Pneumolysin and the bacterial capsule of Streptococcus pneumoniae cooperatively inhibit taxis and motility of microglia.

BACKGROUND: Streptococcus pneumoniae is the cause of a highly lethal form of meningitis in humans. Microglial cells in the brain represent the first line of defense against pathogens, and they participate in the inflammatory response. The cholesterol-dependent cytolysin pneumolysin and the bacterial capsule are key pathogenic factors, known to exacerbate the course of pneumococcal meningitis. METHODS: We utilized live imaging and immunostaining of glial cells in dissociated and acute brain slice cultures to study the effect of pneumococcal factors, including the cholesterol-dependent cytolysin pneumolysin and the pneumococcal capsule, on microglial motility and taxis. RESULTS: In brain tissue, primary microglia cells showed an enhanced response towards lysates from bacteria lacking capsules and pneumolysin as they moved rapidly to areas with an abundance of bacterial factors. The presence of bacterial capsules and pneumolysin cumulatively inhibited microglial taxis. In mixed cultures of astrocytes and microglia, the motility of microglia was inhibited by capsular components within minutes after exposure. The reduced motility was partially reversed by mannan, a mannose receptor inhibitor. The effects on microglia were not mediated by astrocytes because pure microglial cells responded to various pneumococcal lysates similarly with distinct cell shape changes as seen in mixed cultures. CONCLUSIONS: Our data indicate that microglia possess the capacity for a very agile response towards bacterial pathogens, but key pathogenic factors, such as pneumococcal capsules and pneumolysin, inhibited this response shortly after a bacterial challenge. Furthermore, we demonstrate for the first time that the bacterial capsule affects cellular behaviors such as motility and taxis.

The inflammatory response and neuronal injury in Streptococcus suis meningitis.

BACKGROUND: Many of the currently used models of bacterial meningitis have limitations due to direct inoculation of pathogens into the cerebrospinal fluid or brain and a relatively insensitive assessment of long-term sequelae. The present study evaluates the utility of a Streptococcus (S.) suis intranasal infection model for the investigation of experimental therapies in meningitis. METHODS: We examined the brains of 10 piglets with S. suis meningitis as well as 14 control piglets by histology, immunohistochemistry and in-situ tailing for morphological alterations in the hippocampal dentate gyrus and microglial activation in the neocortex. RESULTS: In piglets with meningitis, the density of apoptotic neurons was significantly higher than in control piglets. Moreover, scoring of microglial morphology revealed a significant activation of these cells during meningitis. The slight increase in the density of dividing cells, young neurons and microglia observed in piglets suffering from meningitis was not statistically significant, probably because of the short time frame between onset of clinical signs and organ sampling. CONCLUSIONS: The morphological changes found during S. suis meningitis are in accordance with abnormalities in other animal models and human autopsy cases. Therefore, the pig should be considered as a model for evaluating effects of experimental therapeutic approaches on neurological function in bacterial meningitis.

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.

Glucose and Oxygen Levels Modulate the Pore-Forming Effects of Cholesterol-Dependent Cytolysin Pneumolysin from Streptococcus pneumoniae.

A major Streptococcus pneumoniae pathogenic factor is the cholesterol-dependent cytolysin pneumolysin, binding membrane cholesterol and producing permanent lytic or transient pores. During brain infections, vascular damage with variable ischemia occurs. The role of ischemia on pneumolysin's pore-forming capacity remains unknown. In acute brain slice cultures and primary cultured glia, we studied acute toxin lysis (via propidium iodide staining and LDH release) and transient pore formation (by analyzing increases in the intracellular calcium). We analyzed normal peripheral tissue glucose conditions (80 mg%), normal brain glucose levels (20 mg%), and brain hypoglycemic conditions (3 mg%), in combinations either with normoxia (8% oxygen) or hypoxia (2% oxygen). At 80 mg% glucose, hypoxia enhanced cytolysis via pneumolysin. At 20 mg% glucose, hypoxia did not affect cell lysis, but impaired calcium restoration after non-lytic pore formation. Only at 3 mg% glucose, during normoxia, did pneumolysin produce stronger lysis. In hypoglycemic (3 mg% glucose) conditions, pneumolysin caused a milder calcium increase, but restoration was missing. Microglia bound more pneumolysin than astrocytes and demonstrated generally stronger calcium elevation. Thus, our work demonstrated that the toxin pore-forming capacity in cells continuously diminishes when oxygen is reduced, overlapping with a continuously reduced ability of cells to maintain homeostasis of the calcium influx once oxygen and glucose are reduced.

Astrocytic tissue remodeling by the meningitis neurotoxin pneumolysin facilitates pathogen tissue penetration and produces interstitial brain edema.

Astrocytes represent a major component of brain tissue and play a critical role in the proper functioning and protection of the brain. Streptococcus pneumoniae, the most common cause of bacterial meningitis, has a high lethality and causes serious disabilities in survivors. Pneumolysin (PLY), a member of the cholesterol-dependent cytolysin group and a major S. pneumoniae neurotoxin, causes deterioration over the course of experimental S. pneumoniae meningitis. At disease-relevant sub-lytic concentrations, PLY produces actin and tubulin reorganization and astrocyte cell shape changes in vitro. In this article, we show that sub-lytic amounts of PLY remodel brain tissue and produce astrocytic process retraction, cortical astroglial reorganization and increased interstitial fluid retention, which is manifested as tissue edema. These changes caused increased tissue permeability to macromolecules and bacteria. The pore-forming capacity of PLY remained necessary for these changes because none of the nonpore-forming mutants were capable of producing similar effects. We suggest that PLY can increase the permeability of brain tissue toward pathogenic factors and bacteria in the course of meningitis, thus contributing to the deterioration caused by the disease.

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.

Easy to build cost-effective acute brain slice incubation system for parallel analysis of multiple treatment conditions.

BACKGROUND: Acute brain slices represent a powerful tool for analysis of brain function in physiology and pathology. Commercial systems and custom-build solutions with carbogen (95% O2/5% CO2) aeration, but they are expensive, have a high working volume requiring large amount of substances, and only limited options for treatment in parallel are possible. NEW METHOD: We developed a novel cost-effective incubation system using materials available in every laboratory, allowing parallel incubation of several treatment conditions, thus also reducing the number of experimental animals. Our system incubation parameters were optimized for cortical neuron observation. RESULTS: We tested several different options using 6, 12 or 24 standard culture well plates, combining them with cell strainer baskets inside. The system was placed in a pre-warmed incubator at 37 °C. Carbogen was injected through a 22 gauge needle, positioned between the basket and the wall of the well. Best results were achieved in a 6-well plate. In 12 and 24-well plates bubbles accumulated beneath the basket, displacing it upwards, making it unsuitable for our purposes. The gas oxygenized the medium without mechanically disturbing the slices, protected within the strainer basket, but still allowing optimal diffusion through the 100 µm pores. In a 6-well plate, six simultaneous treatments were possible in parallel. LDH/Cytotoxicity tests showed an acute toxicity of less than 7%. The system lost about 2.5% per hour of the fluid through evaporation, which was replenished every 2 h. Up to 6 h after treatment, however, this evaporation was excellently tolerated by the neurons even without fluid replenishment, most probably due to the anti-swelling effect of the mildly hypertonic medium. We performed two staining procedures, working excellently with this experimental setup, namely - a modified DiI staining and a slice silver impregnation method, both confirming the intact neuronal morphology. Preserved CA3 calcium influx and removal response following KCl depolarization confirmed the normal physiology of the pyramidal neurons 6 h after exposure in the system. COMPARISON TO EXISTING METHODS: The proposed system is much cheaper than the commercial solutions, can be constructed in any lab, allows up to 6 different treatments in parallel, which none of the existing systems allows. Antibiotic presence in the incubation medium and adequate evaporation control is required if longer incubation (> 6 h) is needed. Lower incubation volumes (3-6 ml) allow sparing expensive reagents. Our procedure was optimized for cortical neurons, further fine tuning to meet other specific requirements is possible. CONCLUSIONS: The system we propose allows filling the gap for budget solutions for short to mid-term incubation of acute brain slices.

Pneumolysin boosts the neuroinflammatory response to Streptococcus pneumoniae through enhanced endocytosis.

In pneumococcal meningitis, bacterial growth in the cerebrospinal fluid results in lysis, the release of toxic factors, and subsequent neuroinflammation. Exposure of primary murine glia to Streptococcus pneumoniae lysates leads to strong proinflammatory cytokine and chemokine production, blocked by inhibition of the intracellular innate receptor Nod1. Lysates enhance dynamin-dependent endocytosis, and dynamin inhibition reduces neuroinflammation, blocking ligand internalization. Here we identify the cholesterol-dependent cytolysin pneumolysin as a pro-endocytotic factor in lysates, its elimination reduces their proinflammatory effect. Only pore-competent pneumolysin enhances endocytosis in a dynamin-, phosphatidylinositol-3-kinase- and potassium-dependent manner. Endocytic enhancement is limited to toxin-exposed parts of the membrane, the effect is rapid and pneumolysin permanently alters membrane dynamics. In a murine model of pneumococcal meningitis, mice treated with chlorpromazine, a neuroleptic with a complementary endocytosis inhibitory effect show reduced neuroinflammation. Thus, the dynamin-dependent endocytosis emerges as a factor in pneumococcal neuroinflammation, and its enhancement by a cytolysin represents a proinflammatory control mechanism.

Dendritic spine loss deep in the neocortex and dendrite distortion with diffusion disturbances occur early in experimental pneumococcal meningitis.

Introduction: Streptococcus pneumoniae (pneumococcus) meningitis is a serious disease with substantial lethality and long-term disability in survivors. Loss of synaptic staining in the superficial layers of the neocortex in rodent models and in humans, and pneumolysin (a major pneumococcal toxin)-dependent dendritic spine collapse in brain slices have been described. It remains unclear how deep in the neocortex more discrete changes are present, how soon after disease onset these changes occur, and whether other properties of dendrites are also affected. Methods: Using a mouse model of pneumococcal meningitis, we studied changes in the neocortex shortly (3-6 h) after the onset of clinical symptoms via modified Golgi-Cox silver staining. Results: Dendritic changes were present in areas with otherwise unchanged cell numbers and no signs of necrosis or other apparent neuronal pathology. Mature dendritic spines were reduced in the pyramidal neurons running through layers 1-5. Additionally, spine morphology changes (swelling, spine neck distortion), were also observed in the deeper layers 4 and 5 of the neocortex. Immature spines (filopodia) remained unchanged between groups, as well as the dendritic arborization of the analyzed neurons. In a third of the animals with meningitis, massive mechanical distortion of the primary dendrites of most of the pyramidal neurons through layers 1-5 was observed. This distortion was reproduced in acute brain slices after exposure to pneumolysin-containing bacterial lysates (S. pneumoniae D39 strain), but not to lysates of pneumolysin-deficient bacteria, which we explain by the tissue remodeling effect of the toxin. Experimental mechanical dendrite distortion in primary neural cultures demonstrated diminished FRAP diffusion of neuronally-expressed enhanced green fluorescent protein (eGFP), indicative of disturbed dendritic diffusion. Discussion: Our work extends earlier knowledge of synaptic loss in the superficial cortical layers during meningitis to deeper layers. These changes occurred surprisingly early in the course of the disease, substantially limiting the effective therapeutic window. Methodologically, we demonstrate that the dendritic spine collapse readout is a highly reliable and early marker of neural damage in pneumococcal meningitis models, allowing for reduction of the total number of animals used per a group due to much lower variation among animals.

Meningitis-associated pneumococcal serotype 8, ST 53, strain is hypervirulent in a rat model and has non-haemolytic pneumolysin which can be attenuated by liposomes.

Introduction: Streptococcus pneumoniae bacteria cause life-threatening invasive pneumococcal disease (IPD), including meningitis. Pneumococci are classified into serotypes, determined by differences in capsular polysaccharide and both serotype and pneumolysin toxin are associated with disease severity. Strains of serotype 8, ST 53, are increasing in prevalence in IPD in several countries. Methods: Here we tested the virulence of such an isolate in a rat model of meningitis in comparison with a serotype 15B and a serotype 14 isolate. All three were isolated from meningitis patients in South Africa in 2019, where serotype 8 is currently the most common serotype in IPD. Results and Discussion: Only the serotype 8 isolate was hypervirulent causing brain injury and a high mortality rate. It induced a greater inflammatory cytokine response than either the serotype 15B or 14 strain in the rat model and from primary mixed-glia cells isolated from mouse brains. It had the thickest capsule of the three strains and produced non-haemolytic pneumolysin. Pneumolysin-sequestering liposomes reduced the neuroinflammatory cytokine response in vitro indicating that liposomes have the potential to be an effective adjuvant therapy even for hypervirulent pneumococcal strains with non-haemolytic pneumolysin.

Rapid microtubule bundling and stabilization by the Streptococcus pneumoniae neurotoxin pneumolysin in a cholesterol-dependent, non-lytic and Src-kinase dependent manner inhibits intracellular trafficking.

Streptococcus pneumoniae is the most frequent cause of bacterial meningitis, leading to permanent neurological damage in 30% and lethal outcome in 25% of patients. The cholesterol-dependent cytolysin pneumolysin is a major virulence factor of S. pneumoniae. It produces rapid cell lysis at higher concentrations or apoptosis at lower concentrations. Here, we show that sublytic amounts of pneumolysin produce rapid bundling and increased acetylation of microtubules (signs of excessive microtubule stabilization) in various types of cells--neuroblastoma cells, fibroblasts and primary astrocytes. The bundling started perinuclearly and extended peripherally towards the membrane. The effect was not connected to pneumolysin's capacity to mediate calcium influx, macropore formation, apoptosis, or RhoA and Rac1 activation. Cellular cholesterol depletion and neutralization of the toxin by pre-incubation with cholesterol completely inhibited the microtubule phenotype. Pharmacological inhibition of Src-family kinases diminished microtubule bundling, suggesting their involvement in the process. The relevance of microtubule stabilization to meningitis was confirmed in an experimental pneumococcal meningitis animal model, where increased acetylation was observed. Live imaging experiments demonstrated a decrease in organelle motility after toxin challenge in a manner comparable to the microtubule-stabilizing agent taxol, thus proposing a possible pathogenic mechanism that might contribute to the CNS damage in pneumococcal meningitis.

CSF-1 receptor inhibition as a highly effective tool for depletion of microglia in mixed glial cultures.

BACKGROUND: A breakthrough in the microglia and macrophages field was the identification of the macrophage colony stimulating factor-1 (CSF-1) as a pro-survival factor. Its pharmacological inhibition in animals depletes rapidly all microglia and macrophages. Microglial depletion in mixed glial cultures has always represented a challenge and none of the existing approaches delivers satisfactory results. NEW METHOD: We applied a CSF-1R inhibitor (PLX5622) in primary mouse glial cultures, analyzing microglial dose-responses, starting at different time-points and incubating for various periods of time. RESULTS: We used two treatment modalities with 10 µM PLX5622 to deplete microglia: i) immediately after brain homogenization and ii) at day in vitro 12. The application of the inhibitor immediately after cell preparation depleted microglia to 8% at 1 week, to 2% at 4 weeks and to 0.5% at 6 weeks (half-time 3.5 days). When mixed glial cultures were treated starting at day in vitro 12, microglia depletion was slower (half-time 6 days) and not complete, indicating a decreased sensitivity to CSF-1. The remaining astrocytes preserved their proliferation ability, their migration in a scratch wound assay, and their pro-inflammatory (IL-6) response towards lipopolysaccharide. COMPARISON TO EXISTING METHODS: The proposed approach for microglial depletion in mixed glial cultures is more effective than other existing methods and is non-toxic to non-microglial cells. CONCLUSIONS: CSF-1R inhibitors are effective tools for depleting microglia in mixed glial cultures. Longer maturation of the cultures leads to a diminished sensitivity of microglia towards CSF-1. Thus, the treatment should start as early as possible after glial culture preparation.

Missing elimination via membrane vesicle shedding contributes to the diminished calcium sensitivity of listeriolysin O.

The lytic capacity of cholesterol-dependent cytolysins is enhanced in the extracellular calcium-free environment through a combination of limited membrane repair and diminished membrane toxin removal. For a typical neurotoxin of the group, pneumolysin, this effect has already been observed at reduced (1 mM) calcium conditions, which are pathophysiologically relevant. Here, we tested another neurotoxin of the group, listeriolysin O from L. monocytogenes, active in the primary vacuole after bacterium phagocytosis in host cells. Reduced calcium did not increase the lytic capacity of listeriolysin (in contrast to pneumolysin), while calcium-free conditions elevated it 2.5 times compared to 10 times for pneumolysin (at equivalent hemolytic capacities). To clarify these differences, we analyzed membrane vesicle shedding, known to be a calcium-dependent process for toxin removal from eukaryotic cell membranes. Both pneumolysin and listeriolysin initiated vesicle shedding, which was completely blocked by the lack of extracellular calcium. Lack of calcium, however, elevated the toxin load per a cell only for pneumolysin and not for listeriolysin. This result indicates that vesicle shedding does not play a role in the membrane removal of listeriolysin and outlines a major difference between it and other members of the CDC group. Furthermore, it provides new tools for studying membrane vesicle shedding.

Application of simple photobleaching microscopy techniques for the determination of the balance between anterograde and retrograde axonal transport.

The directionality of axonal transport represents an important question in neurophysiological and neuropathological research. Various approaches such as videomicroscopy, radioisotopic and fluorescence-based techniques are used. Recently, a novel FRAP-based (fluorescent recovery after photobleaching) technique using synaptophysin-EGFP expression in primary neurons was applied, allowing reliable and sensitive evaluation of gross axonal transport changes using confocal live-imaging microscopy. Here, we describe a novel FLIP-based (fluorescence loss in photobleaching) approach using a synaptophysin-EGFP probe that allows the differential evaluation of the ante- and retrograde transport parameters. Furthermore, we improved the sensitivity of the probe by substituting EGFP with an ECFP/VenusYFP fusion FRET (fluorescence resonance energy transfer) pair. The use of this FRET couple improves the precision of axonal transport measurements by combining FLIP and FLAP (fluorescence localization after photobleaching) techniques and eliminating the need for pre-bleaching images and thus pixel shifts between various exposures, and by reducing the deleterious effect of photobleaching.

Distinct Neurotoxicity Profile of Listeriolysin O from Listeria monocytogenes.

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.

Magnesium therapy improves outcome in Streptococcus pneumoniae meningitis by altering pneumolysin pore formation.

BACKGROUND AND PURPOSE: Streptococcus pneumoniae is the most common cause of bacterial meningitis in adults and is characterized by high lethality and substantial cognitive disabilities in survivors. Here, we have studied the capacity of an established therapeutic agent, magnesium, to improve survival in pneumococcal meningitis by modulating the neurological effects of the major pneumococcal pathogenic factor, pneumolysin. EXPERIMENTAL APPROACH: We used mixed primary glial and acute brain slice cultures, pneumolysin injection in infant rats, a mouse meningitis model and complementary approaches such as Western blot, a black lipid bilayer conductance assay and live imaging of primary glial cells. KEY RESULTS: Treatment with therapeutic concentrations of magnesium chloride (500 mg·kg-1 in animals and 2 mM in cultures) prevented pneumolysin-induced brain swelling and tissue remodelling both in brain slices and in animal models. In contrast to other divalent ions, which diminish the membrane binding of pneumolysin in non-therapeutic concentrations, magnesium delayed toxin-driven pore formation without affecting its membrane binding or the conductance profile of its pores. Finally, magnesium prolonged the survival and improved clinical condition of mice with pneumococcal meningitis, in the absence of antibiotic treatment. CONCLUSIONS AND IMPLICATIONS: Magnesium is a well-established and safe therapeutic agent that has demonstrated capacity for attenuating pneumolysin-triggered pathogenic effects on the brain. The improved animal survival and clinical condition in the meningitis model identifies magnesium as a promising candidate for adjunctive treatment of pneumococcal meningitis, together with antibiotic therapy.

Breakdown of axonal synaptic vesicle precursor transport by microglial nitric oxide.

The mechanism of axonal injury in inflammatory brain diseases is still unclear. Increased microglial production of nitric oxide (NO) is a common early sign in neuroinflammatory diseases. We found by fluorescence correlation spectroscopy that synaptophysin tagged with enhanced green fluorescence protein (synaptophysin-EGFP) moves anterogradely in axons of cultured neurons. Activated microglia focally inhibited the axonal movement of synaptophysin-EGFP in a NO synthase-dependent manner. Direct application of a NO donor to neurons resulted in inhibition of axonal transport of synaptophysin-EGFP and synaptotagmin I tagged with EGFP, mediated via phosphorylation of c-jun NH2-terminal kinase (JNK). Thus, overt production of reactive NO by activated microglia blocks the axonal transport of synaptic vesicle precursors via phosphorylation of JNK and could cause axonal and synaptic dysfunction.

Neuronal injury mediated via stimulation of microglial toll-like receptor-9 (TLR9).

Innate immune cells express toll-like receptor-9 (TLR9) and respond to unmethylated, CG dinucleotide motif-rich DNA released from bacteria during infection or endogenous cells during autoimmune tissue injury. Oligonucleotides containing CG dinucleotide (CpG-DNA) mimic the effect of unmethylated DNA and stimulate TLR9. CpG-DNA was cytotoxic to neurons in organotypic brain cultures. Neurotoxicity of CpG-DNA was mediated via microglial cells and started primarily from neurites as determined by time-lapse imaging of enhanced green fluorescent protein (EGFP)-transfected neurons. Cultured brain microglial cells expressed TLR9 and responded to CpG-DNA by production of the inflammatory mediators nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha). Blockade of NO synthase and TNF-alpha prevented damage of neurites and neurotoxicity of CpG-DNA. The data suggest that stimulation of microglia via TLR9 and subsequent release of NO and TNF-alpha is a major source of neurotoxicity in bacterial and autoimmune brain tissue injury.

Direct transmembrane interaction between actin and the pore-competent, cholesterol-dependent cytolysin pneumolysin.

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.

Changes in astrocyte shape induced by sublytic concentrations of the cholesterol-dependent cytolysin pneumolysin still require pore-forming capacity.

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.