Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue

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.

Bacterial Cytolysin during Meningitis Disrupts the Regulation of Glutamate in the Brain, Leading to Synaptic Damage

“…Glutamate release was completely blocked under calcium-free buffer conditions, as the observed cell lysis was identical or even greater compared with the cell lysis in 2 mM calcium conditions (Fig. 6B), which we have described previously [24]. The calcium-free experiments confirmed that permeabilization alone was not sufficient to increase the glutamate levels around the astrocytes and that calcium was required for this effect.…”

“…To clarify whether PLY alone can cause the observed synaptic changes, we studied the role of PLY at non-lytic, disease-relevant concentrations in acute mouse brain slice cultures as described previously [24]. We chose 12–14 day-old infant mice as the tissue source because they can be sliced with minimum cytotoxicity (<5%) and be maintained for up to 24 h in an oxygenized environment without losing viability, while already demonstrating signs of maturation (such as myelination) that resemble the normal tissue environment in human meningitis.…”

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

“…To confirm the pathophysiological relevance of the injected amounts, samples from the CSF were taken within 2 h of the injection of PLY and/or 1% Evans Blue dye (Sigma) to show the complete distribution of the injected fluid in the CSF. The final concentration of PLY was 0.2 μg·mL −1 (Wippel et al, ), which was comparable to the toxin amounts observed in the CSF during pneumococcal meningitis (Spreer et al, ). Equimolar amounts of albumin Fr.…”

“…Higher calcium concentrations mildly ameliorated swelling in our brain slice system (Supporting Information Figure S1C). The mechanisms involved here was most likely associated with the modulation of membrane toxin binding by calcium (Wippel et al, ). High calcium, however, is not an alternative to the magnesium treatment due to the multiple secondary calcium‐mediated effects such as enhanced cell dysfunction, excitotoxicity and accelerated cell death (Amagasa et al, ; Ravens et al, ; McGinnis et al, ; Lorget et al, ), but it further confirms the critical sensitivity of the PLY molecule to divalent ions.…”

Red Blood Cell Susceptibility to Pneumolysin

“…Membrane Modification and Cell Susceptibility to PLY-It has previously been shown that reduction of extracellular calcium strongly enhances the lytic capacity of PLY due to increased membrane binding (97). One of the main findings of this study suggests that cell susceptibility to PLY also critically depends on the oxidative and/or other chemical stress the membrane has been subjected to before interaction with the toxin (Fig.…”