From the Biomembrane Structure Unit, Department of
Biochemistry, University of Oxford, South Parks Road, Oxford OX1
3QU, the § Department of Microbiology and Immunology,
University of Leicester, Leicester LE1 9HN, and the
Institute of Biomedical and Life Sciences, Division Infection
and Immunity, University of Glasgow, Glasgow G12 8QQ, United
Kingdom
Received for publication, June 13, 2000, and in revised form, October 19, 2000
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ABSTRACT |
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Pneumolysin, a major virulence factor of the
human pathogen Streptococcus pneumoniae, is a soluble
protein that disrupts cholesterol-containing membranes of cells by
forming ring-shaped oligomers. Magic angle spinning and wideline
static 31P NMR have been used in combination with
freeze-fracture electron microscopy to investigate the effect of
pneumolysin on fully hydrated model membranes containing cholesterol
and phosphatidylcholine and dicetyl phosphate (10:10:1 molar ratio).
NMR spectra show that the interaction of pneumolysin with
cholesterol-containing liposomes results in the formation of a
nonbilayer phospholipid phase and vesicle aggregation. The amount of
the nonbilayer phase increases with increasing protein concentration.
Freeze-fracture electron microscopy indicates the coexistence of
aggregated vesicles and free ring-shaped structures in the presence of
pneumolysin. On the basis of their size and analysis of the NMR spectra
it is concluded that the rings are pneumolysin oligomers (containing 30-50 monomers) complexed with lipid (each with 840-1400 lipids). The
lifetime of the phospholipid in either bilayer-associated complexes or free pneumolysin-lipid complexes is > 15 ms.
It is further concluded that the effect of pneumolysin on lipid
membranes is a complex combination of pore formation within the
bilayer, extraction of lipid into free oligomeric complexes,
aggregation and fusion of liposomes, and the destabilization of
membranes leading to formation of small vesicles.
Pneumolysin is a major virulence factor from the human pathogen
Steptococcus pneumoniae and is a member of a family of
cholesterol-binding toxins
(CBTs),1 which also includes
perfringolysin from Clostridium perfringens, listeriolysin
from Listeria monocytogenes, and streptolysin from Streptococcus pyogenes. Streptolysin is well known for its
use in cell biology for the permeabilization of cells. As well as pore
formation leading to the breakdown of membrane barriers into cells and
between tissues, these toxins act in a complex manner involving the
activation of intracellular signaling pathways and the induction of
inflammation, among other responses (see Morgan et al. (1)
for review). Understanding the mechanisms by which CBTs act has been
greatly assisted by the determination of a crystal structure for
soluble, monomeric perfringolysin (2) and the use of electron
cryo-microscopy (cryo-EM) to obtain three-dimensional reconstructions
of the oligomeric and pore-forming states of CBTs using pneumolysin
(3). These direct structural insights have been augmented by results
obtained by Tweten and co-workers (4) using fluorescence spectroscopy
(5), which demonstrate that a pendant domain of the CBTs interacts with
phospholipid molecules during pore formation via two Two mechanisms for pneumolysin action in disease are well defined (7).
First, pneumolysin is well known as a pore-forming toxin that binds to
cholesterol in membranes, oligomerizes into ring-shaped assemblies, and
undergoes conformational changes causing it to penetrate and form a
pore within the bilayer (3). Secondly, pneumolysin activates the
complement system in a nonimmunospecific manner as a result of
interaction with IgG-Fc and complement proteins (8, 9).
Solid state NMR provides a direct and quantitative way for
investigating lipid-protein interactions in membranes (10-12). Changes in protein-induced phospholipid phase behavior can be observed through
characteristic intensity distribution in the chemical shift anisotropy
(CSA)-dominated lineshapes of wideline 31P NMR spectra.
Characteristic lineshapes have been observed from phospholipid phases
with different modes of molecular motions (Fig. 1 and Refs. 13-15).
Using 31P magic angle spinning (MAS) NMR, the individual
lipid components in the bilayer can be resolved (11), and changes in
their isotropic chemical shift, CSA and full-width at half-height
(FWHH) in response to modulation of the phosphate motions in the
presence of proteins determined (11, 12, 16).
Here results from solid-state NMR and electron microscopy are presented
that provide novel insights into the mechanism of CBTs and complement
data already acquired using other techniques. Suspensions of
cholesterol-containing liposomes are a model system that has already
been used for analysis of pore formation by pneumolysin using
small-angle neutron scattering (9) and cryo-EM (3).
Sample Preparation--
Large unilamellar vesicles (LUVs) of
egg-yolk phosphatidylcholine (PC), cholesterol, and dicetylphosphate
(DCP) in a 10:10:1 molar ratio were prepared in 100 mM Tris
buffer, pH 7.5, at 5 mM concentration as previously
described (3). Multilamellar vesicles (MLVs) used in a control
experiment were prepared by five cycles of rapid freezing of hydrated
lipid mixtures followed by 15 min equilibration at 40 °C.
Pneumolysin was expressed from recombinant Escherichia coli
and purified as previously described (17). Pneumolysin was mixed with
liposomes at a variety of molar ratios to cholesterol and incubated at
37 °C for 10 min prior to data acquisition.
NMR Spectroscopy--
Phosphorus-31 wideline and MAS NMR
measurements were carried out on a CMX Infinity 500 spectrometer at a
proton frequency of 500 MHz. Typically 5 µmol of lipid dispersion
were used in a 4-mm rotor using an HX Apex probe. A single 90° pulse
was used for detection with broadband decoupling at the proton
frequency during acquisition. The 90° pulse length was 4 µs, and
the strength of the proton decoupling field was 20 kHz. The dwell time
used was 40 µs, and 2048 points were collected in each experiment. Between 300 and 3000 transients were averaged for each free induction decay (FID) during MAS experiments and between 5000 and 10,000 during
wideline experiments with a 5-s delay (exceeding
5T1) between acquisitions in all cases. The
sample rotation speed was maintained at 12 kHz during acquisition of
the MAS spectra, and the temperature was set to 30 °C for all experiments.
High speed MAS lineshape analysis was performed by fitting simulated
Lorentzian lines to the experimental spectra using Spinsight (Chemagnetics, Fort Collins, CO) and Felix (MSI, Cambridge). The intensity of the individual lines was estimated from the integral of
the simulated spectra. All 31P chemical shifts are measured
relative to 0 ppm for 10% v/v phosphoric acid. All spectra were
obtained with 50 Hz linebroadening for the wideline spectra and 10 Hz
for the MAS spectra.
Electron Microscopy--
Samples of LUV suspensions from NMR
experiments were prepared for freeze-fracture by equilibration at room
temperature, followed by a rapid immersion into liquid butane at its
solidification point (18). Fracture of the immobilized frozen droplets
was performed at Lipid Dynamics and Phase Behavior-Phosphorus-31 Wideline
NMR--
Wideline 31P NMR was used to monitor changes in
the phospholipid phase behavior induced by the addition of pneumolysin.
LUVs of egg yolk PC, cholesterol, and DCP (molar ratio 10:10:1) were incubated with pneumolysin at 30 °C. Spectra from the lipid vesicles alone (see Fig. 2a) and in the presence of pneumolysin at
different concentrations are shown (Fig.
2, b-d). The 31P
NMR spectrum from lipid suspension prior to addition of pneumolysin (Fig. 2a) consists of a single
narrow line. The collapse of the phosphorus CSA is because of fast
rotation of relatively small (<100 nm in diameter) unilamellar
vesicles (19). The correlation times (
Fig. 2b shows the spectrum from the phospholipid dispersion
after addition of pneumolysin at 0.5 mol % with respect to
cholesterol. The spectrum is characteristic of a 31P
CSA-dominated spherically averaged (powder) pattern (20) from phospholipid bilayers in extended structures of average size greatly exceeding 100 nm. The most likely cause of this change in apparent liposome size when compared with protein-free liposomes (Fig. 2a) is vesicle aggregation induced by the interaction of
toxin with the membranes. The motional properties of these complexes indicate that the average size of the lipid aggregates exceeds 1 µm
because their reorientation rate (
The inverted powder-like feature of Fig. 2b is characterized
by a sharp upfield edge, arising from molecules oriented at 90 degrees
with respect to the external field and a downfield extended region from
lipids oriented along the magnetic field. Inverted powder-like patterns
arise from nonbilayer lipid complexes with cylindrical symmetry of
motion like, for example, the lipid tubular structures in inverted
hexagonal (HII) phases (14). The effective CSA, measured
from these patterns (~11 ppm), is less than the expected
half-spectral width from the bilayer environment (~40 ppm) and
exceeds the measured effective CSA of the narrow powder pattern
(associated with the presence of DCP). This suggests that fast rotation
of the phospholipid aggregates in the axially symmetric environment
results in additional reduction in the effective CSA and excludes the
possibility of formation of a cylindrically symmetric lipid
distribution from the lipid environment with small effective CSA by a
simple reduction of spherical to cylindrical symmetry. In the latter
case, the width of the axially symmetric distribution would be derived
as a half of the effective CSA in the related spherical distribution
(14, 15).
Further increase in the pneumolysin concentration results in a relative
increase in the intensity of the cylindrical lipid distribution and a
decrease in the intensity of both powder patterns (Fig. 2, c
and d). This behavior shows that the cylindrical lipid population arises from a pneumolysin-associated lipid environment. The
good spectral definition of the characteristic edges of the lipid
environments indicates well separated phases with none or with very
slow ( Quantification of the Pneumolysin-Lipid Interaction by
Phosphorus-31 MAS NMR--
Phosphorus-31 MAS NMR is used here as a
high-resolution approach to membrane studies for quantitative
characterization of the different phospholipid environments (11). High
speed MAS 31P NMR spectra from different lipid-pneumolysin
mixtures are shown in Fig. 4. The lipid
suspension in the absence of toxin gives rise to a single resonance at
~0 ppm. The observed unusual chemical shift of the resonance line and
the lack of resolution between PC and DCP are most likely because of an
interference of the vesicle tumbling and lateral diffusion of the
phospholipid molecules with the proton-phosphorus decoupling and MAS.
Such interaction may result in inefficient proton decoupling and
incomplete averaging of the 31P CSA under MAS for 100 nm
LUVs (see Ref. 21 for discussion). In a control 31P MAS NMR
experiment an MLV suspension of the same composition produced a strong
resonance at approximately
All 31P MAS NMR spectra in the presence of pneumolysin show
two distinctive resonances, (Fig. 4, b-d), a downfield one
at approximately
The narrow upfield resonance (Fig. 4b) undergoes a
pronounced further upfield change in its isotropic chemical shift, Morphology of the Lipid Phases by Freeze-Fracture
EM--
Freeze-fracture electron microscopy was used to obtain
information about the lipid phase topology in the presence of
pneumolysin. Fig. 5 shows transmission
electron micrographs from the lipid system in the presence of 1.25 mol
% pneumolysin with respect to cholesterol. The micrographs show
aggregated lipid vesicles, with the vesicles varying in size from one
hundred to a few hundred nanometers. Fractures across the vesicles
reveal single or only a few bilayers and thus the absence of
multibilayer structures. Nonaggregated vesicles are also seen in
coexistence with the vesicle aggregates. Multifaceted liposome surfaces
reveal close foam-like aggregation. The surfaces of some liposomes show
the presence of circular structures of ~25 nm internal diameter
(Fig. 5a, arrow p). Previously observed pneumolysin
oligomers have a similar size to these circular structures and
represent, most likely, membrane-incorporated oligomeric toxin pores
(3).
Free ring-shaped structures unassociated with liposomes are also
observed with an inside diameter of ~25 nm and an outside diameter of
about 50 nm (Fig. 5a, arrow r). These appear to be the only axially symmetric structures in the system and are the same
size as the rings seen here on liposomes, and previously on liposomes
in solution (3). Fig. 5b (arrow) shows a
structure of the same size (~25-nm inner diameter) as the rings of
Fig. 5a, localized at the boundary between two liposomes
having a common wall. This suggests that pneumolysin oligomers play a
role in liposome aggregation and fusion. A lipid environment resembling that in the proteolipid rings is associated with the pneumolysin ring
shown in Fig. 5b. This suggests that vesicle aggregation may
be related to the formation of nonbilayer proteolipid structures.
An important aspect of the effect of pneumolysin on membranes is
the destabilizing effect the protein has on the lipid bilayers and the
associated withdrawal of phospholipids into nonbilayer structures. The
wideline 31P NMR spectra (Fig. 2, b-d)
originate from a coexistence of phospholipids in structures with a
spherically averaged CSA corresponding to the CSA of slowly tumbling
MLV's, together with a nonbilayer phase with axially symmetric motion
of the lipid structures. The changes in intensity distribution from a
powder, to a distribution with the 90° edge located upfield with
respect to the 0° extreme, are indicative of the presence of an
additional axis of phospholipid molecular averaging, perpendicular to
the main axis of lipid rotation. This situation is observed, for
example, in the presence of inverted hexagonal (HII) phases
(15, 22). In addition to the inversion of spectral intensity
distribution, the overall width of the CSA-dominated intensity
distribution is expected to decrease by a factor of 2 (see Fig. 1 and
Refs. 13, 15). The width of the spectrum from the pneumolysin-induced
nonbilayer phase (~10 ppm) is reduced further to ~one-fourth of the
spectral width from the lipid powder distribution (~40 ppm). This
suggests the presence of fast axial rotation of the lipids in these
structures and that they are smaller than the average diameter of a
lipid aggregate in an HII phase. The increase in the
fraction of the nonbilayer phase with increasing toxin concentration
strongly indicates that its formation is induced by pneumolysin (Fig.
2, b-d).
The ring structures, shown in the micrographs in Fig. 5 possess all the
characteristics necessary to give rise to the inverted powder component
in the static 31P NMR spectra of Fig. 2, b-d.
Their ring shape results in a preferred axis of motional averaging and
their relatively small size would facilitate fast axial rotation. The
internal diameter of these structures corresponds to the size of
pneumolysin rings observed by negative staining EM and cryo-EM (3, 17).
The most likely explanation for the rings seen here by EM and detected
by a characteristic spectral distribution similar to that of a
nonbilayer phase (Fig. 2) is an oligomeric pneumolysin ring with a
lipid annulus decorating the exterior of the oligomer (Fig.
6). The interior of such structures could
be filled with lipid or empty, because both forms of oligomers have
been observed by cryo-EM
(3).2 In either case the
evolution of such structures into free solution from liposomes, with
which pneumolysin interacts would result in the rapid destruction of
the membrane.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helix bundles
refolded into two
-hairpins. A second terminal domain is known to be
involved in membrane binding via cholesterol. Thus, it seems that the
CBT mechanism involves two domains interacting with the membrane (6, 3).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
100 °C, followed by platinum shadowing at 45°
with respect to the fractured surface and a normal deposition of a uniform carbon film in a Balzers freeze-etching apparatus. Residual sample was removed from the carbon-platinum replicas by cyclic exposure
to potassium hypochlorate and methanol. The carbon-platinum replicas
were transferred onto copper electron microscope grids. A Phillips
CM120 transmission electron microscope was used for grid visualization
at 100-kV accelerating voltage and 45,000 × magnification.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
R) for tumbling of
such vesicles are on the order of 10
4-10
5
s, which exceeds the inverse width of the average 31P CSA
(~40-50 ppm, ~6 kHz) for lipid phosphates in extended
bilayers.
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Fig. 1.
Simulated characteristic lineshapes from
spherically symmetric lipid distribution (bilayer phase,
a) and cylindrically symmetric lipid distribution
(HII phase, b) with
the same effective CSA. Lineshapes were simulated using Maple 6 (University of Waterloo, Canada).
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Fig. 2.
Wideline 31P NMR spectra from egg
PC/cholesterol/DCP LUV dispersions in a molar ratio of 10:10:1
(a) and after addition of 0.5 mol % (b), 1.25 mol % (c), and 1.5 mol % (d) pneumolysin. A dotted and a
dashed line are added to the spectrum in b to
identify the spectral contribution from the bilayer PC (below
dotted line), nonbilayer lipid (between dotted and
dashed lines), and bilayer DCP (above dashed
line). See text for details.
R
1) is
much lower than 6 kHz (the effective CSA of lipid phosphates). The
spectrum of Fig. 2b reveals three overlapping features, a powder pattern between ~27 and
14 ppm (below dotted
line), a second powder pattern with reduced effective CSA from 5 to
2 ppm (above dashed line), and an inverted powder-like
distribution from 3 to about
8 ppm (Fig. 2, between dotted
and dashed lines, see characteristic lineshapes). The two
powder patterns arise from a spherical distribution of lipid molecules
rapidly rotating about their long axes. The downfield intense feature
in the powder patterns corresponds to molecules oriented at 90 degrees
with respect to the external magnetic field and the upfield
distribution of intensity to molecules oriented along the field. The
width of the broader powder pattern (below dotted line) is
characteristic of phosphatidylcholine aggregates undergoing slow
reorientation and is likely to result from liposomes aggregated in the
presence of pneumolysin. The narrow powder pattern (above dashed
line) is generated by the phosphates of DCP in the same lipid
bilayer. These powder distributions can be observed as a result of the interaction between the vesicles in the presence of pneumolysin. The
relative intensity of the powder patterns with respect to each other is
independent of the amount of toxin added to the lipid suspension (Fig.
2, b-d), which suggests that the PC/DCP ratio in the
bilayer remains constant. In a control experiment, an MLV (large
complexes with
R
1
effective CSA)
suspension produced a wideline 31P NMR spectrum (Fig.
3) consisting of two powder patterns of
comparable relative intensity and of similar effective CSA to the
powder patterns observed in the presence of pneumolysin (Fig. 2).
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Fig. 3.
Wideline 31P NMR spectrum from an
egg PC/cholesterol/DCP MLV dispersion in a molar ratio of 10:10:1.
The dotted lines help distinguish the PC powder pattern
(bottom) from the DCP powder pattern (top).
ex
1 < 200 Hz), lipid exchange
between them. The 90° edges from the different lipid environments
remain at the same chemical shift, which suggests that the effective
CSA and, therefore, the motional characteristics of these environments
are independent of pneumolysin concentration.
1.0 ppm, attributed to the PC phosphate
and a weak resonance at 0.1 ppm from DCP (spectrum not shown).
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Fig. 4.
Phosphorus-31 MAS NMR spectra from egg
PC/cholesterol/DCP LUV dispersions in a molar ratio of 10:10:1
(a) and after addition of 0.5 mol % (b), 1.25 mol % (c), and 1.5 mol % (d) pneumolysin. See text for details.
1.0 ppm and an upfield resonance with chemical shift
dependent on the pneumolysin concentration (Table
I). The resonance at
1.0 ppm has the
characteristic chemical shift of phosphatidylcholine in a bilayer
environment (11) and consists of two overlapping resonances, one of
FWHH approximately equal to 1.61 ppm and another of 0.35 ppm FWHH. The
FWHH of the broader resonance increases at higher pneumolysin
concentration, whereas that of the narrower resonance does not show a
clear dependence on toxin concentration. The broader resonance,
therefore, probably arises from pneumolysin-associated lipid within the
lipid bilayer phase in the form of pores (see EM evidence below) or
between partially fused liposomes. A low intensity resonance at 0.1 ppm
arises from DCP in the bilayer. The ratio between its intensity and the
intensity of the PC at
1.0 ppm resonance does not depend on
pneumolysin concentration.
Phosphorous-31 isotropic chemical shifts
p,
f)
from MAS NMR of the non-bilayer and total bilayer lipid resonance and
relative intensity I as a fraction of the total spectral
intensity determined from the spectra in Fig. 2 for different
concentrations of pneumolysin with respect to cholesterol at 30 °C
are shown.
,
with increasing pneumolysin concentration (Table I). The relative intensity of this resonance when compared with the total intensity of
the line at
1.0 ppm increases as the toxin concentration is increased
(Table I). The toxin concentration-dependent increase in
the intensity of this resonance parallels, in a qualitative way, the
increase in the intensity of the reverse powder pattern, observed in
the static spectra (Fig. 2, b-d). Therefore, the upfield resonance most likely arises from lipid associated with pneumolysin in
the nonbilayer environment.
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Fig. 5.
Two freeze-fracture electron micrographs
(a, b) from PC/cholesterol/DCP LUV
dispersions in the presence of 1.5 mol % pneumolysin. The length
of the scale bar is 100 nm. Arrow P shows a
membrane pore and arrow R shows a proteolipid ring
(a). The arrow in b shows a
proteolipid structure at the interface of two fused liposomes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 6.
Schematic illustration of the effects of
pneumolysin on lipid bilayers. Each oligomer is estimated to be
complexed with 840 to 1400 phospholipid molecules. Unilamellar vesicles
are ~100 nm in diameter, and the size of the phospholipid aggregate
is much greater than 100 nm. Exchange of phospholipid between the
structures, if possible, occurs slowly (15 ms).
The relative intensity of the nonbilayer spectral feature increases in comparison to the intensities of both the wide and the narrow powder patterns. The concomitant increase in the intensity of the upfield resonance in the 31P MAS NMR spectra can be used as a measure of the relative amount of nonbilayer phase (proteolipid rings). Thus the quantitation of spectral intensity (Table I) reflects the fraction of the total lipid, segregated into the rings and indicates that a large proportion of the lipid is associated with free oligomers detached from the lipid bilayer. In particular, the values presented in Table I show that an increase in pneumolysin concentration with respect to cholesterol from 0.5 to 1.5 mol % results in an increase of nonbilayer phase from 32 to 60% of the total phospholipid content. Consequently, in the presence of ~1 mol % of pneumolysin, 28 phospholipid molecules are associated with each pneumolysin monomer and the number of phospholipid molecules per oligomer, consisting of 30 to 50 toxin molecules (3), can be estimated to vary between 840 and 1400, respectively.
The insights gained here into the mechanism of CBTs are fundamentally important because they suggest that toxin pores do not remain associated with bilayer membranes or bilayer fragments derived from cells, which have been lysed. On the contrary, they become dispersed in complexes with phospholipids and possibly also cholesterol. However, fluorescence studies on the CBT perfringolysin have been interpreted on the premise that the lipids with which the toxin is complexed following pore formation exist in a bilayer environment as part of permeabilized membranes (4, 5). Our data suggest that this is not a valid assumption to make and that therefore conclusions on the regions of CBTs that interact with membranes cannot simply be based on fluorescence data, because lipids complexed with CBT oligomers apparently exist in a variety of environments where solid state NMR can be used to distinguish and quantitate in an approximate way.
It is possible to obtain a lower limit for the life-time of the existence of proteolipid rings from the 31P MAS NMR spectra. Two well resolved lines, arising from the two distinct lipid populations, are observed at a CS separation of ~2 ppm (Fig. 4, Table I). The FWHH of the resonance from the nonbilayer environment is ~0.3 ppm. This suggests that any putative exchange of lipid molecules between the bilayer and the nonbilayer lipid environments may occur at a rate much slower than 60 Hz (~1.7 ppm at 500 MHz) or the nonbilayer phase is stable over times exceeding 15 ms.
The freeze-fracture electron micrographs of pneumolysin-containing lipid systems (Fig. 5a) are produced by rapid (~104 degrees/second) freezing and reveal the presence of small lipid vesicles derived from the initial suspension, interacting in a toxin-independent fashion with the remaining lipid. These small vesicles appear in the immediate vicinity of the other lipid structures, trapped between larger lipid aggregates, indicating that such toxin-free structures coexist along with other vesicles or liposome-containing toxin. Bearing in mind that the location of these liposomes is within the pneumolysin-induced aggregate, it is reasonable to deduce that they result from the effect of pneumolysin on membranes. The data suggest that the mechanism of pore formation firstly involves oligomerization on or in a bilayer but that later the oligomeric rings enter free solution, withdrawing a large fraction of the lipid membrane into the rings (Fig. 6).
A number of conductance experiments carried out on CBTs indicate that they create pores of a range of sizes and lifetimes in membranes, the smallest of which may be blocked using divalent cations (24). This observation indicates that pores do indeed form within membranes. What our results indicate is that the precise nature of such pores is still uncertain, and may involve lesions formed at the interfaces of nascent oligomeric structures and lipids (25) and discrete protein complexes within membranes (3) but also large holes in the bilayer alone. Furthermore, effects downstream of oligomerization and pore-formation involving large scale redistribution of membrane components are indicated by our data. It is suggested that the lytic action of pneumolysin on lipid bilayers is a complex combination of pore formation within the membrane, extraction of lipid into free oligomeric complexes, aggregation and fusion of membranes, and membrane destabilization leading to formation of small vesicles. These observations suggest that CBTs may not only permeabilize cell membranes but also cause them to bleb, to aggregate, and to fuse.
The appearance of a large proportion of the lipid and protein as free
oligomer-lipid complexes is also important for understanding the
noncytolytic mechanisms associated with pneumolysin. As mentioned above, pneumolysin has the ability directly to activate the complement system in a nonimmunospecific manner. This occurs by the
classical pathway as a result of the interaction of pneumolysin,
IgG-Fc, and complement components (8). Toxin oligomers are known to be
capable of complement activation (8), and the existence of large
numbers of free oligomers in solution might explain this facility.
Although streptolysin has been reported to activate complement in the
presence of anti-streptolysin antibodies (26), nonimmunospecific
activation of complement has not been demonstrated so far for other
CBTs. The oligomers of streptolysin were also found to have a
hyperactivating effect on complement compared with monomeric toxin.
Such a phenomenon may also occur with pneumolysin, because the domain
through which pneumolysin activates complement (the C-terminal domain)
has a fold very similar to IgG-Fc, with which it interacts during
complement activation (8, 2, 17). IgG itself has a hyperactivating
effect on complement when oligomerized (27), which suggests the
possibility that the lipids associated with free oligomers may
themselves elicit an immune response because nonbilayer (nonplanar)
lipid structures have been shown to be highly immunogenic (28). The
immunogenicity of such structures is thought to result from their high
curvature (28).
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ACKNOWLEDGEMENT |
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We would like to thank David Shotton for his advice on freeze-fracture EM.
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FOOTNOTES |
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* This work was supported by BBSRC Grant 43/SF09211, MRC Grant 99627959MB, and JREI awards from the HEFCE/BBSRC in 1996 and 1997.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Dr., Oxford OX3 7BN, UK.
** A BBSRC Senior Research Fellow. To whom correspondence should be addressed. Tel.: 44 1865 275 268; Fax: 44 1865 275 234; E-mail: awatts@bioch.ox.ac.uk.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M005126200
2 R. J. C. Gilbert, J. L. Jimenez, S. Chen, and H. R. Saibil, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
CBT, cholesterol-binding toxin;
MAS, magic angle spinning;
FWHH, full width
at half-height;
CS (), isotropic chemical shift;
p
and
f, chemical shift of the toxin-dependent
and the toxin-free phospholipid environment;
CSA, chemical shift
anisotropy;
EM, electron microscopy;
LUV, large unilamellar vesicle;
MLV, multilamellar vesicle;
ex
1, rate of
lipid exchange between different phases or environments;
R
1, rate of rotational motion;
PC, phosphatidylcholine;
DCP, dicetylphosphate.
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REFERENCES |
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