Relative roles of pneumolysin and hydrogen peroxide from Streptococcus pneumoniae in inhibition of ependymal ciliary beat frequency

Abstract

Ciliated ependymal cells line the ventricular system of the brain and the cerebral aqueducts. This study characterizes the relative roles of pneumolysin and hydrogen peroxide (H(2)O(2)) in pneumococcal meningitis, using the in vitro ependymal ciliary beat frequency (CBF) as an indicator of toxicity. We have developed an ex vivo model to examine the ependymal surface of the brain slices cut from the fourth ventricle. The ependymal cells had cilia beating at a frequency of between 38 and 44Hz. D39 (wild-type) and PLN-A (pneumolysin-negative) pneumococci at 10(8) CFU/ml both caused ciliary slowing. Catalase protected against PLN-A-induced ciliary slowing but afforded little protection from D39. Lysed PLN-A did not reduce CBF, whereas lysed D39 caused rapid ciliary stasis. There was no effect of catalase, penicillin, or catalase plus penicillin on the CBF. H(2)O(2) at a concentration as low as 100 microM caused ciliary stasis, and this effect was abolished by coincubation with catalase. An additive inhibition of CBF was demonstrated using a combination of both toxins. A significant inhibition of CBF at between 30 and 120 min was demonstrated with both toxins compared with either H(2)O(2) (10 microM) or pneumolysin (1 HU/ml) alone. D39 released equivalent levels of H(2)O(2) to those released by PLN-A, and these concentrations were sufficient to cause ciliary stasis. The brain slices did not produce H(2)O(2), and in the presence of 10(8) CFU of D39 or PLN-A per ml there was no detectable bacterially induced increase of H(2)O(2) release from the brain slice. Coincubation with catalase converted the H(2)O(2) produced by the pneumococci to H(2)O. Penicillin-induced lysis of bacteria dramatically reduced H(2)O(2) production. The hemolytic activity released from D39 was sufficient to cause rapid ciliary stasis, and there was no detectable release of hemolytic activity from the pneumolysin-negative PLN-A. These data demonstrate that D39 bacteria released pneumolysin, which caused rapid ciliary stasis. D39 also released H(2)O(2), which contributed to the toxicity, but this was masked by the more severe effects of pneumolysin. H(2)O(2) released from intact PLN-A was sufficient to cause rapid ciliary stasis, and catalase protected against H(2)O(2)-induced cell toxicity, indicating a role for H(2)O(2) in the response. There is also a slight additive effect of pneumolysin and H(2)O(2) on ependymal toxicity; however, the precise mechanism of action and the role of these toxins in pathogenesis remain unclear.

FIG. 1

(A) Effect of D39 (10⁸ CFU/ml) on ependymal CBF in the absence (■) or presence (□) of catalase (2,000 EU/ml). ∗, statistically (P < 0.05; paired t test) increased compared with D39. (B) Protective effect of catalase (2,000 EU/ml) (□) on PLN-A induced inhibition (■) of ependymal CBF. ∗, statistically (P < 0.05; paired t test) increased compared with PLN-A. All data are mean and standard error of the mean of four independent experiments.

FIG. 2

Effect of penicillin (1 mg/ml)-lysed pneumococci (10⁸ CFU/ml) in the presence (□) or absence (■) of catalase (2,000 EU/ml). (A) D39; (B) PLN-A. There were no statistical differences in the data (mean and standard error of the mean of four experiments).

FIG. 3

(A) Dose-dependent hydrogen peroxide inhibition of ependymal CBF. (B) Abolition of H₂O₂ inhibition by coincubation with catalase (2,000 EU/ml). All data are mean and standard error of the mean of five or six individual experiments. ∗, statistically (P < 0.05; paired t test) inhibited compared with no H₂O₂.

FIG. 4

(A) The supernatant from D39 (■) at 10⁸ CFU/ml shows a time-dependent increase hemolytic activity, whereas PLN-A (□) at 10⁸ CFU/ml shows no hemolytic activity in the supernatant over 60 min at 37°C. ∗, statistically (P < 0.05; paired t test) increased compared with 0 min. (B) Additive action of pneumolysin and H₂O₂ on ependymal CBF (■, control; □, 10 μM H₂O₂; ●, 1 HU of pneumolysin per ml; ○, 10 μM H₂O₂ plus 1 HU of pneumolysin per ml). ∗, statistically (P < 0.05, paired t test) inhibited compared with pneumolysin alone. All data are mean and standard error of the mean of five individual experiments.

FIG. 5

Scanning electron micrographs of ependymal cilia on a brain slice cut into the floor of the fourth ventricle. (A) Healthy cilia. (B and C) The cilia show an increasing amount of morphological alterations from normal at 60 min with higher concentrations of H₂O₂ (100 μM [B] and 1 mM [C]). (D) Catalase (100 μM H₂O₂ plus 2,000 EU of catalase per ml) protects the cilia from this disruption by H₂O₂. Bar, 4 μm (A) and 3.5 μm (B to D).