor. Surprisingly, the genome of C. jeikeium contained only one gene coding for a cell wall channel of the PorA/PorH type found in other Corynebacterium species. The possible evolutionary relationship between the heterooligomeric channels formed by certain Corynebacterium strains and the homooligomeric pore of C. jeikeium is discussed. KW - antibiotics KW - detergents KW - anions KW - corynebacterium diphtheriae KW - membrane potential KW - corynebacteria KW - cell walls KW - permeability Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129989 VL - 8 IS - 10 ER - TY - JOUR A1 - Bárcena-Uribarri, Iván A1 - Thein, Marcus A1 - Maier, Elke A1 - Bonde, Mari A1 - Bergström, Sven A1 - Benz, Roland T1 - Use of Nonelectrolytes Reveals the Channel Size and Oligomeric Constitution of the Borrelia burgdorferi P66 Porin JF - PLoS ONE N2 - In the Lyme disease spirochete Borrelia burgdorferi, the outer membrane protein P66 is capable of pore formation with an atypical high single-channel conductance of 11 nS in 1 M KCl, which suggested that it could have a larger diameter than ‘normal’ Gram-negative bacterial porins. We studied the diameter of the P66 channel by analyzing its single-channel conductance in black lipid bilayers in the presence of different nonelectrolytes with known hydrodynamic radii. We calculated the filling of the channel with these nonelectrolytes and the results suggested that nonelectrolytes (NEs) with hydrodynamic radii of 0.34 nm or smaller pass through the pore, whereas neutral molecules with greater radii only partially filled the channel or were not able to enter it at all. The diameter of the entrance of the P66 channel was determined to be \(\leq\)1.9 nm and the channel has a central constriction of about 0.8 nm. The size of the channel appeared to be symmetrical as judged from one-sidedness of addition of NEs. Furthermore, the P66-induced membrane conductance could be blocked by 80–90% by the addition of the nonelectrolytes PEG 400, PEG 600 and maltohexaose to the aqueous phase in the low millimolar range. The analysis of the power density spectra of ion current through P66 after blockage with these NEs revealed no chemical reaction responsible for channel block. Interestingly, the blockage of the single-channel conductance of P66 by these NEs occurred in about eight subconductance states, indicating that the P66 channel could be an oligomer of about eight individual channels. The organization of P66 as a possible octamer was confirmed by Blue Native PAGE and immunoblot analysis, which both demonstrated that P66 forms a complex with a mass of approximately 460 kDa. Two dimension SDS PAGE revealed that P66 is the only polypeptide in the complex. KW - radii KW - hydrodynamics KW - SDS polyacrylamide gel electrophoresis KW - molecular mass KW - outer membrane proteins KW - single channel recording KW - blue native polyacrylamide gel electrophoresis KW - borrelia burgdorferi Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129965 VL - 8 IS - 11 ER - TY - JOUR A1 - Tessmer, Ingrid A1 - Kaur, Parminder A1 - Lin, Jiangguo A1 - Wang, Hong T1 - Investigating bioconjugation by atomic force microscopy JF - Journal of Nanobiotechnology N2 - Nanotechnological applications increasingly exploit the selectivity and processivity of biological molecules. Integration of biomolecules such as proteins or DNA into nano-systems typically requires their conjugation to surfaces, for example of carbon-nanotubes or fluorescent quantum dots. The bioconjugated nanostructures exploit the unique strengths of both their biological and nanoparticle components and are used in diverse, future oriented research areas ranging from nanoelectronics to biosensing and nanomedicine. Atomic force microscopy imaging provides valuable, direct insight for the evaluation of different conjugation approaches at the level of the individual molecules. Recent technical advances have enabled high speed imaging by AFM supporting time resolutions sufficient to follow conformational changes of intricately assembled nanostructures in solution. In addition, integration of AFM with different spectroscopic and imaging approaches provides an enhanced level of information on the investigated sample. Furthermore, the AFM itself can serve as an active tool for the assembly of nanostructures based on bioconjugation. AFM is hence a major workhorse in nanotechnology; it is a powerful tool for the structural investigation of bioconjugation and bioconjugation-induced effects as well as the simultaneous active assembly and analysis of bioconjugation-based nanostructures. KW - nanorobot KW - biosensors KW - nanomedicine KW - nanolithography KW - nanoelectronics KW - bioconjugation KW - nanotechnology KW - atomic force microscopy (AFM) KW - DNA origami KW - single molecule Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129477 VL - 11 IS - 25 ER - TY - JOUR A1 - Hupp, Sabrina A1 - Förtsch, Christina A1 - Wippel, Carolin A1 - Ma, Jiangtao A1 - Mitchell, Timothy J. A1 - Iliev, Asparouh I. T1 - Direct Transmembrane Interaction between Actin and the Pore-Competent, Cholesterol-Dependent Cytolysin Pneumolysin JF - Journal of Molecular Biology N2 - The eukaryotic actin cytoskeleton is an evolutionarily well-established pathogen target, as a large number of bacterial factors disturb its dynamics to alter the function of the host cells. These pathogenic factors modulate or mimic actin effector proteins or they modify actin directly, leading to an imbalance of the precisely regulated actin turnover. Here, we show that the pore-forming, cholesterol-dependent cytolysin pneumolysin (PLY), a major neurotoxin of Streptococcus pneumoniae, has the capacity to bind actin directly and to enhance actin polymerisation in vitro. In cells, the toxin co-localised with F-actin shortly after exposure, and this direct interaction was verified by Förster resonance energy transfer. PLY was capable of exerting its effect on actin through the lipid bilayer of giant unilamellar vesicles, but only when its pore competence was preserved. The dissociation constant of G-actin binding to PLY in a biochemical environment was 170–190 nM, which is indicative of a high-affinity interaction, comparable to the affinity of other intracellular actin-binding factors. Our results demonstrate the first example of a direct interaction of a pore-forming toxin with cytoskeletal components, suggesting that the cross talk between pore-forming cytolysins and cells is more complex than previously thought. KW - pore-forming toxin KW - cholesterol-dependent cytolysin KW - actin KW - membrane KW - pneumolysin Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-132297 VL - 425 IS - 3 ER - TY - JOUR A1 - Aistleitner, Karin A1 - Heinz, Christian A1 - Hoermann, Alexandra A1 - Heinz, Eva A1 - Montanaro, Jacqueline A1 - Schulz, Frederik A1 - Maier, Elke A1 - Pichler, Peter A1 - Benz, Roland A1 - Horn, Matthias T1 - Identification and Characterization of a Novel Porin Family Highlights a Major Difference in the Outer Membrane of Chlamydial Symbionts and Pathogens JF - PLoS ONE N2 - The Chlamydiae constitute an evolutionary well separated group of intracellular bacteria comprising important pathogens of humans as well as symbionts of protozoa. The amoeba symbiont Protochlamydia amoebophila lacks a homologue of the most abundant outer membrane protein of the Chlamydiaceae, the major outer membrane protein MOMP, highlighting a major difference between environmental chlamydiae and their pathogenic counterparts. We recently identified a novel family of putative porins encoded in the genome of P. amoebophila by in silico analysis. Two of these Protochlamydia outer membrane proteins, PomS (pc1489) and PomT (pc1077), are highly abundant in outer membrane preparations of this organism. Here we show that all four members of this putative porin family are toxic when expressed in the heterologous host Escherichia coli. Immunofluorescence analysis using antibodies against heterologously expressed PomT and PomS purified directly from elementary bodies, respectively, demonstrated the location of both proteins in the outer membrane of P. amoebophila. The location of the most abundant protein PomS was further confirmed by immuno-transmission electron microscopy. We could show that pomS is transcribed, and the corresponding protein is present in the outer membrane throughout the complete developmental cycle, suggesting an essential role for P. amoebophila. Lipid bilayer measurements demonstrated that PomS functions as a porin with anion-selectivity and a pore size similar to the Chlamydiaceae MOMP. Taken together, our results suggest that PomS, possibly in concert with PomT and other members of this porin family, is the functional equivalent of MOMP in P. amoebophila. This work contributes to our understanding of the adaptations of symbiotic and pathogenic chlamydiae to their different eukaryotic hosts. KW - cell wall KW - protochlamydia amoebophila KW - escherichia coli KW - matrix protein porin KW - gram negative bacteria KW - single channel analysis KW - developmental cycle KW - mycobacterium smegmatis KW - monoclonal antibodies KW - signal peptides Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131176 VL - 8 IS - 1 ER - TY - JOUR A1 - Zhao, Bo A1 - Zhang, Keya A1 - Bhuripanyo, Karan A1 - Choi, Chan Hee J. A1 - Villhauer, Eric B. A1 - Li, Heng A1 - Zheng, Ning A1 - Kiyokawa, Hiroaki A1 - Schindelin, Hermann A1 - Yin, Jun T1 - Profiling the Cross Reactivity of Ubiquitin with the Nedd8 Activating Enzyme by Phage Display JF - PLoS ONE N2 - The C-terminal peptides of ubiquitin (UB) and UB-like proteins (UBLs) play a key role in their recognition by the specific activating enzymes (E1s) to launch their transfer through the respective enzymatic cascades thus modifying cellular proteins. UB and Nedd8, a UBL regulating the activity of cullin-RING UB ligases, only differ by one residue at their C-termini; yet each has its specific E1 for the activation reaction. It has been reported recently that UAE can cross react with Nedd8 to enable its passage through the UB transfer cascade for protein neddylation. To elucidate differences in UB recognition by UAE and NAE, we carried out phage selection of a UB library with randomized C-terminal sequences based on the catalytic formation of UB similar to NAE thioester conjugates. Our results confirmed the previous finding that residue 72 of UB plays a "gate-keeping" role in E1 selectivity. We also found that diverse sequences flanking residue 72 at the UB C-terminus can be accommodated by NAE for activation. Furthermore heptameric peptides derived from the C-terminal sequences of UB variants selected for NAE activation can function as mimics of Nedd8 to form thioester conjugates with NAE and the downstream E2 enzyme Ubc12 in the Nedd8 transfer cascade. Once the peptides are charged onto the cascade enzymes, the full-length Nedd8 protein is effectively blocked from passing through the cascade for the critical modification of cullin. We have thus identified a new class of inhibitors of protein neddylation based on the profiles of the UB C-terminal sequences recognized by NAE. KW - protein NEDD8 KW - E1 KW - system KW - conjugation KW - pathway KW - complex KW - ligases KW - purification KW - neddylation KW - expression Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-128479 SN - 1932-6203 VL - 8 IS - e70312 ER -