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Institute
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.
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.
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.
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.
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. "