@article{FoertschHuppMaetal.2011, author = {F{\"o}rtsch, Christina and Hupp, Sabrina and Ma, Jiangtao and Mitchell, Timothy J. and Maier, Elke and Benz, Roland and Iliev, Asparouh I.}, title = {Changes in Astrocyte Shape Induced by Sublytic Concentrations of the Cholesterol-Dependent Cytolysin Pneumolysin Still Require Pore-Forming Capacity}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69084}, year = {2011}, abstract = {Streptococcus pneumoniae is a common pathogen that causes various infections, such as sepsis and meningitis. A major pathogenic factor of S. pneumoniae is the cholesterol-dependent cytolysin, pneumolysin. It produces cell lysis at high concentrations and apoptosis at lower concentrations. We have shown that sublytic amounts of pneumolysin induce small GTPase-dependent actin cytoskeleton reorganization and microtubule stabilization in human neuroblastoma cells that are manifested by cell retraction and changes in cell shape. In this study, we utilized a live imaging approach to analyze the role of pneumolysin's pore-forming capacity in the actin-dependent cell shape changes in primary astrocytes. After the initial challenge with the wild-type toxin, a permeabilized cell population was rapidly established within 20-40 minutes. After the initial rapid permeabilization, the size of the permeabilized population remained unchanged and reached a plateau. Thus, we analyzed the non-permeabilized (non-lytic) population, which demonstrated retraction and shape changes that were inhibited by actin depolymerization. Despite the non-lytic nature of pneumolysin treatment, the toxin's lytic capacity remained critical for the initiation of cell shape changes. The non-lytic pneumolysin mutants W433F-pneumolysin and delta6-pneumolysin, which bind the cell membrane with affinities similar to that of the wild-type toxin, were not able to induce shape changes. The initiation of cell shape changes and cell retraction by the wild-type toxin were independent of calcium and sodium influx and membrane depolarization, which are known to occur following cellular challenge and suggested to result from the ion channel-like properties of the pneumolysin pores. Excluding the major pore-related phenomena as the initiation mechanism of cell shape changes, the existence of a more complex relationship between the pore-forming capacity of pneumolysin and the actin cytoskeleton reorganization is suggested.}, subject = {Toxikologie}, language = {en} } @phdthesis{Hupp2012, author = {Hupp, Sabrina}, title = {Modulation of Actin Dynamics by the Cholesterol-Dependent Cytolysin Pneumolysin - a novel mechanism beyond pore formation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70889}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Streptococcus pneumoniae is one of the major causes of bacterial meningitis, which mainly affects young infants in the developing countries of Africa, Asia (esp. India) and South America, and which has case fatality rates up to 50\% in those regions. Bacterial meningitis comprises an infection of the meninges and the sub-meningeal cortex tissue of the brain, whereat the presence of pneumolysin (PLY), a major virulence factor of the pneumococcus, is prerequisite for the development of a severe outcome of the infection and associated tissue damage (e. g. apoptosis, brain edema, and ischemia). Pneumolysin belongs to the family of pore forming, cholesterol-dependent cytolysins (CDCs), bacterial protein toxins, which basically use membrane-cholesterol as receptor and oligomerize to big aggregates, which induce cell lysis and cell death by disturbance of membrane integrity. Multiple recent studies, including this work, have revealed a new picture of pneumolysin, whose cell-related properties go far beyond membrane binding, pore formation and the induction of cell death and inflammatory responses. For a long time, it has been known that bacteria harm the tissues of their hosts in order to promote their own survival and proliferation. Many bacterial toxins aim to rather hijack cells than to kill them, by interacting with cellular components, such as the cytoskeleton or other endogenous proteins. This study was able to uncover a novel capacity of pneumolysin to interact with components of the actin machinery and to promote rapid, actin-dependent cell shape changes in primary astrocytes. The toxin was applied in disease-relevant concentrations, which were verified to be sub-lytic. These amounts of toxin induced a rapid actin cortex collapse in horizontal direction towards the cell core, whereat membrane integrity was preserved, indicating an actin severing function of pneumolysin, and being consistent with cell shrinkage, displacement, and blebbing observed in live cell imaging experiments. In contrast to neuroblastoma cells, in which pneumolysin led to cytoskeleton remodeling and simultaneously to activation of Rac1 and RhoA, in primary astrocytes the cell shape changes were seen to be primarily independent of small GTPases. The level of activated Rac1 and RhoA did not increase at the early time points after toxin application, when the initial shape changes have been observed, but at later time points when the actin-dependent displacement of cells was slower and less severe, probably presenting the cell's attempt to re-establish proper cytoskeleton function. A GUV (giant unilamellar vesicle) approach provided insight into the effects of pneumolysin in a biomimetic system, an environment, which is strictly biochemical, but still comprises cellular components, limited to the factors of interest (actin, Arp2/3, ATP, and Mg2+ on one side, and PLY on the other side). This approach was able to show that the wildtype-toxin, but not the Δ6 mutant (mutated in the unfolding domain, and thus non-porous), had the capacity to exhibit its functions through a membrane bilayer, meaning it was able to aggregate actin, which was located on the other side of the membrane, either via direct interaction with actin or in an Arp2/3 activating manner. Taking a closer look at these two factors with the help of several different imaging and biochemical approaches, this work unveiled the capacity of pneumolysin to bind and interact both with actin and Arp2 of the Arp2/3 complex. Pneumolysin was capable to slightly stabilize actin in an actin-pyrene polymerization assay. The same experimental setup was applied to show that the toxin had the capacity to lead to actin polymerization through activation of the Arp2/3 complex. This effect was additionally confirmed with the help of fluorescent microscopy of rhodamine (TRITC)-tagged actin. Strongest Arp2/3 activation, and actin nucleation/polymerization is achieved by the VCA domain of the WASP family proteins. However, addition of PLY to the Arp2/3-VCA system led to an enhanced actin nucleation, suggesting a synergistic activation function of pneumolysin. Hence, two different effects of pneumolysin on the actin cytoskeleton were observed. On the one hand an actin severing property, and on the other hand an actin stabilization property, both of which do not necessarily exclude each other. Actin remodeling is a common feature of bacterial virulence strategies. This is the first time, however, that these properties were assigned to a toxin of the CDC family. Cytoskeletal dysfunction in astrocytes leads to dysfunction and unregulated movement of these cells, which, in context of bacterial meningitis, can favor bacterial penetration and spreading in the brain tissue, and thus comprises an additional role of pneumolysin as a virulence factor of Streptococcus pneumonia in the context of brain infection.}, subject = {Hirnhautentz{\"u}ndung}, language = {en} } @article{WippelFoertschHuppetal.2011, author = {Wippel, Carolin and F{\"o}rtsch, Christina and Hupp, Sabrina and Maier, Elke and Benz, Roland and Ma, Jiangtao and Mitchell, Timothy J and Iliev, Asparouh I}, title = {Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue}, series = {The Journal of Infectious Diseases}, volume = {204}, journal = {The Journal of Infectious Diseases}, number = {6}, doi = {10.1093/infdis/jir434}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-139356}, pages = {930-936}, year = {2011}, abstract = {Background Streptococcus pneumoniae causes serious diseases such as pneumonia and meningitis. Its major pathogenic factor is the cholesterol-dependent cytolysin pneumolysin, which produces lytic pores at high concentrations. At low concentrations, it has other effects, including induction of apoptosis. Many cellular effects of pneumolysin appear to be calcium dependent. Methods  Live imaging of primary mouse astroglia exposed to sublytic amounts of pneumolysin at various concentrations of extracellular calcium was used to measure changes in cellular permeability (as judged by lactate dehydrogenase release and propidium iodide chromatin staining). Individual pore properties were analyzed by conductance across artificial lipid bilayer. Tissue toxicity was studied in continuously oxygenated acute brain slices. Results  The reduction of extracellular calcium increased the lytic capacity of the toxin due to increased membrane binding. Reduction of calcium did not influence the conductance properties of individual toxin pores. In acute cortical brain slices, the reduction of extracellular calcium from 2 to 1 mM conferred lytic activity to pathophysiologically relevant nonlytic concentrations of pneumolysin. Conclusions  Reduction of extracellular calcium strongly enhanced the lytic capacity of pneumolysin due to increased membrane binding. Thus, extracellular calcium concentration should be considered as a factor of primary importance for the course of pneumococcal meningitis. "}, language = {en} } @article{HuppFoertschWippeletal.2013, author = {Hupp, Sabrina and F{\"o}rtsch, Christina and Wippel, Carolin and Ma, Jiangtao and Mitchell, Timothy J. and Iliev, Asparouh I.}, title = {Direct Transmembrane Interaction between Actin and the Pore-Competent, Cholesterol-Dependent Cytolysin Pneumolysin}, series = {Journal of Molecular Biology}, volume = {425}, journal = {Journal of Molecular Biology}, number = {3}, doi = {10.1016/j.jmb.2012.11.034}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-132297}, pages = {636-646}, year = {2013}, abstract = {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 F{\"o}rster 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.}, language = {en} } @article{WippelMaurerFortschetal.2013, author = {Wippel, Carolin and Maurer, Jana and Fortsch, Christina and Hupp, Sabrina and Bohl, Alexandra and Ma, Jiangtao and Mitchell, Timothy J. and Bunkowski, Stephanie and Br{\"u}ck, Wolfgang and Nau, Roland and Iliev, Asparouh I.}, title = {Bacterial Cytolysin during Meningitis Disrupts the Regulation of Glutamate in the Brain, Leading to Synaptic Damage}, series = {PLoS Pathogens}, volume = {9}, journal = {PLoS Pathogens}, number = {6}, doi = {10.1371/journal.ppat.1003380}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130462}, pages = {e1003380}, year = {2013}, abstract = {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.}, language = {en} } @article{MaurerHuppBischoffetal.2017, author = {Maurer, Jana and Hupp, Sabrina and Bischoff, Carolin and Foertsch, Christina and Mitchell, Timothy J. and Chakraborty, Trinad and Iliev, Asparouh I.}, title = {Distinct neurotoxicity profile of listeriolysin O from \(Listeria\) \(monocytogenes\)}, series = {Toxins}, volume = {9}, journal = {Toxins}, number = {1}, doi = {10.3390/toxins9010034}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-172130}, year = {2017}, abstract = {Cholesterol-dependent cytolysins (CDCs) are protein toxins that originate from Gram-positive bacteria and contribute substantially to their pathogenicity. CDCs bind membrane cholesterol and build prepores and lytic pores. Some effects of the toxins are observed in non-lytic concentrations. Two pathogens, \(Streptococcus\) \(pneumoniae\) and \(Listeria\) \(monocytogenes\), cause fatal bacterial meningitis, and both produce toxins of the CDC family—pneumolysin and listeriolysin O, respectively. It has been demonstrated that pneumolysin produces dendritic varicosities (dendrite swellings) and dendritic spine collapse in the mouse neocortex, followed by synaptic loss and astrocyte cell shape remodeling without elevated cell death. We utilized primary glial cultures and acute mouse brain slices to examine the neuropathological effects of listeriolysin O and to compare it to pneumolysin with identical hemolytic activity. In cultures, listeriolysin O permeabilized cells slower than pneumolysin did but still initiated non-lytic astrocytic cell shape changes, just as pneumolysin did. In an acute brain slice culture system, listeriolysin O produced dendritic varicosities in an NMDA-dependent manner but failed to cause dendritic spine collapse and cortical astrocyte reorganization. Thus, listeriolysin O demonstrated slower cell permeabilization and milder glial cell remodeling ability than did pneumolysin and lacked dendritic spine collapse capacity but exhibited equivalent dendritic pathology.}, language = {en} } @article{HuppRosenkranzBonfigetal.2019, author = {Hupp, Sabrina and Rosenkranz, Maaria and Bonfig, Katharina and Pandey, Chandana and Roitsch, Thomas}, title = {Noninvasive Phenotyping of Plant-Pathogen Interaction: Consecutive In Situ Imaging of Fluorescing Pseudomonas syringae, Plant Phenolic Fluorescence, and Chlorophyll Fluorescence in Arabidopsis Leaves}, series = {Frontiers in Plant Science}, volume = {10}, journal = {Frontiers in Plant Science}, number = {1239}, issn = {1664-462X}, doi = {10.3389/fpls.2019.01239}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-189425}, year = {2019}, abstract = {Plant-pathogen interactions have been widely studied, but mostly from the site of the plant secondary defense. Less is known about the effects of pathogen infection on plant primary metabolism. The possibility to transform a fluorescing protein into prokaryotes is a promising phenotyping tool to follow a bacterial infection in plants in a noninvasive manner. In the present study, virulent and avirulent Pseudomonas syringae strains were transformed with green fluorescent protein (GFP) to follow the spread of bacteria in vivo by imaging Pulse-Amplitude-Modulation (PAM) fluorescence and conventional binocular microscopy. The combination of various wavelengths and filters allowed simultaneous detection of GFP-transformed bacteria, PAM chlorophyll fluorescence, and phenolic fluorescence from pathogen-infected plant leaves. The results show that fluorescence imaging allows spatiotemporal monitoring of pathogen spread as well as phenolic and chlorophyll fluorescence in situ, thus providing a novel means to study complex plant-pathogen interactions and relate the responses of primary and secondary metabolism to pathogen spread and multiplication. The study establishes a deeper understanding of imaging data and their implementation into disease screening.}, language = {en} }