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.

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.

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.

Dose-dependent activation of microglial cells by Toll-like receptor agonists alone and in combination.

Microglial cells express Toll-like receptors (TLRs) recognising exogenous and endogenous ligands. Upon stimulation with agonists of TLR2, TLR4, and TLR9, nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) were released by primary mouse microglial cell cultures. Endotoxin was most potent in stimulating microglia followed by pneumolysin, cytosine-guanosine (CpG) oligodesoxynucleotide (ODN), and Tripalmitoyl-S-glyceryl-cysteine. Maximum stimulation of TLR2, TLR4, and TLR9 resulted in approximately equal amounts of nitric oxide release. Pneumolysin was a potent activator of microglial cells; at high concentrations, it reduced cell viability. No cytotoxicity was noted with the other TLR agonists. Costimulation with maximum concentrations of two TLR agonists did not further increase nitric oxide release. Costimulation with submaximum concentrations was additive or supraadditive, suggesting that even low concentrations of products of infectious agents can lead to microglial activation via TLRs.

Intracochlear perfusion of pneumolysin, a pneumococcal protein, rapidly abolishes auditory potentials in the Guinea pig cochlea.

OBJECTIVE: Bacterial meningitis and chronic suppurative otitis media caused by Streptococcus pneumoniae are associated with considerable otological morbidity. Specifically, sensorineural hearing loss is a permanent sequela in a third of those who contract pneumococcal meningitis. Pneumolysin, a pneumococcal protein, has been implicated as one of the main virulence/cytotoxic factors. Its pathogenicity is intimately dependent on an ability to form transmembrane pores on binding with cholesterol in target tissues. MATERIAL AND METHODS: We perfused wild-type pneumolysin, at a number of different concentrations, into the guinea pig cochlea and used electrocochleography to characterize the effects of this cytolytic exotoxin in the organ of Corti. RESULTS: Intracochlear perfusion of pneumolysin (10 microg/50 microl) reduced the compound action potential of the auditory nerve within seconds. The cochlear microphonics (f1=8 kHz, f2=9.68 kHz) and their distortion product (2f1-f2) were also reduced, albeit in a slightly less dramatic fashion. At lower concentrations (1 microg/50 microl), a selective and earlier effect on inner hair cells was observed. CONCLUSIONS: These results clearly show that significant ototoxicity ensues when sensory cells of the organ of Corti are exposed to pneumolysin (and complete cochlear death when the concentration is high enough). Toxicity is dose-dependent and appears to be site-sensitive. This may have implications for any possible future protective strategies against pneumococcal disease in the ear.

Reduced release of pneumolysin by Streptococcus pneumoniae in vitro and in vivo after treatment with nonbacteriolytic antibiotics in comparison to ceftriaxone.

Pneumolysin, a virulence factor of Streptococcus pneumoniae with cytotoxic and proinflammatory activities, occurs at concentrations from 0.85 to 180 ng/ml in cerebrospinal fluid (CSF) of meningitis patients. In pneumococcal cultures and in a rabbit meningitis model, the concentrations of pneumolysin in supernatant and CSF were lower after addition of nonbacteriolytic bactericidal antibiotics (rifampin and clindamycin) than after incubation with ceftriaxone.