Relative Roles of Pneumolysin and Hydrogen Peroxide from Streptococcus pneumoniae in Inhibition of Ependymal Ciliary Beat Frequency

Abstract

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 μM 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 μM) 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.