Zusammenfassung
The Streptococcus pneumoniae toxin pneumolysin belongs to the group
of cholesterol-dependent cytolysins. It produces rapid cell lysis
at higher concentrations or apoptosis at lower concentrations. In
cell membranes, it forms prepores and pores. Here, we show that sublytic
concentrations of pneumolysin produce rapid activation of Rho and
Rac GTPases and formation of actin stress fibers, filopodia, and
lamellipodia. That Rac1-specific and Rho-associated kinase (ROCK)-specific
inhibitors reverted the formation of lamellipodia and stress fibers,
respectively, identifies RhoA and Rac1 as key toxin effectors. Live
imaging excluded macropore formation (as judged by membrane impermeability
toward calcein) but indicated very early membrane depolarization
as judged by bis-(1,3-dibutylbarbituric acid)trimethine oxanol staining,
indicative of formation of micropores with ion channel properties.
That Rac1-dependent lamellipodia formation was reverted by the voltage-gated
calcium channel inhibitor SKF96365 and by toxin exposure in calcium-free
medium suggests a role for calcium influx via endogenous calcium
channels in the Rac1 activation. Cellular cholesterol depletion by
methyl-beta-cyclodextrin or incubation of the toxin with cholesterol
before cell treatment eliminated its membrane binding and the subsequent
GTPase activation. Thus, that our experiments show small GTPase activation
by a cholesterol-dependent cytolysin suggests a membrane cholesterol-dependent
activation mechanism.
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