Extracellular calcium reduction strongly increases the lytic capacity of pneumolysin from streptococcus pneumoniae in brain tissue

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

Morphologische Veränderungen von Dendriten und Synapsen durch das Neurotoxin Pneumolysin - Einblicke und pharmakologischer Ansatz

Streptococcus pneumoniae (Pneumococcus) is one of the leading causes of childhood meningitis,pneumonia and sepsis. Despite the availability of childhood vaccination programs and antimicrobial agents, childhood pneumococcal meningitis is still a devastating illness with mortality rates among the highest of any cause of bacterial meningitis. Especially in low-income countries, where medical care is less accessible, mortality rates up to 50 % have been reported. In surviving patients, neurological sequelae, including hearing loss, focal neurological deficits and cognitive impairment, is reported in 30 to 50 %. Growing resistance of pneumococci towards conventional antibiotics emphasize the need for effective therapies and development of effective vaccines against Streptococcus pneumoniae. One major virulence factor of Streptococcus pneumoniae is the protein toxin Pneumolysin (PLY). PLY belongs to a family of structurally related toxins, the so-called cholesterol-dependent cytolysins (CDCs). Pneumolysin is produced by almost all clinical isolates of the bacterium. It is expressed during the late log phase of bacterial growth and gets released mainly through spontaneous autolysis of the bacterial cell. After binding to cholesterol in the host cell membranes, oligomerization of up to 50 toxin monomers and rearrangement of the protein structure, PLY forms large pores, leading to cell lysis in higher toxin concentrations. At sub-lytic concentrations, however, PLY mediates several other effects, such as activation of the classic complement pathway and the induction of apoptosis. First experiments with pneumococcal strains, deficient in pneumolysin, showed a reduced virulence of the organism, which emphasizes the contribution of this toxin to the course of bacterial meningitis and the urgent need for the understanding of the multiple mechanisms leading to invasive pneumococcal disease. The aim of this thesis was to shed light on the contribution of pneumolysin to the course of the disease as well as to the mental illness patients are suffering from after recovery from pneumococcal meningitis. Therefore, we firstly investigated the effects of sub-lytic pneumolysin concentrations onto primary mouse neurons, transfected with a GFP construct and imaged with the help of laser scanning confocal microscopy. We discovered two major morphological changes in the dendrites of primary mouse neurons: The formation of focal swellings along the dendrites (so-called varicosities) and the reduction of dendritic spines. To study these effects in a more complex system, closer to the in vivo situation, we established a reproducible method for acute brain slice culturing. With the help of this culturing method, we were able to discover the same morphological changes in dendrites upon challenge with sub-lytic concentrations of pneumolysin. We were able to reverse the seen alterations in dendritic structure with the help of two antagonists of the NMDA receptor, connecting the toxin´s mode of action to a non-physiological stimulation of this subtype of glutamate receptors. The loss of dendritic spines (representing the postsynapse) in our brain slice model could be verified with the help of brain slices from adult mice, suffering from pneumococcal meningitis. By immunohistochemical staining with an antibody against synapsin I, serving as a presynaptic marker, we were able to identify a reduction of synapsin I in the cortex of mice, infected with a pneumococcal strain which is capable of producing pneumolysin. The reduction of synapsin I was higher in these brain slices compared to mice infected with a pneumococcal strain which is not capable of producing pneumolysin, illustrating a clear role for the toxin in the reduction of dendritic spines. The fact that the seen effects weren´t abolished under calcium free conditions clarifies that not only the influx of calcium through the pneumolysin-pore is responsible for the alterations. These findings were further supported by calcium imaging experiments, where an inhibitor of the NMDA receptor was capable of delaying the time point, when the maximum of calcium influx upon PLY challenge was reached. Additionally, we were able to observe the dendritic beadings with the help of immunohistochemistry with an antibody against MAP2, a neuron-specific cytoskeletal protein. These observations also connect pneumolysin´s mode of action to excitotoxicity, as several studies mention the aggregation of MAP2 in dendritic beadings in response to excitotoxic stimuli. All in all, this is the first study connecting pneumolysin to excitotoxic events, which might be a novel chance to tie in other options of treatment for patients suffering from pneumococcal meningitis.Streptococcus pneumoniae ist einer der Hauptauslöser für bakterielle Meningitis, Lungenentzündung und Sepsis. Ungeachtet der Tatsache, dass es heutzutage viele Impfprogramme zur Prävention, sowie Antibiotika zur Behandlung gibt, ist die bakterielle Meningitis im Kindesalter, ausgelöst durch S. pneumoniae, immer noch eine ernstzunehmende Krankheit mit Sterberaten von bis zu 50 %. Bei 30 bis 50 % der Patienten, die die Krankheit überstehen, bleiben teilweise schwere neurologische Störungen zurück. Die steigende Resistenz des Erregers gegenüber herkömmlichen Antibiotika macht zudem die Dringlichkeit zur Entwicklung effektiver Therapieansätze deutlich. Ein Hauptpathogenitätsfaktor von Streptococcus pneumoniae ist das Proteintoxin Pneumolysin (PLY). PLY gehört zu einer Familie strukturell verwandter Toxine; die sogenannten cholesterinabhängigen Cytolysine (CDCs). Das Toxin wird hauptsächlich nach spontaner Autolyse des Bakteriums freigesetzt. Nach Bindung des Proteins an das Cholesterin in den Zellmembranen des Wirtsorganismus, Oligomerisierung von bis zu 50 Toxinmonomeren und Umordnung der Proteinstruktur, bildet das Toxin Poren in der Zellmembran, die in höheren Konzentrationen von PLY zur Zelllyse führen. In niedrigeren Konzentrationen löst das Toxin jedoch verschiedene andere Prozesse, darunter Apoptose und Aktivierung des Komplementsystems, aus. Erste Experimente, die mit einem mutierten Pneumokokkenstamm (unfähig, Pneumolysin zu exprimieren) durchgeführt wurden, konnten eine reduzierte Virulez des Erregers zeigen, was die Beteiligung des Toxins am Verlauf der Krankheit verdeutlicht. Ziel vorliegender Arbeit war, die Beteiligung von Pneumolysin sowohl am Verlauf der bakteriellen Meningitis, hervorgerufen durch Pneumolysin, zu erforschen, als auch dessen Beteiligung an der Entstehung von neurologischen Störungen, wie sie auch nach Rehabilitation von einer Meningitis noch bestehen können. Dafür wurden zuerst die Effekte von sublytischen Konzentrationen des Toxins auf primäre Mausneuronen (transfiziert mit GFP und mit Hilfe eines Konfokalmikroskopes aufgenommen) erfasst. Dabei zeigten sich hauptsächlich zwei morphologische Veränderungen in den Dendriten der mikroskopierten Neurone: Die Entstehung von fokalen Schwellungen der Dendriten (sogenannte „varicosities“) sowie eine Verminderung der Anzahl von dendritischen Dornfortsätzen (sogenannte „dendritic spines“). Um diese Effekte in einem komplexeren System näher untersuchen zu können, entwickelten wir eine reproduzierbare Methode um akute Gehirnschnitte über längere Zeit kultivieren zu können. Mithilfe dieser Methode konnten wir die Veränderungen, die wir schon in der Primärkultur beobachteten, ebenso nachweisen. Die Entwicklung der fokalen Schwellungen der Dendriten konnten mithilfe zweier Antagonisten des NMDA Rezeptors rückgängig gemacht werden, wodurch erstmals eine Verbindung der Effekte mit einer Aktivierung von Glutamatrezeptoren aufgezeigt wurde. Die Verminderung der Anzahl dendritischer Dornfortsätze im Gehirnschnittmodell wurde untermauert von den Ergebnissen, die wir durch Gehirnschnitte von Mäusen, die tatsächlich an pneumokokkaler Meningitis erkrankt waren, erlangen konnten. Durch immunohistologische Färbungen mit einem Antikörper gegen Synapsin I (ein präsynaptisches Protein) konnten wir eine Reduktion dieses Proteins im Cortex erkrankter Mäuse nachweisen. Die Tatsache, dass die morphologischen Veränderungen der Dendriten ebenfalls in calciumfreiem Puffer beobachtet werden konnten, macht deutlich, dass nicht nur der Calciuminflux durch die Pneumolysinpore verantwortlich ist für dessen Neurotoxizität. Diese These wird untermauert durch die Ergebnisse, die wir mithilfe der Calciummikroskopie erhielten: Die Applikation eines Antagonisten des NMDA Rezeptors konnte den Zeitpunkt des maximalen Calciuminfluxes in die Zelle nach Behandlung mit Pneumolysin hinauszögern. Zudem konnten wir die Schwellungen in den Dendriten auch durch einen Antikörper gegen MAP2 (ein neuronenspezifisches Protein des Zytoskeletts) darstellen, was ebenfalls eine Verbindung von Pneumolysin zu „excitotoxicity“ (Toxizität aufgrund einer Übererregung von Glutamatrezeptoren) darstellt, da verschiedene Studien die Aggregation von MAP2 in fokalen dendritischen Schwellungen als Reaktion auf die Einwirkung von „excitotoxischen“ Stimuli nachweisen konnten. Zusammenfassend lässt sich sagen, dass dies die erste Studie ist, die Pneumolysin in Zusammenhang mit einer Überaktivierung von Glutamatrezeptoren bringt, was eine komplett neue Sichtweise darstellt und eventuell neue Möglichkeiten der Therapie für Patienten, die an dieser Form der bakteriellen Hirnhautentzündung leiden, eröffnet

Bacterial cytolysin during meningitis disrupts the regulation of glutamate in the brain, leading to synaptic damage

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

NMDA dependence of the dendritic changes caused by pneumolysin.

<p><i>A.</i> Inhibition of the formation of dendritic swellings caused by treatment with 0.2 µg/ml PLY for 5 h by the application of 10 µM of the non-competitive NMDA-receptor inhibitor MK801. *** p<0.001 vs. all. <i>B.</i> Preserved dendritic spine number following treatment with 10 µM MK801 together with 0.2 µg/ml PLY for 5 h. * p<0.05, ** p<0.01. <i>C.</i> Reversal of the PSD95 density loss by PLY when incubated with 10 µM MK801. <i>D.</i> Complete inhibition of dendritic swelling formation caused by treatment with 0.2 µg/ml PLY for 5 h using a 50 µM of the competitive NMDA-receptor antagonist D-AP5. *** p<0.001. All values represent the mean ± SEM; n = 5–6 slices from at least 3 independent experiments.</p

Dendritic and synaptic changes caused by pneumolysin in acute brain slices.

<p><i>A.</i> Equivalent cell lysis (LDH release) between slices that were mock treated or treated with 0.2 µg/ml PLY for 8 h. <i>B.</i> A DiI-stained pyramidal neuron in the neocortex of an acute mouse slice demonstrated a normal spine and dendrite morphology (mock) in contrast to a PLY-treated slice (0.2 µg/ml for 5 h), which showed a reduction in spine number and multiple dendritic enlargements (swellings). Scale bars: 10 µm. <i>C.</i> Magnified dendritic fragments, demonstrating the dendrite configuration and the morphology of the dendritic spines. Scale bars: 10 µm. <i>D.</i> Increased number of dendritic swellings after exposure to 0.2 µg/ml PLY for 5 h. *** p<0.001. <i>E.</i> Decreased number of dendritic spines following 5 h of exposure to 0.2 µg/ml PLY. ** p<0.01. <i>F.</i> Reduced number of PSD95-positive fluorescent puncta in the neocortices of slices treated with 0.2 µg/ml PLY for 5 h. * p<0.05. <i>G.</i> Unchanged number of synapsin I-positive fluorescent puncta in the neocortices of slices treated with 0.2 µg/ml PLY for 5 h. <i>H.</i> Western blot analysis of the protein levels of synapsin I, PSD95 and actin in acute mouse brain slices treated with 0.2 µg/ml PLY for 5 h or in mock-treated slices. <i>I.</i> Unchanged protein expression levels of synapsin I and PSD95 in acute mouse brain slices (normalized to the corresponding levels of actin). <i>J.</i> The delta6 non-pore forming mutant of PLY did not produce varicosity increase and dendritic spine loss. All values are presented as the mean ± SEM; n = 6 slices from at least 3 independent experiments.</p

Reduced synaptic density in human postmortem pneumococcal meningitis neocortical brain tissue samples.

<p><i>A.</i> Schematic representation of the analyzed neocortical regions. <i>B</i>, <i>C.</i> Decreased synapsin I (<i>B</i>) and the PSD95 (<i>C</i>) staining densities in layers I–II of the frontal neocortex of human post-mortem samples from <i>S. pneumoniae</i> meningitis cases (S. pneumoniae) <i>vs.</i> post mortem samples of cases who experienced rapid non-neurological death (Non-meningitis). <i>D.</i> Representative tissue samples (layer II) with anti-synapsin I immunohistochemistry. Scale bar: 10 µm. <i>E.</i> There was no difference in the number of TUNEL-positive nuclei in neocortical layers I–II between non-meningitis and meningitis samples. All values represent the mean ± SEM, and samples from 5 to 6 cases per group were analyzed.</p

Clinical histories of the individual patients from the <i>Streptococcus pneumoniae</i> meningitis histology group.

<p>Note: the synaptic/cell death analysis in our tissue samples involved non-necrotic tissue areas.</p

Kinetics of toxin tissue binding.

<p>Measurement of the fluorescence intensity of GFP-tagged PLY (PLY) in the medium following incubation of brain slices (6 slices per well) challenged with either 0.5 or 2 µg/ml PLY-GFP. The initial toxin concentration in the medium was high but rapidly (within minutes) decreased due to tissue binding. In the enlarged diagram (upper right), a rescaled y-axis fragment of the 0.5 µg/ml PLY experiment is presented.</p

Reduced synaptic density in mouse pneumococcal meningitis neocortical brain tissue samples.

<p><i>A.</i> Reduced synapsin staining in layers I–II of the neocortex in animals with meningitis by PLY-producing bacteria <i>vs.</i> all other groups 36 h after injection. * p<0.05. (D39) indicates the group of mice injected intracerebrally with the pneumolysin (PLY)-producing D39 <i>S. pneumoniae</i> strain; (PLY-) mice indicates those infected with the PLY-deficient D39 strain. <i>B.</i> Reduced staining was observed for PSD95 in layers I–II of the frontal neocortex of mice injected with the PLY-producing strain <i>vs.</i> the PLY-deficient D39 strain animals after 36 h. All values are presented as the mean ± SEM. There were 5 animals in the mock group and 10–13 in the meningitis group. <i>C.</i> Representative tissue sample images with anti-synapsin I immunohistochemistry of layers I–III with magnification of equivalent areas of interest in layer I. Scale bar: 15 µm. <i>D.</i> Representative images of the TUNEL-FITC staining of equivalent areas in layers I/II of the neocortex of mice infected with D39 and PLY-deficient pneumococcal strain, where no TUNEL-positive cells are present. All nuclei were counterstained with propidium iodide (PI). TUNEL-negative control (enzyme missing) and TUNEL-positive control (pretreatment with DNAseI) are presented for staining validation. Scale bar: 20 µm.</p

Increased glutamate release and calcium changes caused by pneumolysin.

<p><i>A.</i> Representative sample of three experiments demonstrating increased neocortical glutamate content (via electrochemical detection in an acute slice; a diagram of the electrode is presented) following 0.2 µg/ml PLY exposure. <i>B.</i> Elevation of glutamate release on the surface of a monolayer of mouse astrocytes by a treatment with 0.1 µg/ml PLY in buffer containing 2 mM extracellular calcium (Ca-rich) <i>vs.</i> unchanged glutamate levels in calcium-free buffer (Ca-free). A permeabilization diagram (propidium iodide-positive cells) is presented above the glutamate release diagram. The values are presented as the means ± SEM; n = 3–5 experiments. <i>C.</i> Increase in cytosolic calcium (Fura-2-loaded mouse astrocytes) following treatment with 0.1 µg/ml PLY and a 10 µM ionomycin control at 800 s in 2 mM calcium-containing buffer (representative experiment). The experiments were repeated 5 times with identical results. <i>D.</i> Unchanged cytosolic calcium concentration following an identical incubation as in <i>C.</i>, but under calcium-free extracellular buffer conditions. <i>E.</i> Preserved glutamate uptake in brain slices following 0.2 µg/ml PLY challenge for 4 h; n = 3 experiments.</p