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Infection and Immunity, March 2000, p. 1557-1562, Vol. 68, No. 3
Department of Child Health, University of
Leicester, Leicester Royal Infirmary, Leicester LE2
7LX,1 Department of Microbiology and
Immunology, University of Leicester, Leicester LE1
9NN,2 and Division of Infection and
Immunity, Institute of Biomedical and Life Sciences, University of
Glasgow, Glasgow G12 8QQ,3 United Kingdom
Received 8 September 1999/Returned for modification 21 October
1999/Accepted 6 December 1999
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 (H2O2) 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 108 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. H2O2 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 H2O2 (10 µM) or
pneumolysin (1 HU/ml) alone. D39 released equivalent levels of
H2O2 to those released by PLN-A, and these
concentrations were sufficient to cause ciliary 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 lysis of bacteria dramatically
reduced H2O2 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 H2O2, which contributed to the
toxicity, but this was masked by the more severe effects of
pneumolysin. H2O2 released from intact PLN-A
was sufficient to cause rapid ciliary stasis, and catalase protected
against H2O2-induced cell toxicity, indicating
a role for H2O2 in the response. There is also
a slight additive effect of pneumolysin and
H2O2 on ependymal toxicity; however, the
precise mechanism of action and the role of these toxins in
pathogenesis remain unclear.
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Relative Roles of Pneumolysin and Hydrogen Peroxide
from Streptococcus pneumoniae in Inhibition of Ependymal
Ciliary Beat Frequency
*
Corresponding author. Mailing address: Departments of
Child Health, University of Leicester, Robert Kilpatrick Clinical
Sciences Building, Leicester Royal Infirmary, P.O. Box 65, Leicester
LE2 7LX, United Kingdom. Phone: 0116 2523269. Fax: 0116 2523282. E-mail: rahirst{at}hotmail.com.
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