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

Ciliated ependymal cells line the ventricular system of the brain and the c erebral aqueducts, This study characterizes the relative roles of pneumolys in and hydrogen peroxide (H2O2) in pneumococcal meningitis, using the in vi tro ependymal ciliary beat frequency (CBF) as an indicator of toxicity. We have developed an ex vivo model to examine the ependymal surface of the bra in slices cut from the fourth ventricle, The ependymal cells had cilia beat ing at a frequency of between 38 and 44Hz. D39 (wild-type) and PLN-A (pneum olysin-negative) pneumococci at 10(8) CFU/ml both caused ciliary slowing, C atalase protected against PLN-A-induced ciliary slowing but afforded little protection from D39, Lysed PLN-A did not reduce CBF, whereas lysed D39 cau sed rapid ciliary stasis, There was no effect of catalase, penicillin, or c atalase plus penicillin on the CBF, H2O2 at a concentration as low as 100 m u M caused ciliary stasis, and this effect was abolished by coincubation wi th catalase, An additive inhibition of CBF was demonstrated using a combina tion of both toxins. A significant inhibition of CBF at between 30 and 120 min was demonstrated with both toxins compared with either H2O2 (10 mu M) o r pneumolysin (1 HU/ml) alone. D39 released equivalent levels of H2O2 to th ose released by PLN-A, and these concentrations were sufficient to cause ci liary stasis, The brain slices did not produce H2O2, and in the presence of 108 CFU of D39 or PLN-A per ml there was no detectable bacterially induced increase of H2O2 release from the brain slice. Coincubation with catalase converted the H2O2 produced by the pneumococci to H2O. Penicillin-induced l ysis of bacteria dramatically reduced H2O2 production. The hemolytic activi ty released from D39 was sufficient to cause rapid ciliary stasis, and ther e was no detectable release of hemolytic activity from the pneumolysin-nega tive PLN-A. These data demonstrate that D39 bacteria released pneumolysin, which caused rapid ciliary stasis. D39 also released H2O2, which contribute d to the toxicity, but this was masked by the more severe effects of pneumo lysin. H2O2 released from intact PLN-A was sufficient to cause rapid ciliar y stasis, and catalase protected against H2O2-induced cell toxicity, indica ting a role for H2O2 in the response. There is also a slight additive effec t of pneumolysin and H2O2 on ependymal toxicity; however, the precise mecha nism of action and the role of these toxins in pathogenesis remain unclear.